UMI INFORMATION TO USERS

Transcription

1 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6 x 9 black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Arbor MI USA 313/ /

2

3 THE EFFECT OF BREEDER HEN AGE ON REPRODUCTIVE CHARACTERISTICS AND SUBSEQUENT EMBRYO AND HATCHLING DEVELOPMENT AND GROWTH OF TURKEYS AND PEKIN DUCKS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Todd J. Applegate, B.S., M.S. * * * * * The Ohio State University 1999 Dissertation committee: Dr. Michael S. Lilbum, Adviser Approved by Dr. Wayne L. Bacon Dr. Joseph S. Ottobre Dr. Sandra G. Velleman Adviser Animal Science Graduate program

4 UMI Number: UMI Microform Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103

5 ABSTRACT To gain insight into how the age of the hen affects selected reproductive characteristics and subsequent embryonic and posthatching development, experiments were conducted to study; 1) hormonal and initial egg production characteristics as influenced by hen age and age at photostimulation; 2) changes in egg component distribution associated with hen age; 3) effects of hen age on yolk sac lipid mobilization and embryonic growth; 4) hen age effects on post-hatch metabolic homeostasis and intestinal development. Reproductive experiments were conducted with the commercial turkey, whereas embryonic and posthatching experiments were conducted with both the commercial turkey and Pekin duckling as experimental subjects. The results of our initial research demonstrated that embryos from young hens developed more slowly than embryos from older hens during the last week of incubation. We hypothesized that these developmental differences are in part due to a proportional increase in yolk mass in eggs from older hens which allows for a greater mass transfer of yolk lipid to the embryo during the last week of incubation. Hypothetically, developmental differences during incubation may be related to problems that occur during the acclimation period after hatching, especially if hatchlings come from very young hens. u

6 In testing this hypothesis, a metabolic challenge was imposed and intestinal development were measured in hatchlings from different hen production ages. The metabolic challenge was tested with a glucose tolerance assay and intestinal development was evaluated by measuring gross intestinal growth (weight and length) during the first week after hatching. Poults and ducklings from young hens remained hyperglycemic longer when given a glucose challenge, suggesting an early impairment in gluocse regulation. Hen production age did not consistently influence poult or duckling weight or length measures of the small intestine after hatching. As weight and length measures are relatively gross measures, final experiments studied the relationship between hen age, poult villus growth and enterocyte proliferation and migration during the first week after hatching. Enterocyte migration was determined after the labeling of proliferating enterocytes with a thymidine analog, bromodeoxyuridine (BrdU), and subsequent immunostaining for BrdU. Poults from the older hens had significantly longer villi and BrdU labeled enterocyte height at hatch, but not after placement. Even though intestinal villi may have been more developed in poults from the older hens at hatch, post-hatching growth of the intestinal system or the poult was not affected. Age-related changes in enterocyte proliferation support the theory that changes in enterocyte fimction occur due to an accumulation of cells rather than to enterocyte turnover rates. In a final study, comparisons were made between the duckling and turkey poult to determine if rates of small intestine development contributed to marked growth differences between the two species during the first week after hatching. By 7 days of age, the duckling s jejunum and ileum were 3.7X heavier, 1.6X longer, and 2.3X more dense (g/cm) and villi iii

7 were 2 A X longer than in the turkey pouit. This phenomenal growth difference aided the duckling in achieving a body weight nearly twice that of the poult during this initial 7 day growth period. IV

8 ACKNOWLEDGMENTS I would like to extend my most sincere appreciation to Dr. Mike Lilbum, for his invaluable advice and support as my graduate advisor at The Ohio State University. Dr. Lilbum s insights and suggestions have been outstanding and are much appreciated. My appreciation is also extended to Dr. Jerry Sell, for his continued guidance, advice, and support throughout my graduate career. I would also like to thank my committee members. Dr. Wayne Bacon, Dr. Joe Ottobre, and Dr. Sandy Velleman for their insight and suggestions throughout my graduate studies at The Ohio State University. My gratitude is extended to Drs. Bacon, Schanbacher, and Velleman for their willingness to share their fecilities. The assistance in and outside of the lab from John Nixon, Dave Long, and Cindy Coy is greatly appreciated. I would also like to extend my gratitude to Dennis Hartzler and the ferm staff for their superb assistance during my experimental work. Additionally, my appreciation is given to Bob Whitmoyer for his assistance with the preparation of photomicrographic plates. I would like to extend special thanks to Dr. Julia Dibner and Marianne Kitchell of Novus International for their willingness for allowing me to conduct my immunohistochemical staining in their lab. Thanks is also given to Dr. Zehava Uni of the Hebrew University of

9 Jerusalem for her assistance with immunohistochemical staining, t i addition, I would also like to thank Maple Leaf Farms for allowing me to work in their research facilities and for research support. I would like to thank the Maple Leaf Staff for all of their assistance, C. Mike Turk, Dr. Dan Shafer, Rhonda Murdoch, Mark Jeffrey, Lisa Weissert, and Ed Ladwig. I would also like to extend my appreciation to British United Turkeys of America, Cooper Farms, and Cuddy Farms for their generous donation of fertile turkey eggs used in my experiments. I would like to thank the Graduate School for supporting my research by awarding me with the Graduate student Alumni Research Award and for sponsoring my last year of studies with a Presidential Fellowship. The financial support and facilities of the Ohio Agricultural Research and Development Center are appreciated. I would like to thank my fellow graduate students, David Jones, Michael Persia, Jill Yaissle, Lisa Dreidger, Steve Younker, Dr. Shih-Tomg Ding and Dr. Keith Turner. Without all your help, suggestions, and fiiendship, none of this work would have been possible. To my parents and in-laws, Martin, Dene, Ken, and Shirley, thank you for your love and support all these years. Without it, I wouldn t be where I am today. Finally, I would like to thank my wife, Andrea. Your encouragement, willingness to listen, love, and your inspiration have made the completion o f this degree possible. VI

10 VITA May 10, Bom - Council Bluffs, Iowa December, B.S., Iowa State University MS., Animal Science, Iowa State University, Ames, Iowa Graduate Research Assistant, Iowa Sate University, Ames, Iowa 1995-present... Graduate Teaching and Research Associate, The Ohio State University vn

11 PUBLICATIONS Applegate, T.J., D. Harper, and M S. Lilbum, Effect of hen production age on egg composition and embyro development in commercial Pekin ducks. Poultry Sci. 77: Applegate, T.J., and M S. Lilbum, Effect of hen age, body weight, and age at photostimulation. 1. Egg, incubation, and poult characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., and M.S. Lilbum, Effect o f hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., W.L. Bacon, and M.S. Lilbum, Effect of age and body weight on plasma concentrations o f luteinizing hormone in turkey hens before and after photostimulation. Dorn. Anim. Endocrinol. 14(6): Applegate, T.J., and M S. Lilbum, Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci. 75: Applegate, T.J., and J.L. Sell, Effect of dietary linoleic to linolenic acid ratio and vitamin E supplementation on vitamin E status o f poults. Poultry Sci. 75: FIELDS OF STUDY Major Field...Animal Sciences vm

12 TABLE OF CONTENTS Abstract... ü Acknowledgments... iv V ita... vü List of Tables...xii List of Figures... xvi Chapters: Page 1. Introduction... I 2. Effect of age and body weight on plasma concentrations of luteinizing hormone in turkey hens before and after photostimulation...33 Abstract Introduction...33 Materials and Methods...34 Results Discussion Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys...41 Abstract...41 Introduction...41 Materials and Methods Results Discussion...45 IX

13 4. Effect of hen age, body weight, and age at photostimulation. 1. Egg, incubation, and poult characteristics of commercial turkeys Abstract...48 Introduction Materials and Methods Results...49 Discussion Effect of hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys Abstract...54 Introduction Materials and Methods Results...55 Discussion Effect of breeder hen age and egg size. 1. Intestinal growth and glucose tolerance of the turkey poult...60 Abstract...60 Introduction Materials and Methods Results...67 Discussion Effect of breeder hen age and egg size. 2. Intestinal villus growth, enterocyte migration and proliferation of the turkey poult Abstract...88 Introduction Materials and Methods Results...93 Discussion... 95

14 8. Effect of hen production age on egg composition and embryo development in commercial Pekin ducks...i l l Abstract... I ll Introduction...I l l Materials and Methods Results Discussion Effect of hen age on intestinal development and glucose tolerance of the Pekin duckling Abstract Introduction Materials and Methods Results Discussion Summary and conclusions Bibliography Appendix A: Turkey and duck comparisons XI

15 LIST OF TABLES Table Eâgê 2.1 Pattern means of luteinizing hormone parameters in plasma o f turkey hens before and after a 3-d exposure to a long-day photoperiod Pattern means of luteinizing hormone parameters in plasma of turkey hens before and after a 3-d exposure to a long-day photoperiod at 24-25, 27-28, and wk BW and absolute and relative weights of the P. Major and abdominal fat of turkey hens after a 3-d exposure to a long-day photoperiod at 24-25, 27-28, and wk Body weight, relative abdominal fat weight, relative Pectoralis major weight, and relative liver weight of turkey breeder hens fed either mash or pelletted diets Egg weight, yolk weight, relative weight, dry matter, lipid concentration, and albumen weight and relative weight from turkey breeder hens fed either mash or pelletted diets Yolk sac weight, dry matter, lipid concentration, and correlation of yolk sac dry matter with lipid concentration of posthatch poults from turkey breeder hens fed either mash or pelletted diets Egg weight, weight at 25 d of incubation (transfer weight), relative transfer weight, poult weight at hatching, and poult weight relative to initial egg weight Egg weight, weight at 25 d of incubation (transfer weight), relative transfer weight, poult weight at hatching, and poult weight relative to initial egg weight from turkey breeder hens fed either mash or pelletted diets...45 XU

16 Tat)lg Page 4.1 Turkey breeder hen carcass characteristics 3 wk after photostimulation Turkey breeder hen reproductive characteristics 3 wk after photostimulation Egg weight, yolk weight, relative weight, dry matter, albumen weight, and relative albumen weight from turkey breeder hens during the first 10 wk of production Egg weight, weight at 25 d of incubation (transfer weight), relative transfer weight, poult weight at hatching, and poult weight relative to initial egg weight from turkey breeder hens during the first 10 wk of production Egg weight, egg weight at 25 d of incubation (transfer weight), poult weight, and poult weight relative to egg weight at set from turkey breeder hens during the first 10 wk of production Yolk-free embryo weight and relative weight to initial egg weight, and liver weight and relative weight at 21 and 25 d of incubation of embryos from turkey breeder hens during the first 10 wk o f production Yolk-free embryo weight and relative weight to initial egg weight, and liver weight and relative weight at 21 and 25 d of incubation of embryos from turkey breeder hens during the first 10 w k o f production Yolk sac weight and relative weight to initial egg weight at 21 and 25 d of incubation of embryos from turkey breeder hens during their first 10 wk of production Yolk and yolk sac weight, dry matter, and lipid composition of selected yolk sacs from embryos at 21 and 25 d of incubation from different ages of turkey breeder hens The lipid composition o f selected yolk sacs from embryos at 21 and 25 d of incubation from different ages of turkey breeder hens Egg and poult body weight from different egg weight classes and ages of turkey breeder hens. Experiment 1 77 xm

17 Table 6.2 Yolk-free BW o f poults from different egg weight classes and ages of turkey breeder hens. Experiment Yolk-free BW o f poults from different egg weight classes and ages of turkey breeder hens. Experiment Glucose tolerance assay of 4-d-old poults from different egg weight classes and ages of turkey breeders Distal jejunum villus height of poults from different egg weight classes and ages of turkey breeder hens Distal jejunum bromodeoxyuridine labeled enterocyte height from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens Distal jejunum bromodeoxyuridine labeled enterocyte height (% of crypt-villus axis migrated ) from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens Distal jejunum bromodeoxyuridine labeled enterocyte migratory rate from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens Proliferating cell nuclear antigen positive cells from distal jejunal viui from poults from 85 to 90 g eggs from differing ages of turkey breeder hens Egg, yolk, and albumen weights from duck breeder hens during the first 16 wk of production Yolk sac weight and relative weight from duck breeder hens during the first 16 wk of production Yolk sac dry matter concentration and content and lipid concentration and content from embryos from duck breeder hens during the first 16 wk of production Embryo weight (devoid of the yolk sac) and relative weight from duck breeder hens during the first 16 wk of production XIV

18 Table Page 9.1 Egg weight and composition from 32- and 44-week-old Pekin ducks. Experiment Duckling body weight from 32-and 44-week-old hens. Experiment Embryo and duckling body weight, devoid of the yolk sac, from 32- and 44-week-old Pekin ducks. Experiment Yolk sac weight o f embryos or ducklings from 32- and 44-week-old Pekin ducks. Experiment Yolk and yolk sac dry matter and lipid content o f duck eggs and embryos from 32- and 44-week-old Pekin ducks Egg weight, egg weight at transfer, and duckling body weight at hatch and 3 days after hatching from 33- and 49-week-old Pekin ducks. Experiment Glucose tolerance assay of 3-day-old ducklings from 33- and 49-week-old Pekin ducks. Experiment A. 1 Duck and turkey egg weight and egg component comparisons A.2 Duckling and poult yolk sac weight comparisons A. 3 Duckling and poult body weight (devoid of the yolk sac) comparisons A.4 Duckling and poult relative duodenum weight comparisons A. 5 Duckling and poult relative jejunum/ileum weight comparisons A. 6 Duckling and poult body weight (devoid of yolk sac), distal jejunum villus height, and crypt depth comparisons XV

19 LIST OF FIGURES Figure Page 1.1 Days to first oviposition in turkey breeder hens Secretory profiles of LH from three photosensitive hens during a short-day photoperiod and after a 3-d exposure to a long-day photoperiod at 24-25, 27-28, and wk-of-age Relationship between poult weight at hatching and egg size from 36- and 55-wk-old hens Duodenum weight, length, and density (g/cm) of poults from different egg weight classes and ages of turkey breeder hens. Experiment Jejunum and ileum weight, length, and density (g/cm) of poults from different egg weight classes and ages o f turkey breeder hens. Experiment Glucose tolerance assay of 4 and 7-d-old poults from different egg weight classes and ages of turkey breeder hens. Experiment Bromodeoxyuridine (BrdU) labeled enterocyte height, relative BrdU labeled enterocyte height (% of cryt-villus axis migrated), and BrdU labeled enterocyte migratory rate of distal jejunal villi from poults from 27 d of incubation to 7 d of age Distal jejunum villi stained using a monoclonal antibody for bromodeoxyuridine and counter-stained with methyl green from poults at 27 days of incubation and 5 days of age Distal jejunum villi stained using an antibody for proliferating cell nuclear antigen and counter-stained with Hematoxylin and Eosin from poults at 1 and 5 days of age XVI

20 Figure Eâgê 9.1 Duodenum weight, length, and density (g/cm) of ducklings from 32- and 44-week-old Pekin ducks. Experiment Jejunum and ileum weight, length, and density (g/cm) o f ducklings from 32- and 44-week-old Pekin ducks. Experiment Villus height and crypt depth from distal jejunum sections from 32- and 44-week-old Pekin ducks, Experiment Glucose tolerance assay o f 3- and 6-day-old ducklings from 32- and 44-week-old Pekin ducks. Experiment A. 1 Duckling and poult duodenum weight, length, and density (g/cm) comparisons A.2 Duckling and poult jejunum/ileum weight, length, and density (g/cm) comparisons A. 3 Duckling and poult distal jejunum villi comparison at 3 d of age xvu

21 CHAPTER 1 INTRODUCTION Hen age and reproductive development and performance The growth potential of the commercial turkey has changed considerably over the last 30 years (Lilbum, 1994) although the improvement in commercial traits is negatively correlated with egg production and other reproductive traits (for review, Nestor, 1985; Dunnington and Seigel, 1996). This correlation, albeit negative, is not consistent however, as within modem, commercial (growth-selected) turkey strains, considerable variability exists in early (2 wk) egg weights, days to 50 % production, and numbers of eggs laid through through the first 23 wk of lay (Slopes, 1995). For maximal reproductive efisciency, the turkey industry utilizes the photosensitivite nature of the immature hen. The photostimulatory response of birds is different than what is observed in seasonal breeding mammals, in that the eye and the pineal gland do not play a critical role in the response (for review. Sharp, 1993). Nevertheless, photoperiodic cues to the gonadotrophin-releasing hormone (GnRH) pulse generator indicate the onset of sexual maturation resulting in a subsequent increase in circulating baseline concentrations of luteinizing hormone (LH) in sheep (Foster e/ or/., 1985) and turkeys (Bacon and Long, 1995).

22 Photoperiodic cues and induction of sexual maturity, however, are equally dependent on physiologic age (Dunn et a l, 1990), body composition (SoUer et al., 1984), and photoperiodic history (Sharp, 1993). The age of the turkey hen at photostimulation can greatly influence the subsequent rate of reproductive development. In a review of studies over the last 37 years, the average time to first oviposition after photostimulation declined with increasing age of the hen from approximately 37 d at 24 wk of age, to 14 d at 36 wk of age (Figure 1.1; Leighton and Shoffiier, 1961a,b; Sexten and McCartney, 1973; Woodard, 1974; Hocking, 1992; Siopes, 1992). Commercial turkey hens are typically exposed to short photoperiods (8 hr light: 16 hr dark) beginning at approximately 18 wk of age and are photostimulated (daily photoperiod is increased to > 14 hr light per day) between 29 and 31 wk of age. These increases in the daily photoperiod are made at chronologic (weeks) as opposed to physiologic (body weight) ages. These age-specific increases in the photoperiod, are largely reflective of the perception that photostimulating hens at younger ages will have negative effects on overall reproductive performance. The few controlled experiments that have been conducted, utilized strains of turkeys that are much smaller than the modem Large White commercial strains of today and these experiments suggested that hens achieve their optimal reproductive BW at approximately 30 wk of age (Leighton and Shoffiier, 1961a,b; Shoffiier et a i, 1962; Wilson et a i, 1962; Leighton and Potter, 1969; Woodard et a i, 1974). In these studies, an unsatisfactory reproductive response was defined as: delayed onset of sexual maturity, decreased egg production, reduced consistency of early egg production, reduced persistency o f lay during the first cycle, prolonged laying o f small eggs, and reduced hatchablity.

23 While 30 wk may have been the age at which the smaller strains typically achieved a satisfectory reproductive BW 25 to 35 years ago, today s commercial strains, may achieve an optimal target BW well before 30 wk of age. Reproductive performance, however, has been reported to be unsatisfectory if these hens are photostimulated well before 30 wk of age. Siopes (1992) photostimulated hens at 24 or 30 wk of age and the number of eggs laid over the first 20 wk of lay was similar. However, the older hens produced heavier eggs and poults through the first 11 wk of lay. Hocking (1993) photostimulated hens at 24 wk and concluded that at this early age, vent size is inadequate in relation to initial egg size and this contributed to an increased incidence of oviductal prolapse. Hocking (1992a) also reported an increased proportion of poults with splayed legs, unhealed navels, and generally smaller poults when hens were photostimulated as early as 18 or 24 wk of age. As selection for growth has greatly increased, commercial hens of today are capable of reaching an optimal target BW well before 30 wk of age. The industry often implements a wide variety of feed restriction programs during the weeks prior to photostimulation, as excessive weight at photostimulation results in poor nesting behavior, higher incidence of oviductal prolapse, and excessive feed costs (for review, Hester and Stevens, 1990). The best time for photostimulation is a combination of age, age appropriate body weight, body composition and photo-periodic history. After photostimulation the turkey hen s reproductive tract will rapidly develop, such that first oviposition occurs within 2 to 3 wk. The coordination of ovarian and oviductal development, however, may be dependent on factors other than those described for the simple onset of sexual maturity. Renema et al. (1998) reported that the average age to sexual maturity after photostimulation was 29.8 d in

24 unselected Bronze turkeys, but the oviduct weight achieved a mature weight 3 d earlier than did the ovary. Similar results were reported by Melnychuk et al. (1997) using one strain of commercial hens but in hens from a different commercial strain, development of the ovary was accelerated. Melnychuk et al. (1997) concluded therefore that selection criteria (i.e. growth) may result in different rates of ovarian and oviductal development after photostimulation. This supports the data of Lilbum and Nestor (1993) who reported a larger increase in relative oviduct weight during the first 49 d of lay in a line of turkey hens selected for growth compared with a line selected for egg production. Several factors will influence the dynamics of follicle and yolk formation in turkey hens. These include, but are not limited to, selection for growth, age and BW at photostimulation, and age during an egg production cycle. Selection for growth in turkeys increases the number of ovarian follicles in development, atretic follicles, numbers of internal ovulations, and numbers of follicular pairs (double-yolked eggs). Taken together, the total number of settable eggs is significantly reduced (Nestor, 1985). The control of BW during rearing in today s commercial hens may reduce the number of multiple-follicle sets (Hocking, 1992b; Renema et a i, 1995) without negatively affecting shell quality or yolk size (Renema et al., 1995). The endocrine control of follicular development in these particular circumstances has not been extensively studied in the commercial turkey hen. Hocking (1992b) hypothesized that multiple-follicle sets in heavy-strains of turkeys were caused by an increased recruitment of follicles, rather than differences in ovarian gonadotropin-hormone

25 stimulation. This observation was made after observing similarities in relative ovary weights at first oviposition between ad libiitum fed and feed restricted hens, which were photostimulated at 24 or 30 wk of age Hen age and egg composition Hen age at sexual maturity is positively correlated -with egg size and egg size is positively correlated with poult weight (Reinhart and Moran, 1979; Shanawany, 1987). Increased egg weight with advancing hen age, however, is not positively correlated with proportional increases in egg components. Reidy et al. (1994) reported that the weight of eggs from commercial turkey breeders increased approximately 11% between the onset of lay and 24 wk of production. During this time period, yolk weight increased 21% but albumen weight only increased 7%. The magnitude of these changes differed slightly depending upon commercial genotype. In a subsequent comparison of eggs from British United Turkeys of America male- and female-line hens, male-line hens were 11% heavier but no significant differences in yolk weight were observed. In turkey hens from three commercial strains, Siopes (1995) reported similar shifts in yolk-to-albumen ratios from 0.47 to 0.55 between 8 and 20 wk after photostimulation. Similar changes in disproportional yolk and albumen investment into the egg have also been noted in the chicken (Cunningham et al., 1960a,b; O Sullivan et al, 1991). Nestor and Noble (1995) compared egg and egg component weights in a random-bred turkey line (RBC2) and a subline of RBC2 selected for 16 wk BW (F-line). Similar to the results of Reidy et al. (1994), eggs from the F-line hens were 10% heavier but with no corresponding changes in absolute yolk weight.

26 As the hen ages through the first cycle of lay, clutch size is diminished and a greater number of first of sequence eggs are laid (Bacon and Nestor, 1979, Lemer et al., 1993). This difference in first of sequence eggs has been associated with reduced fertility and hatchability of those eggs (Bacon and Nestor, 1979, Lemer et al., 1993). In chickens, first of sequence eggs have larger yolks, contain more albumen and accumulate more shell than other eggs in the sequence (Etches, 1996). Yolk deposition in the egg has been the subject of numerous excellent reviews (GriflBn etal., 1984; Walzem,1996). As summarized in these reviews, all the major plasma proteins that are incorporated into the yolk are synthesized by the liver, with the exception of immunoglobulins. The major yolk protein precursors are either absent or exceptionally low in immature hens and increase significantly with increasing concentrations o f estrogen. Liver lipid concentrations increase 2- to 3-fold prior to first oviposition and is largely accounted for by de novo synthesis of triglycerides. The synthesis of two of the major yolk precursor proteins, vitellogenin and apo-vldl H appear to be completely dependent upon estrogen. After ultra-centrifugation, the yolk will separate into three distinct layers: an upper VLDL layer which is 88 % lipid (mostly triglyceride) and accounts for 65% of yolk dry matter; a middle water soluble layer which contains maternal y-globulins; and a lower granular layer which accounts for 25% of yolk DM and contains vitellogenin. Vitellogenin is a phosphorylated protein which binds Ca-H-, and accounts for the high levels of circulating bound Ca++. The VLDL of laying hens differ greatly from those of immature hens. The VLDL of non-laying hens contain at least six different apo-lipoproteins, whereas laying hens only contain two, apo-

27 VLDL n and apo-b. Although, apo-b is synthesized in immature hens, its concentration is much greater in laying hens than in immature hens or males (Griffin et a i, 1984; Walzem, 1996). Large fenestrae between theca interna endothelia allow for passage of massive amounts of lipoproteins and other yolk components from the circulation. The basal lamina then acts as a coarse filter for interstitial fluid from plasma. The granulosa layer also changes during oocyte maturation from stratified layers to a single cell layer and from columnar to cuboidal epithelium with broad channels between them. Both the apo-b containing VLDL and vitellogenin bind to a 95 kda receptor on the oocyte plasma membrane and are internalized via receptor mediated internalization. Directly above the germinal disk, however, yolk accumulation does not occur as extensively (Griffin et a l, 1984; Walzem, 1996). Hen age and embryo growth and metabolism Yolk lipid has been estimated to supply the chick embryo with 90% of its caloric needs (Freeman and Vince, 1974). Approximately 80% of yolk lipid is mobilized during incubation by the embryonic poult with the largest proportion being utilized during the last week o f incubation (Ding et a i, 1995; Ding and Lilbum, 1996). As reviewed by Noble and Cocchi (1990), the yolk sac membrane of the egg is initially comprised of two inner layers, vitelline and perivitelline layers, and two outer layers, a continuous and an extra vitelline membrane. The inner layers are formed during follicular maturation, whereas the outer layers are formed during passage of shed ovum along the oviduct. At the onset of incubation, the yolk sac membrane is comprised of two structural components, an outer mesoderm containing flattened, supportive cells and an inner endoderm

28 containing simple columnar epithelia. By the fourth day of incubation in the chicken, the inner endoderm develops with the formation of elaborate folds assuming a microvillus structure and a capillary network within the villi. In the early stages of incubation, uptake of yolk droplets occurs via endocytosis from bristle coated pits (Mobbs and McMillan, 1981), whereas uptake later in incubation occurs via non-specific phagocytosis (Lambson, 1970). Yolk lipid droplets initially fuse with intra-cellular yolk droplets for temporary storage. Lipid hydrolysis and reesterification along with re-assembly into new lipoproteins occurs prior to transport into embryonic circulation (Noble and Cocchi, 1990). Noble et al. (1986a) attributed hatchability and livability problems observed in embryos from young broiler breeder hens to ineflbcient mobilization and assimilation of yolk sac lipid by the embryo between 15 and 19 days of incubation. This assertion was based on minimal disappearance of lipid and its major subclasses from the yolk sac and subsequent deposition into the liver in embryos from 25 week-old versus 41 week-old broiler breeder hens. The most notable change they reported was a considerable increase in the proportion of cholesterol esters within the yolk sac membrane in the embryos of young hens. In the chick, the majority of cholesterol estérification occurs within the yolk sac membrane during incubation. (Noble and Connor, 1984). During the period of maximal lipid transfer out of the yolk and into the developing poult, there is a parallel increase in hepatic lipid accumulation, 70% of which is cholesterol esters (Ding et a i, 1995). At 19 days of incubation, chick embryos from young broiler breeder hens have lower plasma lipid concentrations, lower plasma lipoprotein concentrations, lower frver lipid concentrations, and a marked reduction in cytosolic accumulation of lipid in 8

29 the liver (Yafifei and Noble, 1990). This is accompanied by increased in vitro hepatic oxidation of Cj4 labeled palmitic acid at 15 days of incubation by embryos from young hens (Noble et al, 1986b) and increased endogenous hepatic cholesterol biosynthesis at 15 and 19 days of incubation (Vajda et al., 1994). The embryos from young hens, therefore, appear to be overcompensating for reduced lipid mobilization out of the yolk sac and these increased metabolic efforts may negatively impact the energy reserves needed during late incubation and for early posthatch growth. The actual length of the incubation period has also been reported to vary with advancing hen age, i.e. eggs from older hens are thought to hatch earlier than those from younger hens (Crittenden and Bohren, 1962; Smith and Bohren, 1975; Shanawany, 1984). Shanawany (1984) hypothesized that a shortened incubation period associated with eggs from older broiler breeder hens is due to several factors: a shortened clutch length with a higher proportion of first of sequence eggs; more time spent in the oviduct prior to oviposition (Mather and Laughlin, 1979); increased shell porosity leading to increased gas exchange (Christensen et ai, 1996); increased efficiency of transfer of nutrients from the hen into the egg; and/or enhanced nutrient utilization by embryos from older hens. Changes in egg component characteristics have been correlated with changes in embryo metabolism and hatching. For example, Christensen et al. (1996) and Rahn et al. (1981) attributed declines in hatchability beginning at mid-lay to a plateau in eggshell conductance at that stage of lay. Embryonic growth in eggs from hens at mid- to late-lay was also greater, resulting in increased hatching poult weights as a proportion of egg weight.

30 The aforementioned differences in hatching times, embryonic lipid transfer, and carbohydrate and lipid metabolism have been hypothesized to affect overall embryo growth and development. At 18 d of incubation, eggs from 44-wk-old broiler breeder hens produced heavier embryos than eggs o f similar size from 28-wk-old hens (Shanawany, 1984). These embyronic differences, however, did not carry through to hatching. Other researchers have noted positive effects of hen age on embryonic and hatching BW, independent of egg weight differences, in Japanese quail (Yannakopoulos and Tserveni-Gousi, 1987) and turkeys (Yannakopoulos, 1989; Christensen et al., 1996). The direct mechanism of this growth differential during incubation is uncertain. Differences in lipid transfer and metabolism described by Noble e/a/. (Noble era/., 1986a,b; Yaffei and Noble, 1990; Vadjaera/., 1994) were conducted in eggs from young broiler breeders and older broiler breeders, with large differences in egg size. The differences in embryonic lipid assimilation, therefore, could be due to either the age of the hen or to the size of the egg. In addition, differences in relative growth and hatchability of turkey embryos from different hen ages have been associated with differences in hepatic and cardiac glycogen utilization during hatch as mediated by thyroxine and triiodothyronine (Christensen et al., 1996). This difference may be a secondary effect, as differentiation in embryonic growth occurs prior to this time. Ding et al. (1995) reported that in two genetic lines differing greatly in BW and egg weight, the weight of the yolk-free embryo was similar until 22 d of incubation, the age at which rapid mobilization of lipid from the yolk to the embryo begins. 1 0

31 Hen age and post-hatching growth and metabolism Several studies with chickens have reported a positive correlation of egg weight and initial post-hatch growth (for review, Wilson, 1991). Studies with turkeys have shown a similar positive correlation between egg weight and BW at 2 wk of age (Moran, 1990), but the correlation decreases and becomes negligible at older ages (Mussehl and Ackerson, 1934; Payne et al., 1957; Moran and Reinhart, 1981). As with the turkey, duck egg weight is also postively correlated with the weight of the duckling at hatch (Shanawany, 1987). In Pekin hatchlings, Knizetova et al. (1988) reported that when hatching weight was related to egg weight at set, newly hatched ducklings from 95 g eggs were 2.5% heavier than hatchlings from 75 g eggs (61.2 and 58.7 %, respectively). In 80, 90, and 105 g eggs collected from Pekin hens in their sixth month of production, differences in BW were maintained from hatching to 42 and 35 days of age in males and females, respectively, after which the differences declined (Knitzetova et al., 1992). Cerveny et al. (1988) reported that egg weight class influenced duckling BW at 7 wk of age when eggs were collected from hens in their second month of production, but did not affect 7 wk BW when eggs were collected during the sixth month of production. At hatching, the poult is nearly deplete of glycogen (Roseborough et al., 1979), and has a body composition containing nearly 25 to 30 % lipid (dry matter basis. Noble and Cocchi, 1990). The poult is in a gluconeogenic state and will remain so until it consumes exogenous nutrients (Romanoff 1960). Commercial turk^ poults are subjected to numerous hatchery associated processing steps before they are placed and given access to feed. These processing steps include desnooding, toe clipping, vaccination, sexing, and prolonged 11

32 transport times to external growers. Hatchery processing combined with metabolic changes associated with acclimation to an external nutrient sources may contribute to peaks of early poult mortality beginning at approximately 4 d of age (Phelps et al., 1987a). After initial feeding, the poult shifts to glycolytic-based energy metabolism (Donaldson and Liou, 1976). Reductions in glucose-6-phosphatase activities occur within 2 hr of feeding, but lipogenic enyme activities (acetyl-co A carboxylase, fatty acid synthetase and fatty acid desaturase) do not increase until 8 hr after feeding. Some researchers, however, have questioned the poult s ability to adequately make and regulate this shift in metabolism. For example, Latour et al. (1995) concluded that homeostatic mechanisms of metabolism in the chick are incomplete in the chick fi'om 1 to 5 d of age based on non-homeostatic fluctuations in daily plasma concentrations of corticosterone (± 8 ng/ml), triglyceride (± 100 mg/dl), and glucose (± 220 mg/dl). In addition, activation of glycogen synthetase is incomplete in the d-old poult but gradually increases during the first wk of age (Roseborough et a i, 1979). Homeostatic regulation of glucose concentrations also appears to be aberrant when poults are subjected to a short-term exposure to low temperatures (21 C, Donaldson and Christensen, 1991). Other research, however, does not support a hypothesis of inadequate homeostatic mechanisms. Houpt (1958) reported that when chicks were fasted beginning at hatch, 2, 6, 10, or 14 d of age, they were able to regulate their plasma glucose concentrations (±30 mg/dl) for 4 to 6 d, after which time acute hypoglycemia occurred followed by death. 1 2

33 Several authors have suggested that after hatching, the residual yolk sac is a significant source of energy for the poult during its acclimation period prior to being completely resorbed by 5 to 6 d of age (Phelps et al., 1987b). Yolk resorption occurs most rapidly after the onset o f feeding (within 3 to 4 d of age, Romanoff, 1944). At hatch, the yolk sac of the poult contains between 0.6 and 2.5 g of lipid (Ding and Lilbum, 1996). This residual lipid contains approximately 1200 mg of triglyceride (Ding et a i, 1995) supplying only 8 to 9 kcal of metabolizable energy to the poult (Lilbum, 1998). Ding et al. (1995) also reported that the residual lipid contains 400 mg of phospholipid and they hypothesized that the residual yolk sac may serve more as a source of structural lipids than as an energy source p er se. A paucity of data exists relative to maternal influences on the post-hatch acclimation period. McNaughton et al. (1978) reported that chicks from young hens had a higher mortality after hatching compared with chicks from old hens. Latour et al. (1996) reported elevated serum cholesterol and lower glucose concentrations in newly hatched chicks from 26- vs 36- or 48-wk-old broiler breeder hens. Similarly, Daly and Peterson (1990) noted lower plasma glucose concentrations in chicks from 27- vs 60-wk-old hens, but the differences in plasma glucose concentrations were not apparent when chick weight was taken into consideration. Turner (1995) reported a delayed decline in plasma uric acid, delayed production of pancreatic lipase, and delayed production of jejunal maltase in poults from young breeder hens. Maternal influences have been documented to influence survivabuty in wild duck species. In wild Mallards, ducklings from heavy eggs (> 56.4 g eggs) were heavier at hatch than ducklings from small eggs (< 48.0 g eggs) and were able to maintain homeothermy at 13

34 lower temperatures, with a lower metabolic rate (Rhymer, 1988). Dawson and Clark (1996) reported that Lesser Scaup ducklings from larger eggs, hatching later in the breeding season, had a 20 % greater chance of survival through 2 wk of age. Intestinal development During the early posthatch growth and development period, there is a tremendous energetic allocation to gastro-intestinal tract (GIT) growth at the expense of most other body parts (Konarzawski er a/., 1990). If a greater proportion of hatchlings from older hens develop more completely in ovo and/or hatch fractionally earlier, they could have a more completely developed GIT when given access to feed and water and which might minimize the aforementioned metabolic effects encountered within the first days of life. Intestinal development has been the subject of recent reviews, for a more detailed discussion on digestive tract development in poultry, one should review Moran (1985), Dibner et al. (1996), Sell (1996), and Noy and Sklan (1997). Within the embryonic chick intestine, the brush border begins to develop as a network of microfilaments which aggregate to form uniform projections as early as 7 to 11 d of incubation (Chambers and Grey, 1979). The microfilaments elongate to form rootlets between 11 and 15 d and further forms a terminal web and microvilli between 19 d of incubation and 5 d of age. This provides both structure and support to the brush border epithelium (Chambers and Grey, 1979). Other researchers have also noted villi containing short microvilli as early as 16 d of incubation (Overton and Shoup, 1964; Lim and Low, 1977). At hatch, however, portions of the lower small intestine still contain epithelial discontinuities (Bayer et al., 1975). In addition, formation of goblet cell pores and extensive convolution of intestinal villi does not occur until 14

35 approximately 1 wk of age or later (Bayer et al., 1975). In addition to being morphologically immature at hatch, the intestine is also functionally immature in terms of digestive and absorptive capabilities (Holdworth and Hastings Wilson, 1967; Sell et a l, 1991; Obst and Diamond, 1992). During the first wk affer hatching, the poult s small intestine increases in weight 9-fold and doubles in length (Sell et a l, 1991). As a proportion of yolk-fi'ee BW, this represents an increase in relative weight of the small intestine firom 1.6 % at hatch to 6.9 % by 8 d of age (Sell et al., 1991). The intestine of the Pekin duckling has a similar 5.5 fold increase in weight between 1 and 7 d of age (1.4 and 4.5% ofbw, respectively; Baranyiova etal., 1983). In chicks, the small intestine will increase in length over the first 5 d after hatch, even during an imposed fast (Baranyiova, 1972). Increases in the relative weight of the small intestine, intestinal diameter, and villus length only occur after feeding, however (Baranyiova, 1972; Baranyiova and Holman, 1976). Energy for support of rapid GIT growth during the acclimation period is at the expense of all other tissues and organs (Konarzewski et al., 1990). Several authors have described the intestine as a relatively flexible system which can undergo morphological and functional adjustments depending on the demands of the organism (Moran, 1985; Obst and Diamond, 1992; Starck, 1996). For example. Uni et al. (1995) noted a positive correlations of jejunal and ileal villus volumes, and enterocyte density with feed intake in chicks during the first 2 wk of age (r=0.77, 0.67, and 0.62, respectively). Functional maturation o f the small intestine is both a physical and physiological process and is one of the main constraints to early growth after hatching (Konarzewski et al., 1989, 1990; Ricklefs et a i, 1998). Physiological maturation (in terms of digestive and 15

36 absorptive functionality) o f the digestive tract involves the increased production o f pancreatic and intestinal enzymes (Nitsan et al., 1991a,b; Sell et al., 1991; Pinchasov and Noy, 1994) and changes in nutrient transporters (Holdsworth and Hastings-Wilson; 1967; Shehata et al., 1984; Obst and Diamond, 1992). However, the physical development of the gastrointestinal tract, namely the increase in surface area of the small intestine, can be a more limiting factor to early growth than changes in digestion or nutrient transporters (Uni etal., 1995, 1996). In the chick, villus length throughout the small intestine increases over two-fold during the first 5 d after hatch (Dibner et al., 1996), villus volume (mmvcm^) increases 3- to 4- fold from hatch to 10 d of age (Uni et al., 1995), and the apical surface area of jejunal enterocytes increases from 300 to 410 pm^from 1 to 14 d of age (Ferrer et al., 1995). Other morphologic changes include changes in the rates of intestinal crypt cell proliferation and enterocyte turnover (Starck, 1998). Enterocyte turnover rates in the small intestine were originally reported to take 2 d in the chick (Imondi and Bird, 1966) but has been more recently reported to vary from 2.8 to 2.9 d in the hatchling to 8.9 to 10.6 d in adult Japanese quail (Starck, 1996). Delayed access to feed greatly affects intestinal morphology and growth after feeding. Baranyiova and Holman (1976) reported that when chicks were fasted between hatching and 5 d of age, villus height and intestinal diameter did not change. In addition, they noted changes in intestinal epithelia in the zone of extrusion that suggested a reduced turnover rate of intestinal epithelia. Even after chicks are given access to feed, jejunal and ileal villus volume is reduced, crypt depth is reduced and crypt morphology remains abnormal. Using scanning electron microscopy. Uni et al. (1998a) also reported clumping of jejunal microvilli 1 6

37 in fasted chicks as compared with fed control chicks. Short-term fasting after hatching has also been reported to slow or stop GIT epithelium in the G1 phase and slow cells in the S phase of the cell cycle (Cameron et al., 1964). This may also be reflected in morphological changes associated with festing. Short-term changes in villus morphology may not, however, adversely affect subsequent digestion and absorption of nutrients upon refeeding. Transient reductions in feed intake up to 75% of ad libitian intake in 6-wk-old chickens, for example can substantially reduce villus height but does not influence nutrient utilization upon refeeding (Michael and Hodges, 1973). Other morphological changes in villus height have been reportedly influenced by: feed restriction, competition with normal GIT microflora, coccidial infections, dietary fiber content, and changes in growth or productive needs (Moran, 1985). Selection for growth has also been reported to influence a number of intestinal development characteristics. During the first wk after hatching, growth-selected lines of chicks have been reported to have greater increases in small intestine weight (absolute and relative to BW) and length (Mitchell and Smith, 1991), longer villus height and perimeter (Uni etal., 1995), greater crypt size and enterocyte migration rate (Smith et al., 1990) and greater villus volume and enterocytes per villus (Uni et al., 1995, 1996) than unselected or small strains. Several distinct differences should also be noted when comparing avian and mammalian intestinal morphology. Uni etal. (1998b) determined that enterocyte proliferation in 18- to 36-d-old chicks was not confined to the crypt region as in mammals, rather the proportion of cells engaged in proliferation (proliferating cell nuclear antigen-positive cells) were 55, 32, and 8% in the crypt, mid-villus, and upper villus, respectively. Also, distinct 17

38 expression of maltase and sucrase within differing regions was not apparent in the chick as it is in mammals. This suggests that discrete zones of proliferation and differentiation that exist in mammalian villi are not evident in avians (Uni et al., 1998b). Smith and Peacock (1989) reported that microvillus length of chicken enterocytes were much longer (3.3 vs 1.6 pm) and the rate of enterocyte migration much less (4.0 vs 8.8 pm/hr) than values reported for mammals. Recent evidence suggests that the yolk stalk (vitelline diverticulum) can provide a passageway for transport of yolk material into the small intestine up to 72 hours after hatching and this yolk material could be detected as proximal as the duodenum (Esteban et a i, 1991; Noy et n/., 1996). Jeurissen etal. (1991) reported that colloidal carbon injected into the yolk sac of chicken eggs at 10 d of incubation was subsequently absorbed from the yolk sac into the lymphoid tissue around the vitelline diverticulum and was subsequently transported to leukocytes and mononuclear phagocytes. This transport thereby suggests a means for physical passage of yolk-derived antigens which could then aid in early immunological maturation of the intestine. Insulin and IGF-I have been found to be deposited into the yolk by the hen (Scavo et a i, 1989) and both have been demonstrated to significantly influence metabolic, growth, and differentiation processes during early embryonic development (de Pablo etal., 1990). In domestic farm animals, the stimulatory effects of maternal colostrum and milk on intestinal development via various growth factors (insulin, EGF, IGF-I and IGFn) has received considerable attention (Odle et al., 1996 for review). If such growth factors are transported into the intestinal lumen from the yolk sac, they could potentially stimulate intestinal maturation. Uni et al. (1998a) noted that yolk sac ablation o f chicks at hatching 1 8

39 caused a transient decrease in villus volume and crypt depth thoughout the small intestine. Intubation with yolk contents after yolk sac ablation, however, increases body weight, enhanced pancreatic and liver development, and restored relative pancreatic lipase activity when compared with ablated chicks (Nitsan et a i, 1995). 19

40 Experimental Objectives To gain insight into how the age of the hen affects reproductive characteristics and subsequent embryonic and posthatching development, this dissertation research was designed to accomplish the following; 1. Determine if hormonal and egg production characteristics are affected by hen age and age at photostimulation; 2. Characterize changes in egg component investment as the hen ages; 3. Ascertain the effects o f hen age on yolk sac mobilization and embryonic growth; 4. Investigate the posthatching effects of hen age on growth, metabolic homeostasis, and intestinal development. Reproductive characterization experiments were conducted with the commercial turkey, whereas embryonic and posthatching experiments were conducted using the commercial turkey and Pekin duckling as experimental subjects. 2 0

41 Figure 1.1. Days to &st oviposition in turkey breeder hens. Data represent mean days to first oviposition as reported by Leighton and Shoffiier, 1961a (Standard Bronze), b (Standard Bronze/Broad Breasted Bronze/Broad Breasted White/and Jersey Buff cross); Sexten and McCartney, 1973 (Large white); Woodard eta l., 1974 (Broad Breasted White); Hocking, 1992 (medum, British United Turkeys of America Strain 71); and Slopes, 1992 (Large White, Nicholas). 2 1

42 First Oviposition of Turkey Hens [y t o % v = 0.154x^ x = Age at photostimulation (wk) I

43 References Bacon, W i., and D.W. Long, Changes in plasma luteinizing hormone concentration in turkey hens after switching from short-day to long-day photoperiods. Dom. Anim. Endocrin. 12: Bacon, W i., and K.E. Nestor, Reproductive traits of first eggs of clutches vs. other clutch positions in turkeys. Poultry Sci. 58: Baranyiova, E., Influence of deutectomy, food intake and fasting on the digestive tract dimensions in chickens after hatching. Acta Vet. Bmo 41: Baranyiova, E., and J. Holman, Morphological changes in the intestinal wall in fed and fasted chickens in the first week after hatching. Acta Vet. Bmo 45: Baranyiova, E., A. Holub, and E. Ponizilova, Changes in the mass and chemical composition of the gastrointestinal tract and fiver of ducks in the first two months after hatching. Acta Vet. Bmo 52: Bayer, R.C., C.B. Chawan, F.H. Bird, and S.D. Musgrave, Characteristics of the absorptive surface of the small intestine of the chicken from 1 day to 14 weeks of age. Poultry Sci. 54: Cameron, I.L., and G. Clefimann, Initiation of mitosis in relation to the cell cycle following feeding of starved chickens. J. Cell Biol. 21: Cerveny, J., H. Knizetova, and B. Knize, Effect of egg weight on growth and feed conversion of Pekin ducks and geese. Pages in: Waterfowl Production: Proceedings of the Intemational Symposium on Waterfowl Production, the Satellite Conference for the X V m World s Poultry Congress. Intl. Acad. Publ., New York, NY. Chambers, C., and RD. Grey, Development of the stmctural components of the bmsh border in absorptive cells of the chick intestine. Cell Tiss. Res. 204: Christensen, V.L., W.E. Donaldson, and J.P. McMurtry, Physiological differences in late embryos from turkey breeders at different ages. Poultry Sci. 75: Crittenden, L.B., and B.B. Bohren, The effects of current egg production, age of the pullet, and inbreeding on hatchability and hatching time. Poultry Sci. 41: Cunningham, F.E., O.J. Cotterill, and E.M. Funk, 1960a. The effect of season and age of bird 1. On egg size, quality and yield. Poultry Sci. 39:

44 Cunningham, F.E., O J. Cotterill, and E.M. Funk, 1960b. The eflfect of season and age of bird 2. On the chemical composition o f egg white. Poultry Sci. 39: Daly, K.R., and R.A. Peterson, The effect of age of breeder hens on residual yolk fat, and serum glucose and triglyceride concentrations of day-old broiler chicks. Poultry Sci. 69: Dawson, R.D., and R.G. Clark, Effects of variation in egg size and hatching date on survival of Lesser Scaup Aythya qffins ducklings. Ibis 138: Dibner, J.J., M L. Kitchell, C A. Atwell, and F.J. Ivey, The effect of dietary ingredients and age on the microscopic structure of the gastrointestinal tract in poultry. J. Appl. Poultry Res. 5: Ding, S T., K.E. Nestor,and M.S. Lilbum, The concentration of different lipid classes during late embryonic development in a randombred turkey population and a subline selected for increased body weight at sixteen weeks of age. Poultry Sci. 74: Ding, ST., and M S. Lilbum, Characterization of changes in yolk sac and liver lipids during embryonic and early posthatch development of turkey poults. Poultry Sci. 75: Donaldson, W.E., and V.L. Christensen, Dietary carbohydrate level and glucose metabolism in turkey poults. Comp. Biochem. Physiol. 98: Donaldson, W.E., and G.I. Liou, Lipogenic enzymes: parallel responses in liver to glucose consumption by newly hatched chicks. Nutr. Rep. Intl. 13: Dunn, I.e., P.J. Sharp, and P.M. Hocking, Effects of interactions between photostimulation, dietary restriction and dietary maize oil dilution on plasma LH and ovarian and oviductai weights in broiler breeder females during rearing. Brit. Poultry Sci. 31: Dunnington, E.A., and P.B. Siegel, Long-term divergent selection for eight-week body weight in White Plymouth Rock chickens. Poultry Sci. 75: Esteban, S., M. Moreno, J.M. Rayo, and LA. Tur, Gastrointestinal emptying in the final days of incubation of the chick embryo. Brit. Poultry Sci. 32: Etches, RL, The egg. Pages in: Reproduction in Poultry. CAB Intemational, Wallingford, Oxon, UK. 24

45 Ferrer, R., J.M. Planas, and M. Moreto, Cell apical surface area in enterocytes from chicken small and large intestine during development. Poultry Sci. 74; Foster, D i., S_M Yellon, and D.H. Olster, Internal and external determinants of the timing o f puberty in the female. J. Repro. Fert. 75: Freeman, B.M., and M. A. Vince, Development of the Avian Embryo. Chapman and Hall, London, U.K. Griffin, H.D., M.M. Perry, and A.B. Gilbert, Yolk Formation. Pages in'. Physiology and Biochemistry of the Domestic Fowl. Volume 5. Edit. B.M. Freeman, Academic Press, Inc., New York, NY. Hester, P.Y., and R.W.C. Stevens, Feed restriction of turkey breeder hens-a review. Poultry Sci. 69: Hocking, P.M., 1992a. Effects of photostimulation at 18, 24 and 30 weeks of age on the productivity of female turkeys fed ad libitum or restricted until point of lay. Br. Poultry Sci. 33: Hocking, P.M., 1992b. Genetic and environmental control of ovarian function in turkeys at sexual maturity. Br. Poultry Sci. 33: Hocking, P.M., Relationships between egg size, body weight, and pelvic dimensions in turkeys. Anim. Prod. 56: Holdsworth, C D., and T. Hastings-Wilson, Development of active sugar and amino acid transport in the yolk sac and intestine o f the chicken. Amer. J. Physiol. 212: Houpt, T.R., Effects of fasting on blood sugar levels in baby chicks of varying ages. Poultry Sci. 37: Imondi, A.R., and F.H. Bird, The turnover of intestinal epithelium in the chick. Poultry Sci. 45: Jeurissen, S.H.M., D. van Roozelaar, and E M. Janse, Absorption of carbon from the yolk into gut-associated lymphoid tissues of chickens. Devi. Comp. Immunol. 15: Knizetova, H., B. Knize, and J. Cerveny, Size of yolk sac in waterfowl and changes during 24 hours after hatching. Pages in: Water fowl Production: Proceedings of the International Symposium on Waterfowl Production, the Satellite Conference for the X V m World s Poultry Congress. Intl. acad. Publ., New York, NY. 25

46 Knizetova, H., J. Hyanek, and J. Cerveny, Egg size and posthatch growth of Pekin duck. Arch. Geflugelk. 56: Konarzewski, M., J. Kozlowski, and M. Ziolko, Optimal allocation of energy to growth of the alimentary tract in birds. Func. Ecol. 3: Konarzewski, M., C. Lilja, J. Kozlowski, and B. Lewonczuk, On the optimal growth of the alimentary tract in avian postembryonic development. J. Zool. 222: Lambson, R.O., An electro microscopic study of the entodermal cells of the yolk sac o f the chick during incubation and after hatching. Amer. J. Anat. 129:1-20. Latour, M.A., E.D. Peebles, C.R. Boyle, J.D. Brake, and T.F. Kellogg, Changes in serum lipid, lipoprotein and corticosterone concentrations during neonatal chick development. Biol. Neonate 67: Latour, M.A., E.D. Peebles, C.R. Boyle, S.M. Doyle, T. Pansky, and J.D. Brake, Eftects of breeder hen age and dietary fat on embryonic and neonatal broiler serum lipids and glucose. Poultry Sci. 75: Leighton, A.T., Jr., and L.M. Potter, Reproductive performance of turkeys subjected to blackout versus brownout restricted light conditions. Poultry Sci. 48: Leighton, A.T., Jr., and R.N. Shoffiier, 1961a. Effect of light regime and age on reproduction ofturiceys. 1. Effect of 15,24 hour and restricted light treatment. Poultry Sci. 40: Leighton, A.T., Jr., and RN. Shoffiier, 1961b. Effect of light regime and age on reproduction of turkeys. 2. Restricted vs. unrestricted light. Poultry Sci. 40: Lemer, S.P., N. French, D. McIntyre, and C. Baxter-Jones, Age-related changes in egg production, fertility, embryonic mortality, and hatchability in commercial turkey flocks. Poultry Sci. 72: Lilbum, M.S., Skeletal growth of commercial poultry species. Poultry Sci. 73: Lilbum, M S., Practical aspects of early nutrition for poultry. J. Appl. Poultry Res. 7: Lilbum, M.S., and K.E. Nestor, The relationship between various indices of carcass growth and development and reproduction. Poultry Sci. 72:

47 Lim, S., and F.N. Low, Scanning electron microscopy of the developing alimentary canal in the chick. Am. J. Anat. 150: Mather, C>M., and K F. Laughlin, Storage of hatching eggs: the interaction between parental age and early embryonic development. Br. Poultry Sci. 20: McNaughton, J.L., J.W. Deaton, F.N. Reece, and RL. Haynes, Effect of age of parents and hatching egg weight on broiler chick mortality. Poultry Sci. 57: Melnychuk, VL., F.E. Robinson, R.A. Renema, R.T. Hardin, D A. Emmerson, L.G. Bagley, Carcass traits and reproductive development at the onset of lay in two lines of female turkeys. Poultry Sci. 76: Michael, E., and R.D. Hodges, Histochemical changes in the fowl small intestine associated with enhanced absorption after feed restriction, ffistochemie 36: Mtchell, M.A, and M.W. Smith, The effects of genetic selection for increased growth rate on mucosal and muscle weights in the different regions of the small intestine of the domestic fowl (Gallus domesticus). Comp. Biochem Physiol. 99A: Mobbs, I.G., and D.B. M cmillan, Transport across endodermal cells of the chick yolk sac during early stages of development. Amer. J. Anat. 160: Moran, E.T., Jr., Digestion and absorption of carbohydrates in fowl and events through perinatal development. J. Nutr. 115: Moran, E.T., Jr., Effect of egg weight, glucose administration at hatch, and delayed access to feed and water on the poult at 2 weeks of age. Poultry Sci. 69: Moran, E.T., Jr., and B.S. Reinhart, Breeder flock productivity and egg size effects on broiler turkey performance and carcass quality. Poultry Sci. 60: Mussehl, F.E., and C.E. Ackerson, Some observations on humidity and weight loss in the incubation of turkey eggs. Neb. Agri. Exp. Sta. Res. Bull. p. 74. Nestor, K.E., Egg production consequences of improving growth and efflciency in turkeys. Pages in: Poultry Genetics and Breeding. Edit. W.G Hill, J.M. Manson, and D. Hewitt. British Poultry Science, Ltd. Nestor, K.E., and D.O. Noble, Influence of selection for increased egg production, body weight, and shank width of turkeys on egg composition and the relationship of the egg traits to hatchability. Poultry Sci. 74:

48 Nitsan, Z, G. Ben-Avraham, Z Zoref, and I. Nir, 1991a. Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. Brit. Poultry Sci. 32: Nitsan, Z., E.A Dunnington, and P.B. Seigei, 1991b. Organ growth and digestive enzyme levels to fifteen days of age in lines of chickens disering in body weight. Poultry Sci. 70: Mtsan, Z, I. Turro-Vincent, G. Liu, E.A. Dunnington, and P.B. Siegel, Intubation of weight-selected chicks with soybean oil or residual yolk: effect on early growth and development. Poultry Sci. 74: Noble, R.C., and M. Cocchi, Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29: Noble, R.C., and K. Connor, The synthesis and accumulation of cholesteryl esters by the developing embryo of the domestic fowl. Poultry Sci. 63: Noble, R.C., F. Lonsdale, K. Connor, and D. Brown, 1986a. Changes in the lipid metabolism o f the chick embryo with parental age. Poultry Sci. 65: Noble, R.C., J.H. Shand, and K. Connor, 1986b. Effects of parental age on fatty acid oxidation in vitro by the liver and heart of the chick embryo. Proc. Nutr. Soc. 45:103 A Noy, Y., and D. Sklan, Posthatch development in poultry. J. Appl. Poultry Res. 6: Noy, Y., Z. Uni, and D. Sklan, Routes of yolk utilization in the newly-hatched chick. Brit. Poultry Sci. 37: Obst, B.S., and J. Diamond, Ontogenesis of intestinal nutrient transporters in domestic chickens (Gallus gallus) and its relation to growth. Auk 109: Odle, J., R.T. Zijlstra, and S.M. Donavan, Intestinal effects of milkbome growth factors in neonates of agricultural importance. J. Anim. Sci. 74: O Sullivan, N.P., E.E. Dunnington, and P.B. Seigei, Relationships among age of dam, egg components, embryo lipid transfer, and hatchability of broiler breeder eggs. Poultry Sci. 70: Overton, J., and J. Shoup, Fine structures of cell surface specialization in the maturing duodenal mucosa of the chick. J. Cell Biol. 21:

49 de Pablo, F., L A. Scott, and J. Roth, Insulin and insulin-like growth Actor I in early development; peptides, receptors and biological events. Endocrine Rev. 11: Payne, L., P.B. Seigei, and L. Ortman, Correlation of dam, egg, poult and adult weights in broad breasted Bronze turkeys. Poultry Sci. 36: Phelps, P. V., F.W. Edens, and V.L. Christensen, 1987a. The posthatch physiology o f the turkey poult - 1. Growth and development. Comp. Biochem. Physiol. 86A Phelps, P.V., F.W. Edens, and R.P. Gildersleeve, 1987b. The posthatch physiology of the turkey poult - m. Yolk depletion and serum metabolites. Comp. Biochem. Physiol. 87A Pinchasov, Y., and Y. Noy, Early postnatal amylolysis in the gastrointestinal tract of taùiqy ipo\ûx.smeleagris gallopavo. Comp. Biochem. Physiol. 107A: Rahn, H., V.L. Christensen, and F.W. Edens, Changes in shell conductance, pores, and physical dimensions of egg and shell during the first breeding cycle of turkey hens. Poultry Sci. 60: Reidy, R.R., J.L. Atkinson, and S. Leeson, components. Poultry Sci. 73: Strain comparisons of turkey egg Reinhart, B.S., and E.T. Moran, Jr., Incubation characteristics of eggs fi'om older small white turkeys with emphasis on the effects due to egg weight. Poultry Sci. 58: Renema, R.A., F.E. Robinson, V.L. Melnychuk, and R.T. Hardin, The use of feed restriction for improving reproductive traits in male-line Large White turkey hens. 2. Ovary morphology and laying traits. Poultry Sci. 74: Renema, R.A., V.L. Melnchuk, F.E. Robinson, H.L. Classen, and R.D. Crawford, Reproductive organ morphology and carcass traits in unselected naturally mating female Bronze turkeys at onset of lay. Can. J. Anim. Sci. 78: Rhymer, J.M., The effect of egg size variability on thermoregulation of Mallard {Anas platyrhynchos) offspring and its implications for survival. Oecologia 75: Ricklefs, R.E., J.M. Starck, and M. Konarzewski, Internal constraints on growth in birds. Pages in: Avian Growth and Development: Evolution within the Altricial- Precocial Spectrum. J.M. Starck and RE. Ricklefs edit. Oxford University Press, Inc., New York, New York. Romanoff, A L, Avian spare yolk and its assimilation. Auk 61:

50 Romanoff A.L., Biochemistry of the Avian Embryo. Interscience Publishers, New York, NY. Roseborough, R.W., E. Geis, K. Henderson, and L.T. Frobish, Control of glycogen metabolism in the developing turkey poult. Growth 43: Scavo, L., J. Alemany, J. Roth, and F. de Pablo, Insulin-like growth factor I activity is stored in the yolk of the avian egg. Biochem. Biophys. Res. Comm. 162: Sell, J.L., Physiological limitations and potential for improvement in gastrointestinal tract function of poultry. J. Appl. Poultry Res. 5: Sell, J.L., C.R. Angel, F.J. Piquer, E.G. Mallarino, and H.A. Al-Batshan, Developmental patterns of selected characteristics of the gastrointestinal tract of young turkeys. Poultry Sci. 70: Sexten, W.E., and M.G. McCartney, The effect of age at lighting on reproduction in the turkey hen. Poultry Sci. 52: Shanawany, M.M., Inter-relationships between egg weight, parental age, and embryonic development. Brit. Poultry Sci. 25: Shanawany, M.M., Hatching weight in relation to egg weight in domestic birds. World s Poultry Sci. J. 43: Sharp, P. J., Photoperiodic control of reproduction in the domestic hen. Poultry Sci. 72: Shehata, AT., J. Lemer, and D.S. Miller, Development of nutrient transporter systems in chick jejunum. Amer. J. Physiol. 246:G101-G107. ShoflBier, RN., C.R Policy, RE. Burger, and E.L. Johnson, Light regulation in turkey management. 2. Female reproductive performance. Poultry Sci. 41: Slopes, T.D., Effects of age at lighting on reproduction of turkey hens. Poultry Sci. 71: Slopes, T.D., Turkey breeder hen performance by strain during consecutive lay periods. Poultry Sci. 74: Smith, K.P., and B.B. Bohren, Age of pullet effects on hatching time, egg weight, and hatchability. Poultry Sci. 54:

51 Smith, M.W., and M.A. Peacock, Comparative aspects of microvillus development in avian and mammalian enterocytes. Comp. Biochem. Physiol. 93A: Smith, M.W., M.A. Mitchell, and M.A. Peacock, EjBfects of genetic selection on growth rate and intestinal structure in the domestic fowl (Gallus domesticus). Comp. Biochem. Physiol. 97A; Starck, J.M., Intestinal growth in altricial European Starling (Shtmus vulgaris) and precocial Jqjanese quail (Cotuniix cotumixjaponicd). Amorphometric and cytokinetic study. Acta Anatom. 156: Starck, J.M., Structural variants and invariants in avian embryonic and postnatal development. Pages />/: Avian Growth and Development: Evolution within the Altricial-Precocial Spectrum. Edit. J.M. Starck and R E. Ricklefs. Oxford University Press, Inc., New York, NY. Turner, K.A., The effect of reproduction phase of breeder hens, poult hatch time, and fatty acid supplementation of the breeder hen diet on livability, groivth characteristics, and yolk and liver fetty acid composition in posthatch poults. Doctor of Philosophy Dissertation, Iowa State University Library, Ames, la. Uni, Z., Y. Noy, and D. Sklan, Posthatch changes in morphology and function of the small intestines in heavy- and light-strain chicks. Poultry Sci. 74: Uni, Z., Y. Noy, and D. Sklan, Development of the small intestine in heavy and Hght strain chicks before and after hatching. Brit. Poultry Sci. 36: Uni, Z., S. Ganot, and D. Sklan, 1998a. Posthatch development of mucosal function in the broiler small intestine. Poultry Sci. 77: Uni, Z, R. Platin, and D. Sklan, 1998b. Cell proliferation in chicken intestinal epithelium occurs both in the crypt and alog the villus. J. Comp. Physiol. 168B: Vajda, K., J.H. Shand, S.W. West, B.K. Speake, and R.C. Noble, Cholesterol metabolism in the Uvers of chick embryos produced from immature birds. Biochem. Soc. Trans. 431S:22. Walzem, RL., Lipoproteins and the laying hen: form follows function. Poultry Avian Biol. Rev. 7: Wilson, H R., Interrelationships of egg size, chick size, posthatching growth and hatchability. W. Poultry Sci. J. 47:

52 Wilson, W.O., F.X. Ogasawara, and V.S. Asmundson, Artificial control of egg production in turkeys by photoperiod. Poultry Sci. 41: Woodard, A.E., H. Abplanalp, V. Stinnett, and R.L. Snyder, The efifect of age at lighting on egg production and pausing in turkey hens. Poultry Sci. 53: Yafifei, N., and R.C. Noble, Further observations on the association between lipid metabolism and low embryo hatchability in eggs fi om young broiler birds. J. Exp. Zool. 253: Yannakopoulos, AX., The relationship between egg weight and shell quality on poult hatching weight. Zootech. Intl. 3: Yannakopoulos, AX., and A.S. Tserveni-Gousi, Research note: Efifect of breeder quail age and egg weight on chick weight. Poultry Sci. 66:

53 CHAPTER 2 EFFECT OF AGE AND BODY WEIGHT ON PLASMA CONCENTRATIONS OF LUTEINIZING HORMONE IN TURKEY HENS BEFORE AND AFTER PHOTOSTIMULATION T.J. Applegate, W.L Bacon, and M.S. Lilbum The Ohio State University, Ohio Agricultural Research and Development Center, Department of Animal Sciences, Wooster, OH Recaved January 3 1,1 ^ 7 Accepted June 23,1997 The independent effects of age and body weight (BW) on pfaotostmiulaiory lesponsc in turkey breeder hens were studied by measuring changes in plasma luteinizing iummone (LH; ngrinl) before and 3 d after phocostimularion. The study was conducted with hens from two BW groups at 24 25, 27 28, and wit of age. There was approximately a I-kg difference in BW between groups within an age. Six hens per BW and age group were cannnlated (jugular vein) and serially sampled during each of two 6-hr periods. Samples were collected at 10-min intervals. The two sampling periods were the last 5 hr of the short-day photoperiod (SD) and the same period dnring the third long day after photostimuiaiion (LO). The photostimulatory response (PR) or difference between the SD and LD baseline LH concentrations was greatest in the wk-old hens. The PR was unaffected by hen BW at any age. The baseline LH concentration during the SD photoperiod declined as bens aged. After photostimnlanan, baseline LH and LH peak amplimde concentraticns were higher in wk-old hens compared with the older ages. The mnnber of LH peaks increased after photostimuiaiion, but there were no signiricant effects attributable to age or BW within an age. In conclusion, the PR was affected by hen age but not hen BW or BW within a particular age. Elsevier Science Inc ESTRODÜCnON In the 30 plus years since 1963, there have been considerable changes in the growth of commercial ttnkeys (1). Improvements in the genetic potential for body weight gain and increased carcass yield, however, have negatively affected reproductive traits (2 4). Commercial turkey hens are normally exposed to a nonstimulatory lighting schedule (8L:16D) beginning at approximately 18 wk of age and are switched to a photostimulatory lighting scdiedule between 29 and 31 wk of age. These changes are mad^ at age-specific rather than BW-specific points in time and are the result of field data that suggest that photostimulation at younger ages wfll have negative effects on overall reproductive petformance. Some of the reported negative effects include delayed onset of sexual maturity after photostimulation and an overall decline in egg production and hatchability. Where controlled smdies have been performed, they have often been conducted with turkey strains with considerably smaller body sizes than the large white commercial strains prevalent in the industry today. The results of these studies suggested that 30 wk was the age by which hens had achieved sufficient BW for satisfactory reproductive performance (5 10), Commercial hens today are capable of reaching a satisfactory reproductive target BW well before 30 wk, but reproductive performance is still poor if hens are photostimulated (PS) at too young an age. Slopes (II) reported that when hens were PS at 24, 26, 28, or 30 wk, the number of eggs laid over the first 20 wk of production Elsevier Science Inc Æ7/S17.Q0 655 Avenue of the Americas, New York, NY Pll ^7)

54 was similar. The oldest hens (30 wk) laid the heaviest eggs. In addition, poults hnt^h<»h firom hens that were PS at 24 and 26 wk were lighter during the first 11 wk of lay when compared with poults hatched finm older hens. This study did not account for HifFpn'ng BW at the different PS ages. A shorter laying cycle and reduced persistency of lay was also observed in hens PS at 24 wk (12). During early lay, the number of yellow follicles was greater in hens PS at 24 versus 30 wk. However, by 55 wk they had fewer yellow follicles and more atretic follicles than the hens PS at 30 wk (13). The photostimulatory response in birds is different than in seasonal breeding mammak in that the eye and the pineal gland have no critical role in the response (14). An increase in baseline LH is associated with the onset of sexual maturity in sheep (15) and hypothesized to be the case in turkeys (16). Dunn et al. (17) reported that plasma LH in dwarf broiler breeder females increased dramatically 4 d after PS regardless of age (3,.7, 11, 15, and 19 wk). The change in LH at the youngest three ages was 1.7 times greater than at the oldest two ages. The authors concluded that the neuroendocrine pathway needed to convey photoperiodic information was in place as early as 3 wk. Increased oviductal weight 2 wk after PS only occurred in 11-, 15-, and 19-wk hens, however. The objectives of the current smdy were to smdy the independent effects of hen age and BW on the changes in the secretory patterns of LH on PS in modem commercial turkey hens. IVIATERIALS AND METHODS The breeder hens used for this study were firom a commercial strain (British United Turkeys of America, Lewisburg, WV) and were reared at the Ohio Agricultural Research and Development Center (OARDC), Wooster, OH. At 18 wk, hens were subjected to a pfaotoinhibitory lighting schedule (8 hr light: 16 hr dark; 8L:16D). The study was conducted at three ages (24 25, 27 28, wk). Six hens from each of two BW groups were sampled within an age and the mean BW for each group was 12.1 ±.4 (Normal) and 13.1 ±.4 kg (Heavy) before cannulation. These BW groups reflect the range of target body weights of hens at photostimulation by commercial turkey breeders. Three hens were randomly selected from each BW group a week before cannulation and moved to individual cages (60 X 60 X 80 cm) with wood shavings as litter. Only six hens could be carmulated and sampled per week, so three hens per BW group were carmulated on consecutive weeks. Hens were cannulated during the photoperiod I or 2 d before the first serial sampling period as described previously (18). Thirty-six hens were initially carmulated, but a fuu complement of samples was collected from only 30 hens (10 per each age group). One-milliliter samples were collected every 10 min during each sampling period. After collection of each blood sample, 1.0 ml of a solution containing 0.7% sodium chloride, 5 mg/ml sodium citrate, and 0.5 mg/ml gentamicin sulfate was returned to each individual hen. The first serial sampling period was the last 6 hr of the SD photoperiod (08:00 to 14:00 hr). Hens were then switched to a photostimulatory light schedule (16 L:8 D; LD) by adding 8 hr of light to the end of the 8-hr SD photoperiod. The second serial sampling period was during the same 6 hr (08:00 to 14:00 hr) on the third day after photostimulation (i.e.: 2 d after the first blood sampling period). After collection of the LD samples, hens were euthanized with an overdose of soditun pentobarbital. The left Pectoraiis major muscle and abdominal fat (including the fat around the gizzard) were removed carefully and weighed. The ovaries were examined visually for determination of follicular development. The LH radioimmunoassay was conducted as previously validated and reported by Bacon and Long (19) using chicken LH (20) for both iodination and as the standard. 34

55 Anii-cfaicken LH (1:20,000; 20) was used as the primary antisera. Duplicate aliquots of plasma (100 pj) from each sample were assayed. The inter- and intra-assay coefbcients of variation (17.05% and 12.65%, respectively) were calculated using a plasma pool from laying turkey hens (mean concentration 0.99 ng/ml). The sensitivity of the assay was 0.2 ngfrnl plasma. The identifrcation of LH peaks and calculations of baseline and peak amplitude concentrations were derived using the PC PULSAR algorithm (21). The G values for these analyses were: G(l) = 3.8, G(2) = 2.6, G(3) = 1.9, G(4) = 1.5, and G(5) = 1.2. The G values represent the number of assay SD above the baseline that one, two, three, four, or five consecutive samples must achieve to qualify as a peak. The assay SD for the algorithm was calculated firom a random sample of 10% of the experimeatal data. For this calculation, the SD for sample means was calculated and regressed on the sample mean. The calculated assay SD used in the PULSAR algorithm was 6.53X , where X equaled the concentration of LH in a sample. Smoothing time for the algorithm was set at 360 min (36 samples). A repeated-measures analysis of variance was used to determine the effects of PS, BW group, hen age, and their interactions on various components of LH concentration. The General Linear Models (GLM) procedure of SAS (22) was used for the statistical analysis. To characterize the age by photoperiod interaction, the photostimulatory response (PR) was calculated as the difference between baseline LH concentrations before and after PS in the same hen. The PR and carcass characteristics were subsequently analyzed by two-way AKOVA using the GLM procedure of SAS (22). The main effects determined in this model were BW group, ben age, and their interaction. Differences between hen ages in both models were determined using least square means separations. RESULTS Hen BW did not affect LH secretory profiles within an age, between ages, and did not alter the pfaotostimulatory change in baseline concentrations. Observed LH means averaged 1.15 and 1.03 ng/ml for Normal and Heavy hens, respectively. Baseline means of LH were 1.11 and 0.98 ng/ml for Normal and Heavy hens, respectively. The photostimulatory change in baseline concentrations of LH between the SD and LD photopeiiod were 0.97 and 0.89 ng/ml for Normal and Heavy hens, respectively. The secretory profiles of LH changed notably when hens were switched to a LD pfaotoperiod. A representative subsample of LH secretory profiles are shown in Figure 1. The mean values for various LH measures before and just after PS are presented in Table 1. The LH secretory profile after PS was characterized by an overall increase in mean concentration (0.94 ng/ml; P ) and an increase in the baseline mean concentration (0.91 ng/ml; P ^ ). After PS, the number of peaks identified by the PC PULSAR algorithm increased from 0.4 to 1.1 every six hr (P ^ 0.05). Peak amplitude and peak duration were not significantly affected by PS. The age of hen at PS also had a significant effect on the secretory profile of LH (Table 2). During the SD photoperiod, wk-old hens had higher observed and baseline mean concentrations of LH (P ^ 0.05) when compared with the 31-32, but not the 27 28, wk-old hens. After 3 d of exposure to a LD photoperiod, wk-old hens had higher (P 0.05) observed and baseline mean concentrations of LH over the 6-hr sampling period ±an hens from the older two ages. As noted by the differences in baseline LH, the wk-old hens responded more to PS than did the 27 28, but not the wk-old hens. Peak number was unaffected by age at PS, whereas peak amplitude was affected by hen age only during the LD photoperiod. The amplimde of peaks was 0.6 ng/ml greater after PS (P ^ 0.05) in 35

56 Hon # 4 (24-25 wk) s 4 % B Timm (min) Hon # 2 5 ( wk) Timm (mum) 240 Hon # 3 5 ( wk) Timm (min) Figure 2,lSecretory profiles of LH fiom three photosensitive hens during a short-day photoperiod (SD) and after a 3-d exposure to a long-day photopcriod (LD) at (A), (B), and (Q wjc-of-age. Hens were photostimulated by the addition of 8 hr of light to the photoperiocl The profiles start the second hr after dawn. Serial blood samples were collected at 10-min intervals for 6 hr- The hens represented in this figure had profiles closest to baseline concentrations for age group before and after photostimulation. Arrows within the graph indicate the peaks detected with the PULSAR algorithm.. S D ;, SD baseline;, LD; a n d, LD baseline. 36

57 Table 2.1 Pa t ie r k m e a n s o f l u t e in iz in g h o r m o n e (LH) p a r a m e t k s in pla sm a o f t u r k e y h en s b e f o r e AND a fter a 3 - d expo su re TO A LONG-DAY PHOTTOPERIOD Observed Baseline Peak Peak Peak mean^ mean^ nomber amplimde dnranon SEP LD~* S D L D S D L D S D L D S D LD (ng/ml) (#/6 hr) (ngtal) (min) 0.63* I.IO I. l l SEM Somce of Variation Probabiliiy Photopcriod ' Mean of LH concenoarions dnring earfi 6-br serial blood sampling period. ^ Mean of caloilared baseline concenoarions of LH during each 6-hr serial blood sampling period, as calculated by PC PULSAR analyses. * SD, short daylight photopcriod (8-hr light and 16-hr dark per day). '* LD, long daylight photopcriod (16-hr light and 8-hr dark per day). * Means of 30 turkey hens, pooled across two BW groups at three discrent ages. the wk-old hens compared with the or wk-oId hens. Peak length was unaffected by hen age before or after PS. After the LD serial sampling period, hens were euthanized for visual determination of premature follicular development and age-associated changes in carcass measurements. The ovarian follicles of all hens were white and rather small (=^6 mm in diameter), confirming that all cannulated hens had not been previously exposed to external light stimulation. No age-by-bw interactions were apparent, so only age-associated changes in carcass characteristics averaged across BW groups are presented in Table 3. No differences were observed in relative weights of the P. major or ab d om in al fat within or between ages. Age associated changes in absolute or relative weights of the P. major or abdominal fat were indistinguishable. Table 25 Pattern m eans o f luteinizing horm o n e (LH) parameters in PLASMA o f tu rk ey hens before AND AFTER A 3-D EXPOSURE TO A LONG-DAY PHOTOPERIOD AT , ,.AND WK Observed Baseline Baseline Peak Age Mean* M ean' Difference* Number SD* LD* SD LD SD EJd (wk) (ng/ml) (#/6 hr) 156* ^ **" ND* to *-* 2.17* 0.76* 2.13* 27 to ** 1.13* 056**" 1.05* 31 to * 1.42* 0.48* 153* SEM Source of Variation Age Age by Photoperiod -Probability Means in columns with no common superscripts differ significantly (P SO.05). Mean of LH concentrations dining each 6-br serial blood sampling period. * Mean of calculated baseline concentrarions o f LH dining each 6-hr serial blood sampling period, as calculated by PC PULSAR analyses. * Difference between the baseline mean concentrarions o f LH, before and after photostimnlarioil Baseline difference was starisrically analyzed using a two-way ANOVA to determine the effect of hen age, hen BW, and their interactidn. This model was used to further define the age by photoperiod interaction o f the previous model * SD, short daylight pfaotoperiod (8-hr light and 16-hr dark per day). * LD, long daylight photoperiod (16-br light and 8-hr dark per day). * Means of ten turkey hens per age, pooled across two BW groups. ^ ND, no peaks were detected. 37

58 Table 23 BW a n d a b s o l u t e a n d r e l a t i v e w h g h i s o f t h e P. m j o r a n d a b d o m in a l f a t c f t u r k e y h e n s AFTER A 3 -d e x p o s u r e TO A LONG-DAY PHOTOPERIOD AT , , AND WK Age Body Weight Pecwralis major^ Abdominal Fat^ (wk) (kg) (g) (%) (g) (%) 24 to ^ to Z4I 31 to SEM Source o f Vanadoa Probability Age ' Weight of the left P. m ajor muscle. P. m ajor percentage = left P. m ajor weight/body weight x 100. ^Weight of the abdominal fal including that which was disassociaied ftom the gizzard. Abdominal fat percenmgc = abdominal fiit weighi/bw X 100. ^ Means of twelve turkey hens per age, pooled across two BW groups. DISCUSSION The biggest change in LH secretory characteristics associated with PS was the 25-foId increase in baseline concentration over the two day experimental period. This increase in LH baseline is similar to what was observed for other genetic strains of turkeys with very different carcass characteristics (16,18). A dramatic change in luteinizing hormone (LH) secretion on PS has been demonstrated in a strain of turkey hens genetically selected for increased egg production (18). LH secretion shifted from a characteristic low baseline with high amplimde pulses of relatively low frequency to a pattern characterized by a high baseline with few, low-amplimde pulses. This secretory pattern remained until ovulation began approximately 3 wk after PS, at which time an ovulatory surge of LH was superimposed on the relatively high baseline. Bacon and Long (16) reported that this change in the baseline concentration of LH occurred during the first long-day scotoperiod (dark period) with a fruther increase during the second long-day scotoperiod. These observations were made in a random-bred, large-white strain of turkey hens representative of the commercial turkey of Bacon and Long (16) also reported that the change in LH secretion was primarily a function of a shift in baseline concentrations rather than a change in pulse frequency or amplimde. In the current smdy, the SD and LD baseline difference was greatest with the wk-old hens. This was primarily a reflection of a much higher LD baseline LH concentration compared with hens from the two older ages. Bacon (personal communication) also noted a decline in plasma LH concentrations as hens aged firom wk of age. This decline was apparent whether hens were exposed to either a SD (6L:18D) or LD (16L;8D) photoperiod from 10 wk. Selection for growth and carcass related traits in commercial turkeys has increased the relative amounts of abdominal fat and pelvic fat but not vent diameter (23). When commercial turitey hens were PS at 24 wk, vent size in relation to initial egg size was much smaller than hens PS at 30 wk. This change in vent size was then attributed to a higher incidence of oviductal prolapse observed in obese hens (23). In the current smdy, BW at PS or BW within an age of PS had no influence on the photoperiodic response, or difference in baseline LH concentrations. As hens aged, there were no proportional changes in relative or absolute weights of abdominal fat or P. major. In broiler breeder hens, the onset of egg production is correlated better with lean BW rather than fat content However, a minimal amoimt of adiposity may influence the onset of puberty (24). Dunn and Sharp (25) measured changes in LH concentrations while trying to establish the m inim al daylength required to stimulate ovarian and oviductal growth. The photoperiodic response differed between broiler and egg-laying strain of chickens, but there were 38

59 no effects attributable to feed restriction (50 60% intake restriction of hens fed ad libitum). In the broiler strain of hens, feed restriction did reduce ovarian and oviductal growth 2 wk after PS, however. In our study, therefore, whereas BW differences between hens at PS within or between ages did not affect the photoperiodic LH response, there may sthl have been long-term effects on reproductive performance. In a foiiow-up study with commercial tuiiey breeder hens by Applegate and Lilbum (26), egg weight through the first 10 wk of production was significantly affected by hen BW at PS when hens were pfaotostimulaled at 29, but not 31, wk. Hens that were phctostimulated at 31 wk also had heavier ovarian weights, primarily because of an additional follicle of ^20 mm. In conclusion, BW of commercial turkey hens, within or between ages, had no effect on the photostimulatory response. The wk hens had the greatest change in baseline concentrations of LH after PS, suggesting the neuroendocrine pathway involved in a photostimulatory response is active at. this early age. This response, however, was dependent on physiological age and not gross body composition. ACKNOWLEDGMENTS The authors wish to thank- Dr. J. Proudman, USD A. ARS, Beltsvüle, MD for kindly supplying antiserum and LH used in the radioimmunoassay. The authors would also like to thank John Nixon, Cindy Coy, and Dave Long for their technical contributions. Corresponding author: Dr. M. S. Lilbum, Department of Animal Sciences, The Ohio State University, O A JLD.C, Wooster, OH Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, Manuscript ntunber REFERENCES 1. Lilbum MS. Skeletal growth of commercial poultry species. Poult Sci 72: , Hester PY, Stevens RWC. Feed restrictioa of turkey hens-a review. Poult Sci 69: , Nestor KE. Genetics of growth and reproduction in the turkey. 9. Long-term selection for increased 16-week body weight. Poult Sci 63: , Nestor KE, Noble DO. Influence of selection for increased egg production, body weight, and shank width of turkeys on egg composition and the relationship of the egg traits to hatchability. Poult Sci 74: , Leighton AT Jr, Shoffiier RN. Effect of light regime and age on reproduction of tmkeys. 1. Effect of 15,24 hour, and restricted light treatment. Poult Sci 40: , Leighton AT Jr, Shof&ier RN. Effect of light regime and age on reproduction o f turkeys. 2. Restricted vs. unrestricted light. Poult Sci 40: , ShofBier RN, Policy CR, Burger RE, Johnson EL. Light regulation in turkey managemenl 2. Female reproductive performance. Poult S d 41: , I96Z 8. Wilson WO, Ogasawam FX, Asmimdson VS. Artificial control of egg production in turkeys by photoperiod. Poult Sci 41: , Leighton AT Jr, Potter LM. Reproductive performance of turkeys subjected to blackout versus brownout restricted light conditions. Poult Sci 48: , Woodard AE, Abplanalp H. Stinnett V, Snyder RL. The effect o f age at lighting on egg production and pausing in turkey hens. Poult Sci 53: , Slopes TD. Effects of age at lighting on reproduction of turkey hens. Poult S d 71: , Hocking PM. Effects of photostimulation at 18, 24 and 30 weeks of age on the productivity of female tiukeys fed ad libitum or restricted until point of lay. Br Poult S d 33: , 199Z 13. Hoddng PM, Gilbert AB, Whitehead CC, Walker MA. Effects o f age and of early lighting on ovarian function in breeding turkeys. Br Poult S d 29: , Sharp PJ. Photoperiodic control of reproduction in the domestic hen. Poult S d 72: , Foster, DL, Ryan KD, Papkoff H. Ihtcmal and external determinants o f the timing of puberty in the female. J Reprod Pert 75: , Bacon WL, Long DW. Changes in plasma luteinizing hormone concentration in turkey hens after switching from short-day to long-day photoperiods. Domest Animal Endocrin 12: , Dtmn IC, Sharp PJ, Hocking PM. Effects o f interactions between photostimulation, dietary restriction and dietary maize ou dilution on plasma LH and ovarian and oviductal w dghts in broiler breeder females during rearing. Br Poult S d 31: ,

60 18. Chapman DP, Bacon WL, Long DW, Kudina K, Buike WH. Phctosmnuiatioa changes the paoem of luteinizing honncne secretion in tutitcy hens. Gen Comp Endocrin 96:63-74, Bacon WL, Long DW. Secretion of luteinizing hormone during a forced molt in tuiiccy hens. Poult S d 75: , 1996l 20. Krisfanan KA, Proudman JÂ, Bahr JM. PuriiTcarioa and partial characterization of isoforms o f lutemm ng hormone from the chicken pittntaiy gland. Comp Biochem Physiol 108B: , Meniam OR, Wachter KW. Algorithms for the smdy of episodic hormone secretion. Am J Physiol 243Æ , SAS Instimte. SAS* User s Guide: Basics. Version 5 Edition. SAS Institute Inc., Cary, NC, Hocitmg, PM. Relationships between egg size, body wdghc and pelvic dimensions in turteys. Anhn Prod 56: , SoHer M, Eitan Y, Brody T. Effect of diet and early quantitative feed restriction on the minimum weight requirement for onset of sexual maturity in White Rock broiler breeders. Poult S d 63: , Dunn IC, Sharp PI. Photoperiodic requirements for LH release in juvenile broiler and egg-laving strains of domestic chickens fed ad libitum or restricted diets. J Reprod f e t 90: , Applegate TJ, Lilbum MS. Hffrci of hen age, body wdght, and age at photostimulation. 1. Egg, mcubarion, and poult characteristics of conunetdal turkeys. Poult S d (in press),

61 CHAPTERS Independent Effects of Hen Age and Egg Size on Incubation and Poult C haracteristics in Commercial Turkeys^ TODD J. APPLEGATE and MICHAEL S. LELBURN Department o f Animal Sciences, The Ohio State Unioersity, Ohio A griculbanl Research and Deoeiopment Center. Wooster, Ohio ABSTRACT Nicholas hens firam the sam e Sock were studied throughout the growing and laying periods. From 36 to 55 w k of age, 800 eggs w ere collected at approximately 5-wk intervals. At each age, a subsample of loo eggs were randomly selected for egg weight, yolk weight, yolk DM, and yolk lipid (DM). The remaining 700 eggs were individually weighed and incubated. All eggs were re weighed at 25 d (transfer) and poults were individually weighed at hatch. At each sam pling age, 50 poults were selected for analysis of yolk sac weight, yolk sac DM, and yolk sac lipid (DM). From 33 to 45 wk, mean hen BW decreased OS kg. Across all ages, the relative weight of abdominal fat decreased more than (Key words: hen age, egg size, poult, yolk sac, lipid) that of the Pectoral^ major. Relative yolk weight increased and relative albumen weight decreased with age. Yolk sac weight and Upid content (DM) of the poults at hatch increased markedly with age of hen. Across all ages, eggs were divided into weight classes. Initial egg w eight had no effect on proportional moisture loss at 25 d or on relative poult weight at hatch. Egg moisture loss through 25 d was greatest in eggs from 36-wk-old hens compared with hens at 41, 45, 50, or 55 wk. Mean poult w eight increased horn 62.6 g at 36 wk to 64.9 g at 55 w k. Relative poult weight increased 3% from 36 to 55 wk w hen corrections for initial egg size were taken into consideration Poultry Science 75: INTRODUCTION The 1993 commercial turkey grew twice as fast as the commercial turkey of 1963 (Lilbum, 1994) and witii a considerable increase in breast muscle size (lilb u m and Nestor, 1991). Selection for growth, however, can result in increased carcass hit (Bacon ef al., 1986) as well as negative effects on egg production and hatchability (Nestor, 1984; Nestor and Noble, 1995). Turkey hens reach their maximal BW shortly after photostimulation and then go through a period of BW loss during the initial stages of egg production (Krueger et al., 1978; Bacon and Nestor, 1982; Lilbum and Nestor, 1993). Much of this BW loss is as carcass lipid McCartney et al., 1977; Robel, 1984; Ferket and Moran, 1986; Lilbum and Nestor, 1993). Hen age is positively correlated with egg size and egg size is positively correlated witii poult w eight (Reinhart and Moran, 1979; Shanawany, 1987). Increased egg weight, however, is not necessarily accompanied by proportional increases in egg components. Reidy et al. (1994) reported that the w e i^ t of eggs h o m commercial Secaved for pubuotton January 30, Aœepled for publication June 11, ^Salaries and research support provided by State and Federal funds appropriated to the Ohkj AgrlcuituraJ Research and Development Center, The Ohio State University. Manuscript Numtier turkey breeders increased approximately 11% between the onset of lay and 24 w k of production. During tfiis time, yolk weight increased 21% but albumen weight only increased 7%. The m agnitude of these changes differed slightly depending upon commercial genotype. Ih a subsequent comparison of eggs from British United Turkey male and female line hens, male line eggs were 11% heavier but there w ere no significant difierences in yolk w e i^ L N estor and Noble (1995) compared egg and egg component weights in a randombred turkey line (RBC2) and a subline o f RBC2 selected for BW at 16 wk (F line). Similar to the results of Reidy et al. (1994), eggs from F line hens w ere 10% heavier but with no corresponding changes in absolute yodc w eight Reinhart and M oran (1979) compared eggs from a Small White turkey strain at two ages, 48 and 55 wk, and classified eggs according to w eight There were no age effects on moisture loss through 25 d, but poult weight as a percentage of set weight was greater in eggs from younger hens. Increased egg size was associated with decreased moisture loss through 25 d and increased poult weight as a percentage of egg weight In broiler breeders, eggs firom 44-wk-oId hens resulted in heavier embryos at 18 d than eggs of similar size from 28-wk-old hens (Shanawany, 1984). In the latter instance, increased BW at 18 d o f incubation, independent of egg weight differences, m ay be a reflection of inefficient mobilization, transportation, and metabolism of yolk 41

62 Hen age Table 3.1 Sody w eight, iclative abdominal fat weight, relative Peetondis major w eight, and relative II ver weight of turkey breeder hena. fed eidier maah (M) or pelletted (P) diets BW Abdommal Peaomiis major^ Liver^ M P M P M P M P (wk) 19< ^ L J IJ : SEM Age Diet Age X diet J t Abdominal fat percentage - abdominal fat w e i^ t/b W x 100. ip. major percentage = P. major weight/bw x loo. H iver weight percentage = liver weight/bw x 100. tweeic 19, 25, and 30 means based-on 20 hens. SWeek 33, 45, and 55 means based on 15 hens. lipitis by embryos &om young hens (Noble et al, 1986; Yaffei and Noble, 1990). YoUc lipids atxount for 90% of embryotaic energy needs (Freeman and Vînœ, 1974) and impaired utilization may hinder the availability of energy to embryos from younger hens. Ding and Lilbum (1996) reported that yolk w eight w as similar in eggs from RBC2 and F line hens and em bryo weights were similar through 21 d of incubation. Embryo weights began to diverge at 22 d, the age at which extensive lipid transfer from the yolk sac to the embryo begins. One objective of the current research w as to measure the changes with increasing age of selected carcass traits in turkey breeder hens before and during production. The other objective was to study the effects of hen age on incubation characteristics of eggs from commercial hens with consideration given to age-assocdated changes in egg w eight MATERIALS AND METHODS Nicholas^ hens were reared in commercial growing and breeder facilities at Roll Turkey Faums, Inc, Versailles, OH At 19, 25, 30, 33, 45, and 55 wk of age. a random sample of hens was killed by cervical dislocation for BW and carcass measurements. This included the weight of the abdominal fat p ad including fat around the gizzard and liver, and one-half of the Pectoraiis major (measured at 33, 45, and 55 wk). Hens were photostimulated at 30 wk of age. During the growing and breeder phases of the study, hens were fed either a mash or pelleted d ie t The mash Hsfidiolas Turkey Breeding Paires, Sonoma, CA H eteisiree Inc. Gettysburg, OH feed was mixed on the farm whereas the pelleted feed was custom mixed and purcltased from a conunerdal company. The diets w ere form ulated to similar specifications but analysis by a commercial lab indicated the actual nutrient content to be somewhat different Feed samples taken during the breeder phase had the following analysis: 6.76% frit, 14.18% CP (mash); 5.48% % CP (pellets). Effects due to nutrient composition vs those due to pelleting cannot be distinguished. A t 36, 41, 45, 50, and 55 w k of age, 400 eggs per diet (800 per age) were collected from the Cuddy Farms hatchery in Danville, O H Fifty eggs per diet (100 per age) were individually weighed and hardboiled for albumen and yolk w eight determinations. Ten yolks per diet and hen age were random ly selected for yolk DM determination. Total lipid (TL) was determined on dry yolks after a 2:1 diloroform unethanol extraction (Folch et a l, 1957). Each of the rem aining 700 eggs from each hen age was individually w eighed and set in a Petersime incubator.3 After 25 d of incubation, eggs were individually reweighed and placed in - individual pedigree baskets. At hatch, all poults were individually weighed and 25 poults per diet (50 p er hen age) were randomly selected and euthanatized by decapitation. The yolk sac of die poult was carefully rem oved, weighed, and frozen in liquid nitrogen. The DM content w as determined after lyophilization Total yolk sac lipid was measured as previously described from a subsample of poult yolk sacs of increasing DM. The results w ere analyzed statistically by analysis of variance using the General Linear Models procedure of SAS (SAS fristitute, 1986). The main effects tested were hen age, diet, and the interaction of age and diet. As mentioned previously, one objective of diis experiment

63 Variable Table 3 ^ Egg weight, yoqc w eight tdative weight, iliy matter, Upid tont entiation. and albtm en weight and teladee w eight from cndcey breeder hena fed either m ash fm) or pelletted (F) dietat Egg weight Yoflc Aifauxnen Weight PercenageZ DVP Lipid^A Weight Percentage^ (g) (%) Hen age. wk 36 S&l J " & J S SEM Diet M L7 SZ P SEM Source of variation Age OOOOl OOOOl OOOOl Diet OOOOl Age X diet ^ Means based on 50 eggs per diet (ICO total) p er age, unless indicated otherwise. ^odc percentage = yolk weight/egg weight x loa ^Means based on 10 yolks per diet (20 total) per age *tipid concentration of yolk on a DM basis, ^Albumen percentage = albumen weight/egg weight x 100. was to differentiate between the effects due to egg size from those due to hen age. Therefore, all eggs were assigned to one of eight egg size classes for statistical analysis. Egg weight at setting; transfer w eight (25 d), and poult w eight were analyzed using a model that included egg weight as a main effect in addition to hen age and diet. Regression analyses were done to determfrie the relationships between yolk sac DM and yolk sac TL on 10 yolk sacs of the poults within an age. From 41 through 55 wk, this resulted in a significant R? and the data was used to develop a standard curve for the measurement of percentage lipid in all remaining samples. A t 36 wk, the relationship was not significant; however, so the percentage lipid value represents the average of only those samples actually measured (n = 10). Similarly, egg w e i^ t at transfer, relative transfer weight, poult weight, and relative poult weight were regressed on egg w eight RESULTS Hens in both dietary treatment groups reached their maximal BW at 33 w k (3 w k after photostimulation) and at this age also had their greatest quantities of abdominal fat (Table 1). Over the ensuing 22 wk, the hens progressively lost BW and abdominal fat decreased proportionately. The largest decrease in relative weight of abdominal fet occurred between 33 and 45 wk. There were no significant (P g 0.07) consistent effects of hen age on the relative w eight of the P. major. In measurements m ade during the rearing period (19, 25, and 30 wk) and just prior to the onset of lay (33 wk), hens fed mash had greater relative liver weights than those fed pellets w ithin each o f these ages (P < O.CXll). Diet had no effect on relative liver w eights of hens at 45 or 55 wk. There w as a decline in relative liver weight between 25 wk and photostim ulation (30 wk) with an increase observed thereafter a t 30, 45, and 55 wk. There was a significant diet by age m teraction (P S 0.001) for relative liver weight. This interaction was primarily due to the aforementioned increase in hens fw. mash prior to the onset of pnaduction w ith no observable diet effects at 45 or 55 wk. fri the randomly sam pled eggs used for yolk and albumen m easurem ents, there was a decline in egg weights a t 36 and 41 w k as compared w ith egg weights at 45, 50, and 55 w k (Table 2). At 36 and 41 wk, mean egg w eight was lighter than that of the remaining 700 eggs used for the incubation part of the experiment. Relative yolk weight increased with hen age from 28.2% at 36 w k to 34.0% at 55 w k (P S 0.001). During this same time period, relative album en weight decreased from 60.2 to 533% (P S 0.001). Eggs firom hens fed pellets were heavier with increased absolute weights of the yolk and album en (P S 0.(X)1). The relative weight of the yolk (P < 0.04) and album en (P S 0.06) w ere l i f t e r and heavier, respectively, in hens fed pellets. Yolk DM percentage was increased in eggs firom hens fed mash (P S 0.001), b u t there w ere no differences to yolk lipid percentage. Poults sam pled for yolk sac measurements were heavier at 45, 50, and 55 w k than those sampled at 36 43

64 Vaiiabte Table 3 J Yolk u c weight, d iy u u tte i; lipid concentration, and correladon of yolk aac dry m atter wilfa lipid concentration of posthatch poults from turkey breeder hens fed either mash (M) o r pelletted (F) diets^ weight Yolk sac Poult U pid weight Weight DM Upidz weight ig; - y JgJ» Yolk sac DM: Spid RZ P > F Hen. age. w k 36 8& ^ x S ZOO SEM Diet M P SEM Source of variation Age Diet Age X diet tmeans represent 25 eggs and poults per diet (50 total! per age. ^lipid concentration of yolk sac of poults on a dry matter basis. and 41 wk (P < 0.001; Table 3), Poults selected from hens fed pellets were heavier (P < 0.001) than those from hens fed mash. There was a progressive increase in yolk sac weight (P < 0.001), yolk sac DM percentage (P S 0.(X)1), and yolk sac lipid weight (P < 0.004) of the poults w ith increasing age of hen. Within an age, variability precluded any significant age effects on yolk sac lipid (DM), but there was considerably less lipid (DM) in yolk sacs of the poults associated w ith the yoim gest hens (36 wk). There were no significant diet effects on yolk sac weight, DM percentage, lipid percentage, o r absolute lipid w eight A t all ages except 36 wk, there was a significant (P S 0.01), positive correlation betw een DM percentage and lipid concentration of poult yolk sacs. There was no correlation between egg yolk DM percentage and lipid percentage in the fresh eggs sampled for albumen and yolk measurem ents (data not shown). When the egg weights for all ages were divided into egg weight classes, there was still a «unall b u t significant increase in egg weight with increasing hen age (P S 0.001; Table 4). This increase in egg w eight resulted in a significant age by egg size interaction (P S 0.001). The small egg weight differences were reflected in slight but significant age effects on transfer w eight at 25 d, transfer percentage, poult weight, and poult percentage (P S 0.001). In almost all cases, the 36-wk data accounted for m ost of the age effects. Egg weight and transfer weights were at most 1 to 2% different w hen the youngest (36 wk) and oldest data were compared, but p o ult w eight and relative poult weight at 55 wk were 4 and 3% higher in the older hens, respectively. The egg size by hen age interaction effect on poult weight is show n in Figure 1. Larger eggs resulted in greater transfer weights and heavier poults (P < 0.001) b u t there were no significant effects on transfer percentage or poult percentage. This result suggests that m oisture loss during incubation between eggs of different w eights was not different in eggs of differing sizes. Egg weight was positively correlated w ith transfer weight (R > 0.86, P < 0.001) a n d poult w eight (R & 0.65, P < 0.001) for each hen age. There were sign ificant diet effects on all egg and incubation measurements except for transfer w eight (Table 5). The dietary effiects were small and w ould appear to predude their biological or practical relevance. 74 I I 12 O a. 38 WK 55 WK U.4 Egg W eight (g) Figure 3.1 Relationship between poult w egju at hatdung and egg size tioizi 36" and 55-wk*old hens. 44

65 Table 3.4 w eight weight at 25 d of incobatiaa (fzsufer w e^ht), relative tnn*h>r w eight poult weight at hatching, and ponit weight relative to initiai egg «rexgfat Variable weight* Transfer weight Poult weight^ - ( g ) C%)3 (8) (%)* Hen age, wk S SEM Egg nze, gj S to to to to to to Z ' 712 SEM Source of variation Age Egg size Age X egg size ^%g weight, transfer weight, and transfer percentage means based on 350 eggs per diet (700 total) per age. ^Poult weight and poult percentage means are based on 64,65, 66.65, and 72% hatchability from hens at 36, ,50, and 55 weeks of age, respectively. ^Transfer percentage = egg weight at 25 d incubation/egg weight at set x 100. <Pouit percentage = poult weight at hatch/egg weight at set x 100. ^%g size means based on 256, 300, , 575, 505, 397, and 447 eggs for each weight dass, respectively. DISCUSSION Hens reached maximal BW after photosdmulation and just prior to the onset of lay (33 wk). The peak in BW just prior to lay has been reported previously (Harper, 1950; Krueger et al., 1978; Ferket and Moran, 1986; Lilbum and Nestor, 1993). After the onset of egg production there was a progressive 15% loss in BW through 55 w k that w as paralleled by a 25 to 40% reduction in the relative w eiÿrt of abdominal fet. This large decrease in carcass lipid associated with BW loss during the early stages of production condnns previous data in the literature (McCartney ef al, 1977; Robel, 1984; Ferket and Moran, 1986; Lilbum and Nestor, 1993). The minimal decline in relative w eight of abdominal fet after 45 wk supports the conclusion by Lilbum and Nestor (1993) that the first 10 w k of production are the most critical with respect to positively or negatively efiecting carcass composition of turkey breeder hens. During this same time period, the relative w e i^ t of the P. nmjor did not change significantly and this finding emphasizes the importance of carcass lipid as opposed to carcass protein Tabic 3.5 Egg w eight, w eight i t 25 d of incubitton (tzansfer weight), lelative transfer weight, poult wefgitt i t hatching and poult w eight leiative to initial egg w e i^ t 6nm turkey breeder hens fed either mash (M) o r pelletted (F) diets Diet weight* Transfer weight Poult weight^ M P SEM Source of variation Diet Diet X age Diet X egg size (g) S IJ 81.6 OJ (% ) Probability ^Bgg weight, transfer weight, and transfer percentage means represent 1J50 eggs per diet. % u lt weight and poult percentage means are based on 66% hatchabili^. 45 Cg) (%)

66 as the prim ary nutrient reserve. Ferket and Moran (1986) reached similar conclusions using the subjective measures of carcass Snish and fleshing as t h ^ indices of carcass lipid and protein, respectively. The relative w eight o f the yolk increased with age of hen at the expense of albumen. This age effect increased the yolk to album en ratio from 0.47 to 0.63 from 36 to 55 w k of age. and consistent with previous observations in chickens (Cunningham et al ah; O'Sullivan et al., 1991) and turkeys (Reidy et a i, 1994). Moran and Reinhart (1980) also reported a significant increase in yolk percentage in eggs from 55- vs 48-wk-old Small W hite turkey hens. The significant effect of age on yolk DM percentage in the current study w as prim arily due to the increase at 55 w k com pared with the relative consistency observed at. the four earlier ages. M oran and Reinhart (1980) also reported a s l i ^ t b u t significant increase in yolk DM percentage in 55- vs 48-wk-old hens. Reidy et al. (1994) reported a significant increase in yolk DM percentage with age in one of the two strains studied and only after 24 w k compared with 3 and 12 wk of lay. This observation is similar to the 55-wk data reported herein. In broiler breeders, O'Sullivan et al. (1991) observed increased yolk DM percentage in eggs from older hens, but the data were som ew hat biased by the values from the two oldest ages, 39 and 41 wk of age. The conclusion from all these reports is that if there is an age effect on yolk DM percentage, it does not appear to be linear. The w eight of the residual yolk sac of the poult increased w ilh hen age and with increasing w e i^ t of the p o u lt This result is similar to those of Daly and Petersen (1990) and O'Sullivan et ol (1991) with broiler chicks and those of Skewes et al. (1988) with Bobwhite quail The results are contrary to the results of Moran and Reinhart (1980); however, those authors used eggs from a Small White turkey strain and their comparisons were m ade at two older ages when differences in egg w eight w ere minimal. Transfer weights of eggs after 25 d of incubation relative to initial set weights were notably lower for eggs from 36-wk-old hens compared with eggs from older hens. This result confirms earlier studies by Christensen and McCorkle (1982) and Lemer et ol (1993). W hen eggs w ere categorized by size, however, the percentage moisture loss at 25 d was similar among the different weight classes. This finding is in contrast to the results of Reinhart and Moran (1979), who reported that relative egg w eight loss at 25 d was less for heavier eggs. These conflicting results may be a function of different incubation conditions in each of the two studies together with strain related differences in egg size. D ing and Lilbum (1996) reported that 22 to 28 d of incubation is when the greatest amount of yolk sac lipid is transferred to the developing turkey embryo. Eggs from the youngest hens (36 wk) had the smallest fresh yolks and poult yolk sac weights, along with absolute and percentage reductions in embryonic yolk sac lipid. These data suggest that age associated increases in yolk and residual lipid in the embryonic yolk sac are reflective o f changes in lipid metabolism. Alteration in lipid metabolism in embryos from turkey hens is consistent with other studies with chickens ^ofale and Conner, 1984; Noble, 1987; Yaffei and Noble, 1990; O'Sullivan et al., 1991). D ing and Lilbum (1996) have hypothesized that posthatch, yolk sac lipid m ay be more im portant as a precursor source of cellular phospholipids than as an energy source. The weight of poults as a percentage of setting weight was considerably higher in this study than in the data presented by Wilson (1991). The positive, independent effects of hen age on poult weight are sim ilar to that reported for embryos of broilers (Shanawany, 1984) and Japanese quail (Yannakopoulos an d Tserveni-Cousi, 1987). This effect may be related to the im provem ents in lipid metabolism associated with embryos from older hens. fri conclusion, hen BW loss w ith the onset of production was reflected mainly in the relative loss of abdominal fat, rather than the relative loss of P. major weight. As hens aged, relatively more yolk was deposited in the egg at the expense of album en. This change w as reflected in an increased residual yolk sac w eight in poults at hatching. Independent of egg size, poult w e i ^ t at hatching w as greater as hens aged firom 36 to 55 w k of age. ACKNOWLEDGMENTS The authors wish to thank C uddy Farms, Inc, Danville O H for the generous donation of hens and fertile eggs used in tiiis experim ent We would also like to express our appreciation to Roll Turkey Farms, fric, Versailles, O H for their help in obtaining the carcass data and for supplying the fertile e g ^. REFERENCES Bacon, W. L., and K. E. Nestor, Body weight changes during ttie reproductive period in four strains of turkey hens. Poultry Sd Bacon, W. I_, K. E. Nestor, and P. A. Rermer, The influence of genetic increases in body weight and shank width on the aixlominal fat pad and carcass composition of turkeys. Poultry Sd Christensen, V. L, and F. M- McCorkle, Characterization of incubational egg weight losses in tfiree types of turkeys. Poultry Sci Cunningfiam, F. E., O. J. CotterilL and E. M. Funk, 1960a. The effect of season and age of bird 1. On egg size, quality and yield. Poultry Sd Cunningham, F. E, O. J. CotterilL and E M. Fimk, 1960b. The effect of season and age of bird 2. On the chemical composition of egg wtiite. Poultry Sd Daly, K. R., and R. A. Peterson, The effect of age of breeder hens on residual yolk fat. and serum glucose and triglyceride concentrations of day.old broiler chicks. Poultry Sd. 69:

67 Ding, S. T., and M. S. Liibum, Oiaracterizaaon of changes in yolk sac and liver lipids during embryonic and early posthatch develoomenc of turkey poults. Poultry Sd. 75:4T8-!83. Serket. P. R., and E. T. Moran, Jr., Effect of plane of nutrition from starting to and through the breeder pericxi on reproductive performance of hen turkqis. Poultry Sd. 65: rblch, J., M. Lees, and G. H. Slone-Stanley, A simple method for the isolation and purification of total Uplds ffom animal tissues. J. Biol. Chenu rreeman, B. M- and M. A. Vince, Page 163 on Development of the Avian Embryo. Chapman and HaB, London. UK. Harper, J. A, Changes in body weight and conformation of broad breasted bronze turkeys during the breeding season. Poultry Sd. 29: Krueger, K. K, J. A Owen. C E. Krueger, and T. M. Ferguson Effect of feed and light regimens during the growing pericxi on subsequent reprtxiudive performance erf Broad- Breasted White turkeys fed two protein levels. Poultry Sd Lemer, S. P...M. French. D. McIntyre, and C. Baxter-fcmes, Age-related changes In egg prcxlucnion, fertility, embryonic mortality, üid hatchability in commercial turkey flocks. Poultry Sd. 72: Lilbum, M. S Skeletal growth of commercial poultry species. Poultry Sd. 73: Lilbum, M. S., and K. E. Nestor Body weight and carcass development in different lines of turkeys. Poultry Sd. 70; Lilbum. M. S., and K E Nestor, The relationship between various indices of ctcrcass growth and development and repnxiuction in turkey hens. Poultry Sd. Th McCartney, M. G., D. C. Borron, and H. B. Brown, Repniductlve performance of tiukey females as affikted by growth anci pre-breeder diets. Poultry S d 56: Moran, E T., Jr., and B. E Reinhart, Poult yolk sac amount and composition upon placement: effect of breeder age, egg weight, sex, and subsequent change with feeding or ksting. Poultry S d 59: Nestor, K E, Genetics of growth and reproduction in the turkey. 9. Long-term selection for increased 16-week body weight. Poultry S d Nestor, K. E, and D. O. Noble, Influence of selection tor increased egg production, body weight, and shank width of turkeys on egg composition and the relationship of tfie egg trails to hatchability. Poultry S d 74: Noble; R. C, Lipid metabolism in the chick embryo: Some recent ideas. J. Exp. ZooL l(suppl): Noble, R. C, and K. Connor, Lipid metabolism in the chick embryo of the domestic fowl (Grflirs domesticus). World's Poult S d J. 40: Noble, R. C, F. Lonsdale. K. Cormor, and D. Brown, Changes in the lipid metabolism of the chick embryo with parental age. Poulffy S d 65: O'Sullivan, N. P., E E Duimington, and P. B. Seigel, Relationships among age of dam, egg components, embryo lipid transfer, and hatchability of broiler breeder eggs. Poultry S d Reidy, R. E, J. L Atkinson, and 6 Leeson, Strain comparisons of turkey egg corrmonents. Poultry S d 73: Reinhart B. S.. and E T. Moran. Jr., Incubation characteristics of eggs fiom older small white turkeys with emphasis on the efferhs due to egg weight Poultry S d 58: Robel. E J., Weight dass as related to brxdy composition in aging white turkey hens. Poultry S d 63: SAS Institute, 1986 SAS User's Guide: Statistks Edition. SAS Institute Inc, Cary, NC. Shanawany, M. M., Inter-relationship between egg weight parental age and embryonic development Br. Poult S d 25: Shanawany, M. M., Hatching weight in relation to egg weight in domestic birtfs. World's Pmilt Sd. J. 43: Skewes, P. A, H. E WEson. and F. B. Mather, Correlation among egg weight, diick weight and yolk sac weight in Bobwhite cpiail {Colimis oirgmianus). Florida Sd 51: Wilson, H. E, Interrelationships of egg size, chick size, posthatching growth and hatchability. World's Poult S d J Yaffd, N.. and E C Noble Further observations on the association between lipid metabolism and low embryo hatchability in eggs from young brrhler birds. J. Exp. ZooL Yannakopoulos. A E. and A S. Tserveni-GousL Resarrch Note: Effect of breeder cjuail age and egg weight on chick weight Poultry S d 66:

68 CHAPTER 4 Effect of Hen A ge, Body W eight, and Age a t P hotostim ulation. 1. Egg, Incubation, a n d Poult Characteristics o f Com m ercial Turkeys^ T. J. APPLEGATE and M. S. LILBURN2 Deçaitment of Arrimai Sciences, The Ohio State Uaivetstty, Ohio A plcuituial Research and Deve&pment Center, Wooster. Ohio ABSTRACT Turkeys from two BW groups (which averaged 11.3 and 12.9 kg. Normal and Heavy, respectively) w ere photosdmulated at either 29 o r 31 w k o f age. to d aennin e w hat esects age-associated changes in hen carcass characteristics, egg weight, an d egg components have on subsequoit poult weight at hatching during the Erst 10 w k of production. Carcass m easurem ents w ere daerm ined from a subsample o f hens 3 w k after photostimuiaiion (PS). Subsamples o f eggs from each PS age and BW group were selected fo r yolk and album en measurements. All other eggs from these hens were individually weighed and incubated a t 2-wk intervals during the Erst 10 w k of lay (4 to 14 w k after PS). All incubated e g ^ were reweighed at 25 d (transfer) and poults w ere individually weighed a t hatch. Hen BW at PS had no effect on ovarian w eight and follicular size or num ber 3 wk later. Hens photostim u lated at 31 w k had 18.7 g heavier ovaries 3 w k after K than hens photosdm ulated a t 29 w k (P < 0.06), primarily due to one additional follicle o f > 20 mm (P 0.04). During the first 10 w k of lay, egg w eight increased 7.8 g, 4.4 g of which w as due to increased yolk w d g h t Eggs firom Normal BW hens w eighed significantly less than those firom Heavy BW hens only w hen h a is were subjected to PS a t 29 wk. Egg w eight a t transfer relative to egg weight a t set w as significantly increased firom hens 4 to 6 w k after PS com pared w ith the four older production ages (6 to 8, 8 to 10,10 to 12, and 12 to 14 wk after PS). There was a significant increase in poult weight with increasing hen production age bm no changes in poult weight relative to egg w eight at set In conclusion, hen BW at PS had m inim al effects on egg component weights and subsequent poult weight at hatching. (Key words: turkey, hen aige, hen weight, egg, photostimulation) 1998 Poultry Science 77: INTRODUCTION Conunerdal turkey hens are typically exposed to a photoinhibitory lighting schedule (8 h UgbtrlS h dark) beginning at approximately 18 w k of age. They are subsequently photostimulated ^ 14 h light: PS) between 29 and 31 w k of age. Within the industry, there exists a concept that PS at younger ages will have negative effects on reproductive performance, including delayed onset of smcual maturity after PS. decreased egg production, and decreased hatchabüity. In the few controqed studies thar have been reported, noncommerd al turkey strains have been used and these strains weigh considerably less than the Large White coiiunerd al strains prevalent in the industry today. The results of these studies suggest that hens achieve their optim al reproductive BW at approximately 30 wk of age Received for pubucaoon March 3, Accepted for publication October tsalaiies and research support provided by State and Federal funds appropriated to the Ohio Agricultural Reseandi and Development Center. Manuscript Number rfo whom correspondence should be addressed: lubum.ieoau.edu (Leighton and ShofBier, 1961a,b; Shoffiier e t al. 1962: Wilson fit ai, 1962; Leighton and Potter, 1969; Woodard ef ai. 1974). Present-day commercial turkey hens can reach their reprodutztive BW target w ell before 30 w k of age. yet reprodutxive performance is still poor if hens are photostimulated at too young an age. Slopes (1992) reported tliat when hens w ere PS a t 24 vs 30 w k of age, egg production was sim ilar during the first 20 w k of lay, but age at PS w as positively correlated w ith egg weight and poult w eight during the first 11 w k. There was no mention of hen BW in this study, however. Hocking (1992) reported that hens photostim ulated at 18 or 24 wk of age produced a higher proportion of poults with splayed 1 ^ and unfiealed navels than hens photostimulated at 30 w k of age. After tfie onset of egg production, there is a positive correlation of hen age w ith egg w eight and egg weight with poult w eight (Reinha rt and Moran, 1979; Shanawany, 1987). Breeder age and hatchling weight may not sfiare the sam e degree of relatedness, however Abbreviatioa PS = phocostim aladm. 48

69 (McNaugfaton et ai, Applegate and Lilbum. 1996a). W hai adjustm ents w ere m ade for initial egg weight. A p p l^ ate and Lilbum (1996a) reported that poults from young turkey hens (36-wk-old) weighed less than those fmm older hens (55-wk-old). They attributed this reduced w eight partly to differences in the maternal investment in the form of yolk deposition. Li that stuc^, the yolk to album en ratio increased from 0.47 to 0.63 between 36 an d 55 w k of age. The hypothesis o f the current experiment was that hen age at M and hen BW at PS have independent effects on selected carcass traits 3 w k after PS. To furtha" test this hypothesis, w e also studied the subsequent effects of fia i age a t PS and hen BW at PS on poult w eight at hatching w ith consideration given to age associated changes in egg weight through the first 10 w k of production. MATERIALS AND METHODS Commercial turkey h a ts (Bntish United Turkeys of America.^ 1995) w ere fratched and reared a t the Ohio A gricultural Research an d D evelopm ent C enter (OARDC). Wooster, O H AH hens were exposed to a photoinhibitory lighting period (8 h Iight:16 h dark) beginning a t 18 w k of age. The experimental design included tw o BW classes at PS (NotrnaL kg; Heavy ± 0.5 kg) and two ages at PS (29 and 31 w k of age). These BW groups reflect the range of target BW of hens at PS by commercial turkey breeders, and were created by e le c tin g hens within these ranges from a larger population of hens. A t each of the two PS ages. 11 iiens ^ m each BW treatm ent were allocated to three tireeder pens w ith litter floors (total n = 33 hens per BW class and PS age). Three weeks after PS, five hens per BW treatm ent and PS age were randomly selecmd for BW and carcass measures, including the weight of the Pectoraiis mafor and Pectoraiis m inor musdes. abdominal 6 t pad weight, including & t around the gizzard, and w eight of the ovary. The numfjer and diam eter of vitellogenic follicles ^ 8 mm) w as also recorded. Hens w ere inseminated weekly commencing 2 w k after PS w ith sem en from a randombred Large White strain (RBC-3, Noble et ai. 1995). Eggs were collected twice daily by pen and saved in 2-wk intervals prior to setting. Iherefore. the hen production age data are reported in relation to PS as follows: 4 to 5. 6 to 8. 8 to to 12. an d 12 to 14 w k after PS. Prior to setting. 5 eggs per pen (15 total per hen BW and age at PS) were individually weighed and fiard-cooked in shell for albumen and yolk measurements and yolk DM determinations. O ther eggs were individually weighed prior to setting in a Robbins incubator* the day after tfie last day of the 2-wk egg collection period. Incubator tem perature B ritish United Turkeys of America. Lewisburg, WV irnbbiits Incubator Co.. Denver. CO w as m aintained at 37.5 C and 60% RH and hatcher tem perature maintained a t 36.9 C and 80% RH. After 25 d of incubation, eggs w ere reweigfied and placed witfiin individual pedigree b ask as. A t hatch, poults were individually weighed, w ing-banded, and brooded. Mortality data were th a i recorded through 2 w k of age^ The d ata were analyzed by analysis of variants using the General Linear M odels procedure of SAS (SAS Institute. 1986). The m ain effects tested were age a t PS. BW a t PS. hen production age. and their interactions, w h e e b y a pen of hens represaited an experimental unit. Analysis of percentage data was done after arc sine transformation. One objective o f tins eiqperiment w as to dlffaentiate baw een th e effects due to egg size vs hen production age. At the a i d of the study, all eggs were assigned to one of eight egg w eight classes for further statistical analyses. Egg w eight at set. transfer w eight (25 d). and poult w eight w ere reanalyzed using a model th at included egg w eight class, hen production age. and their interactions. RESULTS Age a t PS had no effect on hen BW, P. major weight. P. mfoor weight, or abdom inal tel w eight 3 wk after PS (Table 1). Hen BW w as still significantly di& rent baw een the two BW groups 3 w k after PS (averaged across ages at K. P S 0.002). Neither the afnolute nor relative w eight of th e P. major o r P. m inor were statistically different b a w e a i Normal and Heavy BW hens. Heavy BW bens, however, had 71 g more abdominal fat (P 0.03} tfian Normal hens. Ovarian weights w k after PS) w ere 18.7 g or 15% heavier (P S 0.06) w hen hens w ere PS a t 31 vs 29 wk (Table 2). This w as largely a function of one additional follicle o f > 20 mm in hens PS at 31 wk. Hen BW a t PS had no effect on afaolute or relative ovary weight or on the size or num ber o f follicles 3 wk after PS. Over the 10-wk production period, there w as a significant increase in th e weigfrt of the eggs randomly selected for yolk an d album en measurements (P ; Table 3). prim arily due to a significant increase in yolk w eight (4.4 g; P < ). Yolk DM percentage significantly decreased (P 0.001) over the course of the first 10 w k of lay. A bsolute altiumen weight did not significantly ciiange w ith hen production age; tfius there w as a significant decline in albumen w eight relative to egg w eight (P < ). W hen hens w ere PS at 31 wk of age. eggs selected for y o lk and albumen measurements w ere heavier, had heavier yolks (afrsoluie and relative weight), and had relatively lighter albumen weight over tfie 10-wk production period (P S 0.03). Hen BW at PS fiad no consistent effects on egg weight or egg com ponent weights. With increasing fien production age. tfiere were significant increases in egg w eight (P S ). transfer egg w eight (P < ). an d poult w eight (P S ) and a significant d ed in e in transfer w eight relative to 49

70 Table 4.1 Turkey breedo' ben cairaan cfaaracteristks 3 w k afler pbom adm nlaüan Age at phocosormrlatloa Hen BW» Hen BW P n fjimtff m a jas^ rvffna-atff /ni'rvua Abdominal 6c* W O (kg) (e) (36) (g) (36) <g) (36) 29 Normal 11.87! Heavy Normal U Heavy SEM Source of variadon Age ac pbofnsümu^rioo H en BW Age X BW Main effect meatw Age ac pfaotosttmujacion 29 w k w k H en BW ac pliotosrimulaüon Norm al H eavy ^Average ben BW ac pbocusnmuiaüon: N orm al => 11.8 kg. H eavy 129 kg. zpecoralû mgon P. m apr percentage = (left P. nsgar weighc/bw ) x 100. ^Lelt Peaonslis m im r P. nânar percentage = (left P. aauor weight/bw ) x Abdominal includes the fat disassociated &om the gizzard. Abdominal percentage = (abdominal Die weigfit/bw) x 100. SMeans represent five hens per BW group and age at photostiinulatton. egg w eight at set (P < ; Table 4). There w ere no significant différences in poult weight as a percentage o f initial egg weight. When egg w eight fiom all hen produtnian ages w ere stratified into egg w eight classes, there w as a significant increase in egg w eight a t 25 d transfer (P S ). transfer w eight relative to egg w eight at set (P g 0.004), and poult w eight a t hatch (P g ), but there w as a significant decline in relative poult w eight (P < , relative to egg w eight at set) w ith increasing egg w eight Age at PS had no effect on initial egg w eight or transfer w eight (Table 5). Relative transfer egg w eight (P g ), absolute poult w eight (P < 0.05), and relative poult weight (P S 0.03) w ere increased w hen hens were Table 4.2 Turkey breeder ben reproductive cfaaraclerrsrlcs 3 w k alter photustim ulatloa Age at photostimolatioa Hen BW* OvaryZ Follicles 28 mm >20 mm (wk) (g) (36) 29 Normal Heavy Normal Heavy SEM Source of variatioo Age at photostimulatian Hen BW Age X BW \ 6 m effect means Age ac photoatfmulatlon à wk w k Hen BW at photostlmulatkm Normal Heavy ^Average ben BW at pbomstbnulaüon: Norm al = 11.8 kg. Heavy 129 kg. io vaty pacenrage = (ovary weighc/bw) x 100. h e a rts lerrresent Ove hens per BW group and ape ac photosrhnulatlon. 50

71 Table 4 3 weight, yolk w eight, relative w eig h t dxy matter, album en w e ig h t rehtive albumen w eight firom t u d ^ breeder bens during the first 10 w k o f prodncdoo^ Variafafe Egg w eight YoDt weight Relative yoflt weight Y olk D M Album en w eight R elative albumen w eight - (ÿ DM) <g) m Weeks after phocostlmujadon 4 to 6 wk to S wk S ID 10 wk to 12 w k to 16 w k Age at photostimulattoa 29 wk wk Hen BW«Normal Heavy SEM Soares of vartation Hen age Age ac pfaotostlmtilatlon Hen BW 0J ^Means based on 15 e g g p e r hen BW and age a t photostimuiadoa (60 total) p e r age. ZYoDc percentage = w elght/egg weight) x 100. ^Albumen percentage = (albumen weight/egg welghc) x 100. ^Average hen BW at photostinmiation: Normal = 113 Heavy = 123 kg. Table 4.4 Egg weight, w eight at 25 d o f incubation (transfer w eight), relative transfer weight, poult w eight a t baching, and poult w eight relative to initiai egg w eight &om tn r k ^ breeder hens during the first 10 wk o f prodocdoa Variable weight^ Transfer weight Relative transfer w eight P oult w eight Relative poult weight - w P6) (g> C6) Weeks after photostbntiiazkm 4 to 6 wk to 8 wk to 10 wk to 12 wk to 14 wk SEM Egg sise, g< S to to to to to to & SEM Source of variacloa Hen age Egg sizb weight, transfer weight, and transfer percentage based on 860, 777,530, 555, and 455 eggs for each production age, respectively. tra n s fe r percentage = (egg weight ac 25 d Incuhation/egg weight at set) x 100.? o u lt w eight and poult percentage means based on an d 280 p o u te fo r each production age. respective^. Poult percentage = (poult weight a t batch/egg w ei^it a t set) x 100. ^Egg size means based on , and 58 gggt fo r each w eight class, respectively. 51

72 Age at photostlmuiatlon Table 4 J Kgg w eigu. egg w eigm a t S <1 o f inciuatloa (baib ter weignt). poult wdgdc and poult weight relative to egg w eight a t set horn tuthey bteeda beta dating the Dist ID w it o f prortnidlnrf Ifen BW2 Egg weight T ransfer w eight Rejattve tra n sfe r w eight Poult weight fe ) W (& 06) 23 Nonnal Heavy Nonnal Heavy 8 4 J SEM Relative poult weight Source of variation Age ac phocosomulacloa Hen BW Age X BW ODT Main efiecc oieaas Age at phocosumuiation 23 wk 84J wk Hen BW ac pbocostimuladaa Normal Heavy legg w eight and transfer w eight means based o n at least TOO eggs per ben BW and age at photostim ufadon. Poult w eight m eans based on at least 400 poults per hen BW and age at phocosilmufahon. ^Average ben BW. at photostlmuiatlon: Notmal = l U kg. Heavy = 119 kg. itransfer perceniage = (egg weight at 25 d incubadon/egg weight at set) x IQG. ipoult weight = (poall w eight/egg weight at set) x 100. PS at 29 w k of age. Egg w eight at set from Heavy hens PS a t 29 w k of age w as increased and this resulted in an overall effect of hen BW a t PS (P 0.056). There w ere no significant difierences in percentage transfer weight, poult weight, or penrentage poult weight due to hen BW at PS, The aforementioned increase in egg w eight by Heavy hens PS at 29 w k resulted in a hen PS age by BW at PS interaction (P s 0.07). Poult mortality through 2 wk of age was unaffected by hen production age, hen age at PS. or hen BW at PS (data not shown). DISCUSSION Hens th at were PS a t 31 wk of age had enhanced ovarian development prim arily due to an additional large follicle (> 20 mm). Applegate et al. (1997) reported that baseline luteinizing hormone concentrations were increased in hens E*S at 24 to 25 w k of age compared with hens PS at 27 to 28 o r 31 to 32 w k of age. Appl%ate et al (1997) concluded th at the differences in luteinizing horm one response to PS was as dependent upon chronologic age as it w as on difierences in body composition and supports the age-associated responses in ovarian development rqxjrted here. As egg weight increased during the initial 10 w k of production, there w as a concomitant increase in the yolk to album en ratio from 0.50 to Similar changes in the relative proportion of egg components with increasing hen production age have also been reported in chickens (Cunningham et al a.b; O'Sullivan ef al, 1991) and in previous studies with turkeys (Moran and Reinhart, 1980; Reidy et a l, 1994: Applegate and Lilbum, 1996a). While studying h en production age-associated changes in egg w e i^ ts Grom Nicholas hens, Applegate and Lilbum (1996a) repotted an increase in the yolk to albumen ratio from 0.53 to 0.62 from their earliest two hen productian ages (36 to 41 wk of age) with only a 0.1-g increase in egg w eight over this production period, approximately the period o f lay reported in the current experiment. The differences reported in hen production age-associated changes in egg w eights and egg component weights between these studies m ay be largely a reflection of measuring difierent strains over chfiering hen production periods. Egg transfer w eight percentage w as greatest during the hen production period 4 to S w k after PS. The similarity in transfer percentages over the last four hen production ages, however, is in contrast to other reports in the literature, in w hich transfer w eight percentage increased with increasing hen production age (Christensen and McCorkle. 1982; L em er et a l. 1993; Applegate and Lilbum. 1996a). A pplegate and Lilbum (1996a) had previously reported th at in eggs collected from Nicholas hens over an entire 19-wk production period and stratified according to eg g weight, there was also no difference in transfer w eig h t percentage due to differences in initial set w eight. In the report by A pplegate an d Lilbum (1996a). poults from 55-wk-old h en s were significantly heavier (3%) than poults from younger hens (36-wk-old) when initial egg w eight w as acçustecl They attributed part of this w eight difference to differences in yolk size and 52

73 yolk sac vveight together w ith proportional teductians in residual yolk sac Upid a t hatch, hi the current study, poult w eight at hatch increased during the S ist 10 w k of lay, but relative poult w eight did not change. A s the present experiment covered less than half of the production cycle w ith a d ls a e n t strain of hen, realistic comparisons w ith Applegate and Lilbum (1990a) are not appropriate It is interesting, though, th at the w eight of the poult as a percentage o f egg w s g h t in the current study is sim ilar to the d ata presented by Wilson (1991) but considerably low er than that reported by Applegate and Lilbum (1998a). Absolute and relative yolk-hee embryo w eight a t ^ d did increase w ith age of hen (Applegate ef al, 1996b), so there is som e m erit to the hypothesis th at hen production age w ill influence embryonic developm ent independent of dis aences in initial egg weight. In conclusion, hens PS at 31 w k had increased ovarian developm ent prim arily due to one additional large follicle ^ 20 mm) and these eggs had larger yolks than eggs of hens PS at 29 w k of age. Only w hen hens were PS at 29 w k of age did BW have any bearing on egg weight during the Srst 10 w k o f lay. Hen BW at PS had no consistent effect on egg component w eights, incubation measures, or new ly hatched poult weight. With increasing hen production age, there w as proportionately m ore yolk deposited in the egg a t the expense of albumen, although this did not appear to influen ce the poult w eight at hatch relative to egg w eight a t s e t ACKNOWLEDGMENTS The authors wish to thank British U nited Turkeys of America. Lewisburg, WV for their generous donation o f fertile eggs used in this experim ent The authors gratefully acknowledge the technical assistance of John Nixon and the farm staff at the OARDC Turkey Research Center. REFERENCES Applegate. T. J.. W. L. Bacon, and M. S. Ulbum Effect of age and body weight on plasma concentrations of luteinizing hormone in turkey hens before and after photostimulation. Dom. Anim. EndocrinoL Vol 14(6):(ln press). Applegate. T. J.. and M. S. Ulbum. 1996a. Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci 75: Applegate. T. J., J. E. Nixon, and M. S. LUbum. 1996b. Effect of turkey hen age, body weight, and age at photostimuiaiion on egg and embryonic characteristics. Poultry Sd. 75(Suppl. 1):75. (Abstr.) Christensen. V. L, and F. M. McCorkle Characterization of incubational egg weight losses In three qpes of turkeys. Poultry Sd. 61: Cunningham. F. E.. O. J. CotterilL and E M. Funk. 1960a. The effect of season and ageof birrl 1. On egg size, quality and yield. Poultry Sd. 39: Cunningham. F. E. O. J. CotterilL and E M. Funk. 1960b. The effect of season and age of birrl 2. On the chemical composition of egg white. Poultry Sd. 39: Hocking, P. M.. 199E Effects of photostimulatian at I I 24 and 30 weeks of age on the praductivity of female turkeys fed atf UUtum or restricted until point of lay. Hr. PoulL Sd. 33: Leighton. A. T.. Jr- and L. M. Potter Reproductive performance of turkq^ subjected to blackout vasus brownout restricted light conditioos. Poultry Sd. 48: Leighton. A. T- Jr- and R- N. ShoSher. 1961a. Fffwt of light regime and age on reproduction of turkeys. 1. PFTwr of hour, and restricted light treatment. Poultry Sd. 40: Leighton. A. T- Jr- and R. N. Shoffiier. 1961b. Effect of light regime and age on reproduction of turkqts. 2. Restricted vs. unrestricted light. Poultry ScL 40: Lerncr. S. P- N. French. D. Mclniyre. and C. Baxter- Jones Age-related changes in egg production, fertilily. embryonic mortality, and hatchability in commercial turkey flocks. Poultry Sd. 72: McNaughton. J. L. J. W. Deaton. F. N. Reece, and E C. H ^nes Effect of age of parents and hatching egg weight on broiler chick mortality. Poultry Sd. 57: Mtxan. E T- Jr- and B. E Reinhart Poult yolk sac amount and composition upon placemenc effect of breeder age, egg weight, sex. and subsequent change with feeding or fasting. Poultry Sd. 59: Noble. D. O- D. A. Emmersoa and K. E Nestor The stability of three randomized control lines of turkeys. Poultry ScL 74: O'Sullivan. N. P- E A. Ourunngton. and P. B. Seigd Relationships among age of dam. egg components, embryo lipid transfer, and hatchability of broiler breeder eggs. Poultry Sd. 70: Reidy, E R- J. E Atkinson, and S. Leeson Strain comparisons of turkey egg components. Poultry ScL 73: Reinhart. B. S.. and E T. Moran. Jr Incubation characteristics of eggs from older small white turkeys with emphasis on the effects due to egg weighl Poultry Sd. 58: SAS Institute SAS* User s Guide: Statistics Edition. SAS Institute Inc. Cary. NC. Shanawany. M. M., Hatching weight in relation to egg weight in domestic birds. World's PoulL Sd. J. 43: Shoffner. E N.. C. E Poiley. R. E Burger, and E E Johnson Light regulation In turkey managemenl Z. Female reproductive performance. Poultry Sd. 41: Slopes. T. D Effects of age at lighting on reproduction of turkey hens. Poultry Sd Wilson. H. R Interreltttionships of egg size, chick size, posthatching growth and hatchability. World's PoulL Sd. J. 47:5-20. Wilson. W. O.. F. X. Ogasawara. and V. S. Asmundson Ardfldal control of egg production in turkeys by photoperiod. Poultry Sd. 41: Woodard, A. - H. Abplanalp. V. SüimetL and E E Snyder The dfect of age at lighting on egg production and pausing in turkey hens. Poultry Sd. 53:

74 CHAPTERS Effect of Hen Age, Body Weight, and A ge a t Photostim ulation. 2. Embryonic C haracteristics of Com m ercial Turkeys^ T. J. APPLEGATE and M. S. LILBURM2 Dqxa ln ia il o f Animal Sciences, The Ohio State Universitif, Ohio Agricultural Research and Development Center, Wooster, Ohio ABSTRACT Turkey hens &om two BW groups (which averaged 11.8 and 12.9 kg; N orm al and Heavy, respectively) were photosdm ulated a t either 29 or 31 w k of age to determine how changes in egg weight and egg component weights with hen age affect subsequent embryonic grow th and yolk sac lipid mobilizadon. A t 2-wk intervals during the first 10 w k of lay, all eggs w ere collected, individually weighed, and incubated. A subsample of eggs from each photostimulation (PS) age and BW group w ere randomly selected for yolk and albumen w e i^ t determinations and embryo weight, liver weight, and yolk sac measurements at 21 and 25 d of incubation. Yolk and yolk sac lipid measurements were done on sized eggs selected a t 4 to 6 and 12 to 14 w k zifter PS. Yolk-firee embryo weight, liver weight, and yolk sac w e i^ t at 21 and 25 d of incubation increased during the (Key words: turkey, hen age, embryo, hpid, yolk sac) first 10 w k of lay. Neither hen age nor BW at PS had any consistent effects on yoflc-fiee embryo weight, liver weight, o r yolk-sac w eight When simflar-sized eggs (80 to 85 g) w ere selected for analyses, yolk lipid content did not change w ith hen production age. The lipid content of the yolk sac was 0.97 g greater in 21-d embryos from hens 12 to 14 w k after PS than firom hens 4 to 6 w k after PS. Differences in yolk sac residual lipid and lipid subclass characteristics were not evident after 25 d of incubation. In conclusion, hen BW at PS or age at PS h ad minimal affects on embryonic growth during the last week of incubation, and m ost differences in embryonic grow th w ere attributed to differences in youc sac lipid mobilization between hen production ages hrdependent of egg size Poultry Science 77: INTRODUCTION The age of a hen during reproduction can significantly influence embryo development and hatchling growth, and these effects can be independent of differences in egg weight (McNaughton et al, 1978; Shanawany, 1984; Yarmakopoulous and Tserveni-Gdusi, 1987; Applegate an d Lilbum, 1996). hr the latter report (Applegate and Lilbum, 1996), both absolute and relative poult weights (relative to egg w eight at set) w ere greater in eggs from 55-wk-old hens than in eggs firom 36-wk-old hens. Effects due to hen production age can partially be explained by greater deposition of yolk in eggs from older hens. Embryos from older hens also have more residual yolk remaining after hatching. Photostimulating hens at older ages can increase the hatching weight of poults (Slopes, 1992) as well as im prove poult quality (Hocldng, 1992). Received for publicadan March 3, Accepted for pubiicaddn Novenitxr 1, tsalaries and research support provided by state arid federal funds appropriated to the Ohio Agricriltural Research and Development Center, Hie Ohio State University. Manuscript Number ^ o whom correspoi tdence should be addressed: hlbuiill@o6u.edu Noble et al (1986) attributed the reduced hatchability and livability observed in embryos fimm young broiler breeder hens to inefficient yolk sac lipid mobilization and assimilation into the onbryo. This assertion was based on the lack of disappearance of lipid and its major subclasses firom the yolk sac and subsetpient deposition into the em bryo and liver during 15 to 19 d of incubation of eggs from 25-wk-old hens as compared to 41-wk-old broiler breeder hens. Yaffei and Noble (1990) reported th at in embryos from 23-wk-old hens, there were lower plasm a lipid and lipoprotein concentrations and a marked, reduction in the cytosolic accumulation of hepatic lipid at 19 d of incubation compared with enibryos from 35-wk-old broiler breeder hens. Yolk lipid has been estimated to supply 90% of the energy n œ d s of the embryonic chick (Romanoff, 1960; Freeman a n d Vince, 1974). In the embryonic turkey (poult), nearly 80% of total yolk lipid is mobilized during embryonic development (Ding et ol, 1995; Ding and Lilbum, 1996). More than half of this is mobilized Abbreviatioa Key: CE = cholesterol ester; NL = lœutiul lipid; PS = photostimulatian; TC» tnacylglycgrol 54

75 Table 5.1 YoOc-bcee embryo weight and relative weight to tnihal egg weight; and lir a weight and relative we%ht at 21 and 25 d of incabation of embryos from tnrlcey breeder hens dnring the first 10 wlc of prrxirretian Weeks after photostimulatûn Embryo weight (yoflc-nee)^ Liver weight^ 21 d 25 d 21 d 25 d (wk) cs) (%) (g) (%) (g) (%) Cs) (%) 4 to Ô ST 6 to 8 29j S to 10 29i L to O S to SEM D4 QÜ2 QM Probability of hen age efect tembryo and liver weight means were based on 60 embryos per hen age. Embryo percentage = Yolk-fiee embryo weight at (sampimg/egg weight at set) % 100. ^Lrvcr parentage = (E va weight/yolk-fiee embryo weight at sampling) x 100. during die final week of incubation (Ding et ul, 1995). The rapid transfer of lipid out of the yolk is positively correlated w ith increased hepatic lipid, the majority of which (76% a t hatch) is in the form of cholesterol esters (Ding et ol, 1995). One objective of the current research was to determine, during the first 10 w k of lay, the independent effects of hen age and BW at photostimulation (PS) on embryonic development at 21 and 25 d of incubation. The other objective was to study the independent effects of hen production age on changes in the major lipid classes in the yolk sac w hen adjustments were m ade for differences in egg weight. MATERIALS AND METRODS British United Turkeys of America^ hens, hatched December, 1995, were reared at the Ohio Agricultural Research and Development Center (OARDC), Wooster, OH All hens were exposed to a photoinhibitory lighting schedule (8 h lighfcl6 h dark) beginning at 18 w k of age. Eleven hens from each of two BW groups w ere assigned to each of three pens at PS (16 h lig h ts h dark), either at 29 or 31 w k of age. Mean hen BW at both PS ages were 11.8 ± 0.5 kg (Normal) and 1Z9 ± 0 5 kg (Heavy). These BW groups reflect die range of target BW of hens at PS by commercial turkey breeders, and w ere created by selecting hens within these ranges from a larger population of hens. Hens were inseminated weekly beghming 2 w k after PS with semen from a randombred, large white strain (RBC-3, Noble et al., 1995). A t 4 to 6,6 to 8,8 to 10,10 to 12, and 12 to 14 w k after PS, all eggs were collected, individually weighed and set in a Robbins incubator.'* Incubator temperature was m aintained at 375 C and 60% RH and hatcher tem perature was maintained a t 365 C and 80% RH. Prior to setting, one 80 to 85 g egg firom each pen was weighed and hard-cooked in shell for yolk measurement and yolk DM determinatian. After 21 and 25 d of incubation, respectively, five eggs per pen (15 total per hen BW and age at PS) w ere randomly selected and the embryos were removed and euthanatized by decapitation. The yoflc sac of the poult was carefully removed, weighed, and frozen in liquid nitrogen. The yolk-firee embryo weight was recorded and the liver was subsequently excised and weighed. O n Day 25 of incubation, all remaining eggs were reweighed and placed within individual pedigree baskets. The DM content of the yoflc sac was determined after lyophilization. The yoflc sac firom one egg per pen that had an initial set weight of 80 to 85 g was selected for total lipid analysis. Total lipid was determined on dry yolks and yoflc sacs after extraction w ith chloroform; methanol (2:1) as reported by Folch et ol (1957) and modified by MarcheseUi and Bazan (1990) and Ding et al. (1995). Lipid extracts were applied to a silica chromatography column^ and the neutral lipid (NL) and phospholipid fractions were eluted firom the column w ith diethyl ether and methanol, respectively. The cholesterol ester (CE) and triacyiglycerol ftg) fiactions of total NL were subsecjuently determined by the HPLC pnacedure described by Bacon et ol (1982) as modified by Ding et al. (1995) by comparison w ith a pure standard containing cholesteryl palmitate and tripalmitin. The results were analyzed statistically by analysis of variance using the General Linear Models procedure of SAS (SAS Institute, 1986). The effects tested were the age a t PS, hen BW at PS, hen production age, and their interactions, whereby a pen of hens represented an experimental u n it Analysis of percentage data was done after arc sine transformafion. B ritish United Turkeys of.america, Lewisburg WV ^Robbins Eicubator Co., Denver, CO scatalog num ber 3092S0: AElech Associates, Etc, Deerfield. IL RESU LTS O ver the 10-wk production period, yolk-free embryo weight at 21 and 25 d of incubafion increased signifi 55

76 Table 5 ^ Yolk> ree onbiyo weight and relative w eight to initial egg w eight and liver weight and relative weight at ZL and 25 d of incabation of emhryoe from turkey breeder hena during the S ist 10 wit o f production Age at photostiixxzilation Hen BWi Fnibryn weight (yodc-ftee)^ Liver weight^ 21 d 25 d 2L d 25 d (W K ) Cg) (%) (g) (%) (g) (%) (g) (%) 29 Normal S I S O S L78 Heavy 2&7 35J} 4 6 J S Normal I S Heavy S LS SEM OOl Source of vanatum Age at photcstzninlatxcn Hen BW 0J O S Z Age X BW Main effect means Age at pbotostxxnuiatxon. 29 w k S. 473 S O S wk S 132 O S 131 Hen BW at photostxmxilatian Monnal J I S O S 130 Heavy JD O S I S 034 L78 ^Average hen BW at photosthnuiatioru Normal = ILS, Heavy = 1?.9 kg. -Embryo percentage - (yoik-hee embryo weight at sam pling/egg w eight at set) X loo. ^Lrver percentage = (liver weight/youc-hee embryo weight at sampling) x 100. SHmbryo and liver means based on 75 embryos per hen BW and age at photostnnolation. cantly (P < 0.003, Table i). Most of the hen production age effects observed a t 21 d of incubation w ere due to considerably lower embryo weight during the initial 4 to 6 w k after PS period w ithout m any differences between the latter four hen production ages. Similarly at 25 d, the hen production age effects were considerably low er at 4 to 6 and 6 to 8 w k after PS w ith no differences betw een the older three hen production ages. Liver w eight a t 21 and 25 d of incubation also increased with increasing hen production age. The weight of the youc-&ee embryo, relative to egg w eight at set, did not change w ith hen production age at 21 d, whereas there was a significant increase (P < 0.02) a t 25 d. Liver weight relative to yolkfree embryo w eight also increased significantly at 25 d, due primarily to the increased weight observed at 12 to 14 w k after PS. Yolk-firee embryo w eight at 21 d of incubation was heavier w hen hens were photostimulated at 31 w k of age (P S 0.03} and w ere of Normal BW at PS (P S 0.03; Table 2). H en age at PS or hen BW at PS had no affect on yolk-free embryo w eight at 25 d of incubatiorl N either hen age nor hen BW at PS had any consistent effects on relative embryo w eight a t 21 o r 25 d (relative to egg w e i^ t a t set). Absolute liver weight was greater in 25 d embryos from hens photostimulated at 31 w k of age (P < 0.019) but there w ere no significant effects on liver weight relative to yolk-free embryo weight. Absolute and relative yolk sac (relative to egg w e i^ t at set) w eight a t 21 d of incubation were greater at 10 to 12 and 12 to 14 w k after PS compared w ith the three younger hen production ages (P < , Table 3). A t 25 d, the absolute and relative weights of the residual yolk sac w ere heavier only in embryos from the oldest hen production age (12 to 14 w k after PS). A t 21 d, there was a slight increase in the weight of the yolk sac (P < 0.007) in h a s photostimulated at 31 w k of age b u t no effects due to hen BW at PS. There were no âgnificant effects due to hen age at PS or BW at PS on any yolk sac m easures a t 25 d of incubation. W hen 80 to 85 g eggs from, hens 4 to 6 and 12 to 14 w k after PS w ere selected for yolk analyses, eggs from the older hens had significantly heavier yolk w eight (P < 0.001; Table 4). The yolks from the younger hens had a sm all b u t significantly greater DM percentage and lipid (as a percentage of DM), resulting in sim ilar absolute am ounts of yolk lipid in eggs from both hen production ages, fri the eggs from the older hens, residual yolk sac w eight a t 21 d was 16% heavier (P < 0.001), b u t no differences w ere apparent at 25 d. The difference at 21 d was largely accounted for by increases in DM percentage and lipid (as a percentage of DM). A t 25 d. absolute o r proportional differences in yolk sac DM o r lipid did not exist betw een hen production ages. In 80 to 85 g eggs, the percentage yolk sac NL or PL a t either 21 o r 25 d was not affected by hen production age, but absolute amounts of NL (P < 6.02) and PL (P 0.001) w ere greater in embryos from the older hens (12 to 14 w k after PS) at 21 d of incubation (Table 5). Differences in absolute amounts of NL and PL were not apparent at 25 d of incubation between hen production ages. There was an approximate 30% decrease in the percentage of Œ in residual yolk Hpld a t 25 d (P < 0.09) b u t there w as increased percentage of triglyceride (P S 0.02) as hens aged. This latter difference (81.1 vs 85.7%) w as fer less than that observed for percentage CE, however. Significant differences in absolute amounts of 56

77 Table 5 3 Yolk sac w eight a n d relative w eight to initial egg weight at 21 and 25 d o f inczzhatxon o f embryos gom, tnzkey breeder hens dnnng their S o t 10 w k of production. Yolk sac weight^ Variable 21 d 25 d (s) (%) (g) (%) Weeks after ? to to to U to 14 2L Age at photostxmulatmn 3 wk wk Hen BW3 Normal Heavy SEM Probability Source of variation Hen age Age at photostimalation Hen BW ^Yoik sac w eight includes the yolk membrane and the yolk contents. Yolk sac percentage = (yolk sac weight/egg weight at set) x 100. ^ e a n s based on 15 eggs per hen. BW and age at photostimulatioa (60 total) per age. ^Average hen BW at photostimiilatfon: Normal = 11.5 kg, Heavy = 12.9 kg. a lipid subclass observed betw een hen production ages at 21 d were not apparent at 25 d of incubation. DISCUSSION Unpublished data horn the authors' laboratory indicate that egg weight increased w ith hen production age between 4 to 6 and 14 to 16 w k after PS and was largely associated with proportional increases in yolk weight prior to se t fin the cmrrent study, there w ere no further increases in yolk-free embryo w eight after 8 w k after PS. This result suggests that a direcd association between egg weight, yolk weight, and embryo developm ent does not cxanir after the first weeks o f production. The ovarian weights of hens 3 w k after PS were significantly heavier and eggs h ad proportionately more yolk when hens were PS a t 31 vs 29 w k of age (unpublished data). A t 21 d o f incnibation, the largest Weeks after photostûnuiation Table 5.4 Yolk and yolk sac w e i^ t, dry matter, and lipid composition of selected yolk s a a &om embryos at 21 and 25 d of incabation &om different ages of tnzkey breeder hens (wk) (g) (% DM) (DM, s) (lipid, % DM) (lipid, g) 4 to to SEM Probability of hot age effect Yolk sae* 21 d 25 d 21 d 25 d 21 d 25 d 21 d 25 d 21 d 25 d ( g ) (% DM) (DM, g ) (lipid, % DM) (lipid, g ) to 6 wk to 14 wk SEM Probability of hen. age effect w d ^ t a t setting for brcsh egg yolk determinations of Hptd and DM averaged 833 ±0.4 and 83.7 ±0.4 g from hens 4 bo 6 and 12 to 14 wk after photostzmulation. respectively. ^ e a n s represent 12 yolks or yolk sacs per hen age. ^ o lk sac includes the yolk m embrane and the yolk contents. Egg weight a t setting for embryos at 21 and 25 d of incubation selected for yolk sac lipid and DM determinations averaged 31.7 ± 0 3 and 33.6 ± 03 g from hens 4 to 6 and 12 to 14 wk after photostimulation, respectively. Yolk: 57

78 Table 53 T he lip id annpoailiim i o f jeiected^ tooc sao> from, an bxyoa at 21 and 25 d o f Lacnhadan mm different ages of txakey breeder hens Weeks after nhotosthnaiatiqn (wk) 4 CO 6 12 to 14 SEM Probability of hen age effect NL PL TG 21 d 25 d 21 d 25 d 21 d 25 d 21 d 25 d 722* 70S OS ( m 75U) 74U) 0^ S 292 O j (% totai OOl 4 to 6 4^ ^ to 14 4^ SEM S Probability o f hen age e& ct o jm INL = nmitml lipids; PL = phospholipids; CE = dm lpstptol esters; TG = triar^i^ycerois. % gg weight a t setting for g nbryos a t 21 and 25 d of incubation seiected lor your sac lipid determinations averaged 51:7 a: 0 3 and 833 = 0 3 g horn hens 4 to 5 and 12 to 14 w k after photostimaiatitai, lespetdxveiy. ^ o lk sa/* includes the yolk membrane and the yolk contents. %feans represent 12 yolk sacs per hen age. proportion, of yoik lipid has yet to be mobilized by the developing embryo, so one could expect that treatment effeths on the yolk w ould still be refletied in the residual yoik sac The results presented here support the conclusion that age at PS affects egg yolk and 21-d yolk sac weight differences. Between 21 and 25 d, there is considerable yolk lipid mobilization Grom the yolk sac (Ding et ol, 1995); therefore differences in residual yolk sac w eight or lipid a t 25 d are more reflective of physiologic differences in yolk mobilization and less refiettive of yolk characteristics in the egg prior to set. Yolk-dee embryo weight at 25 d, however, was not affected by hen age at PS. The positive effects of hen production age on absolute and relative yolk-firee embryo (relative to egg weight at set) w eight at 25 d of incubation were similar in direction to the data w ith new ly hatched poults reported by A pplegate and LUbum (1996). The results also support data w ith broiler embryos (Shanawany, 1984) and Japanese quail (Yannakopouios and Tserveni-Gousi, 1987). It should be noted, however, that embryo weight from the oldest hens (12 to 14 w k after PS) were somewfiat low er titan those observed during the preceding 4 w k, and this again emphasizes the inherent variability in this type of data. hr the developing poult, more A an 50% of yolk lipid is mobilized during the last week of incubation (Ding et ol, 1995). Ding et ol (1995) also reported tfmt in two genetic imes differing greatly in BW and egg weight, the w eight of the yolk-fiie embryo was similar untd 22 d of incubation, the age at which rapid mobilization of lipid fixim tfie yolk to the embryo begins. In the report by Ding et ol (1995), lipid uptake fixnn the yoik sac from 22 d through hatch was also greater in embryos from the fester grow ing line. In the present study, there was significantly less CE observed in 21 d embryos from the youngest hens (4 to 6 w k after PS) as compared to those from the oldest hens (12 to 14 wk after PS). From a quantitative stan d p o in t CE decreased approximately 31% between 21 and 25 d in the yolk sacs of embryos from the older hens com pared with no change in the yolk sacs of younger hens. There vras also a greater net movement of TG and PL in embryos of the older hens between 21 and 25 d than in embryos of the younger hens. Concomitant w ith this relationship was a significant increase in absolute and relative liver weight of embryos w ith increasing hen production age. The increase in liver w eight is partially due to Πdeposition from lipoprotein rem nants (Dmg et a i, 1995). Taken together, the n et decline in CE in embryos Grom older hens, differences in yolk sac lipid weight a t 21 but not 25 d, and increased liver w eight suggest th at differences in embryonic lipid mobilization due to hen production age exist These observations support the conclusion drawn by Noble et al. (1986) in comparisons of 15- and 19-d embryos from 25- an d 41-wk-old broiler breeder hens. Their m ost notable observation w as the change in NL composition. W ithin the NL fraction, they observed a considerably greater proportion of CE wiihm the yolk sac membrane of embryos a t 15 and 19 d of incubation from 25-wk-old hens. Ih both the developing chick and poult, CH form ation takes place primarily within the yolk sac mem brane (Noble and Moore, 1967; Noble and Connor, 1984). fin conclusion, similar-sized eggs from younger hens did not differ in absolute yolk lipid content prior to setting. Less lipid was transiferred from the yolk to the embryo in embryos from the younger hens, and this m ay have contributed to low er yolk-free embryo weight and liver w eight a t 25 d. These changes observed at 25 d, however, w ere not reflected in differences m poult weight at hatch relative to egg weight at set (unpublished data). 58

79 ACKNOWLEDGMENTS The authors w ish to thank British United Turkeys of America, Lewisfaur^ WV for their generous donation of fertile eggs used in this experim ent The authors gratefully acknowledge the technical assistance of John Nixon and the farm staff at the OARDC Turkey Research Center. REFERENCES Applegate, X. and M. S- LUbum, Lndependent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry ScL 75ri Bacon. W. L., K. L. Nestor, D. W. Long, and D. H. Ohm Divergent selection for body weight and yolk precursor in CotuniÙL japoniai. 2. Correlated responses in plasma lipoproteins and lipids. Poultry ScL 6: Ding, S. T.. and M. S. Lilbum, Characterization of changes in yolk sac and liver lipids daring embryonic and early posthatch development in turkey poults. Poultry ScL 75: Ding, S. T-, K. E. Nestor, and M. S. Lilbum The concentration of different lipid classes during late embryonic development in a randombred turkey population and a subline selected fer mcreased body w e i^ t at sixteen weeks of age. Poultry ScL Folch, J.. M. Lees, and G. H. Sloan-Stanley, A simple method fer the isolation and purification of total lipids broui animal tissues. J. BioL Chem. 226: Freeman, B. M., and M. A. Vmce Development of the Avian Embryo. Chapman and ffell, London, UK. Hocking, P. M., Effects of photostimulation at 18,24, and 30 weeks of age on the productivity of female turkeys fed ad libitimt or restricted until point of lay. Hr. PoulL Sri. 33: Marcheselli, V. and N. G. Bazan, Quantitative analysis of fetty adds in phospholipids, diacylgiycerols, free fetty adds and other lipids. J. Nutr. Biocfaem McNaughton, J. L., J. W. Deaton, F. N. Reece, and R_ C Haynes, EÉect of age of parents and hatching egg wei^it on broiler chick mortality. Poultry ScL Noble, D. O., D. A. Exnmerson, and K E. Nestor, The stability of three randombred control Irnp^ of turkeys. Poultry ScL Noble. R. C., and K Connor, The synthesis and accumulation of cholesteryl esters by the developmg embryos of the domestic fewl Poultry ScL Noble, R. C, and J. H. Moore The partition of lipids between the yolk and yolk-sac membrane during the development of the chick embryo. Can. J. Biochem. 45: Noble. R. C, F. Lonsdale, K. Connor, and D. Brown Qianges in the lipid metabolism of the chick embryo with parental age. Poultry ScL 65: Romano0, A. L., The Avian Embryo. Structural and Functional Development. MacMillan Co., New York, NY. SAS Institute, SAS* User's Guide: Statistics Edition. SAS Institute Inc., Cary, NC. Shanawany, M. M., Ihter-relationship between egg weight, parental age and embryonic development Br. Poult ScL 25: Slopes, T. D., ESects of age at lighting on reproduction of turkey hens. Poultry ScL Smith, K. P., and B. B. Bohren, Age of pullet effects on hatching time, egg weight and hatchability. Poultry ScL 54: Yaffei, N., and R. C. Noble, Further observations on the association between lipid metabolism and low embryo hatchability in eggs from, young broiler birds. J. Exp. ZooL Yannakopoulous, A. L, and A. S. Tserveni-Gousi, Resôrch note: Effect of breeder quail age and egg weight on chick weight Poultry ScL 66:155&

80 CHAPTER 6 EFFECT OF TURKEY BREEDER HEN AGE AND EGG SIZE ON POULT DEVELOPMENT. 1. INTESTINAL GROWTH AND GLUCOSE TOLERANCE OF THE TURKEY POULT Abstract Three experiments were conducted to determine if hen age and egg weight affect poult intestinal development and glucose tolerance during the first week after hatch. Differences in glucose tolerance were not consistent across experiments. In Experiment 1, 4- day-old poults fi-om the younger hens and lightest egg weight class had significantly greater festing plasma glucose concentrations (P < 0.04) and were also higher at 30 and 60 minutes post-iiyection o f250 mg glucose. In Experiment 2, egg weight class had no significant effects on plasma glucose concentrations of 4-day-old poults (after injection of 2.5 mg glucose/g body weight). In Experiment 3, plasma glucose concentrations were not different between groups at 0 or 30 minutes post-injection (3.75 mg glucose/g body weight), however, poults fi-om the younger hens tended to remain hyperglycemic 60 minutes post-injection vs poults fi-om the older hens. Neither egg weight class or hen age consistently affected small intestinal weight, length, or density (g/cm) measures across experiments. 6 0

81 Introduction i^plegate and Lilbum [1] reported that after correction for differences in egg weight, turkey poults produced by older hens (55 week) were significantly heavier than those from younger hens (36 week). This difference was partially attributed to a disproportional increase in yolk deposition with advancing hen age, i.e. the yolk-to-albumen ratio increased from 0.47 to 0.63 between 36 and 55 week of age. In a later study, Applegate and Lilbum [2,3] noted increases in embryonic body weight (BW) gain (relative to egg weight) between 21 and 25 days of incubation in comparisons made at the very beginning of egg production and after 10 weeks of lay. Conversely, yolk sacs of embryos from similar sized eggs contained more neutral lipid and phospholipid at 21 days of incubation, but not at 25 days of incubation in the aforementioned embryos [3]. They attributed the greater mass transfer of major lipid subclasses out of the yolk sac, during this period of rapid growth, to the older hen producing eggs with a higher percentage of yolk. Egg weight is positively correlated with poult BW at hatching [4]. In addition, eggs from young turkey breeder hens produce lighter weight poults relative to egg weight [1, 5, 6]. Several studies with chickens have reported a correlation of egg weight and post-hatch growth, but this relationship is reduced as chicks age [7]. Studies with turkeys have demonstrated a similar correlation of egg weight and BW at 2 weeks of age [8], but the correlation diminishes and becomes negligible at older ages [9, 10, 11]. The physiological systems contributing to the early post-hatching growth differences, especially in small eggs from hens early in their first production cycle, is uncertain. 61

82 Several physiological systems are immature at hatch in precocial avian hatchlings. In chicks and turkey poults, these include, but are not limited to, metabohc homeostasis 12,13] and the digestive and absorptive capabilities of the gastro-intestinal tract [14, 15, 16]. The small intestine of poults undergoes considerable development during incubation, yet at hatching it is still functionally immature in terms of digestive capabilities [16]. Functional maturation of the small intestine involves both morphologic and physiologic changes and is one of the main constraints to optimal early growth of precocial birds [17, 18, 19]. Latour et al. [13] concluded that homeostatic mechanisms of metabohsm are incomplete in the chick from 1 to 5 days of age after observing non-homeostatic fluctuations in daily plasma concentrations of corticosterone, triglyceride, and glucose. Studies on post-hatching metabolic homeostasis have largely measured point-m-time changes in metabolic substrate concentrations or enzyme activities in birds whose prandial state (fed vs fasted) is relatively uncertain. Our hypothesis, in the studies reported herein, is that poults from small eggs and/or hens early in their first production cycle have a slower intestinal growth rate and reduced ability to modulate plasma glucose concentrations after a glucose challenge during the first week after hatching than poults from large eggs and/or hens late in their first production cycle. To test this hypothesis, three experiments were conducted to measure BW and small intestine growth during the first week after hatching in poults from relatively young and mature turkey breeder hens when adjusted for egg weight. In addition, we assessed the response capabilities of poults to a glucose challenge. 6 2

83 Materials and methods Experiment 1 Commercial turkey hens (British United Turkeys o f America, BUTA, Lewisburg, West Virginia) were reared in commercial facilities (Cooper Farms, Inc., Ft. Recovery, Ohio 45846) and photostimulated at 29 week of age. At both 34 and 44 week of age, fertile eggs from this flock were collected (n=600). Eggs were individually weighed and assigned to one of three egg weight classes (75 to 79.9, 80 to 84.9, and 85 to 89.9 g from the 34-week-old hens and 80 to 84.9, 85 to 89.9 and 90 to 94.9 g from the 44-week-old hens). Within each egg weight class and hen age, 10 eggs were randomly selected and hard-cooked in shell for albumen and yolk weight determinations. Yolk dry matter was determined after lyophilization and total lipid was determined on five dry yolks per egg weight class and hen age after extraction with chloroform:methanol (2:1) as reported by Folch et a i [20]. The remaining eggs were individually numbered and set in a incubator (Petersime Inc., Gettysburg, Ohio). At 21,25, and 28 days (hatch) of incubation, five eggs or poults per egg weight class and hen age were randomly selected and the embryos or poults euthanized by decapitation. Yolk-free BW was determined on all embryos and poults. At hatch (28 days of incubation), all poults were individually weighed, wingbanded, and placed in electrically-heated Petersime battery brooder pens. Poults were fed ad libitum, and individual BW recorded at 7 and 14 days of age. Five poults per hen age and egg weight class were randomly selected at 1, 2, 3, 5, and 7 days of age. Yolk-free BW was recorded. The small intestine was excised beginning at the proximal end of the duodenum extending to the ileo-cecal junction. The duodenal loop 63

84 (pancreas removed) and lower small intestine (jejunum and ileum) were separated, flushed with saline, blotted dry, and weighed. The lengths of the two segments were recorded to the nearest tenth of a centimeter. A glucose tolerance assay was implemented at 4 and 7 days of age as described by Turner [21]. Within each poult age, eighteen to twenty-four poults per egg weight class and hen age were fasted for 12 hours and then injected i.m. {Pectoralis major) with 0.5 ml of a 50% (wt/vol) glucose solution in 0.9% saline. At 0, 30, and 60 minutes post-injection, blood samples fi om 6 to 8 poults per time point per hen age and egg weight class were collected into heparinized tubes after decapitation. Plasma was separated after centrifugation and fi-ozen for later determinations of plasma glucose concentrations (Glucose Oxidase, Procedure No. 315, Sigma Chemical Co., St. Louis, Missouri). All results were analyzed by analysis of variance using the General Linear Models procedure of SAS [22]. The main effects tested were egg weight class (as defined by the lower, mid, and upper weight class from each hen age), hen age, and their interaction. A second model differentiated the effect of hen age in the 80 to 84.9 g egg weight class and 85 to 89.9 g egg weight class by orthogonal contrasts. Experiment 2 Commercial turkey hens (BUTA) were reared in commercial facilities (Cooper Farms, Inc.) and photostimulated at 29 week of age. At 32 week of age, fertile eggs (n=200) were collected from this flock and all eggs were individually weighed and assigned to one of two weight classes (75 to 79.9 g and > 80.0 g). All eggs were set in a Petersime incubator. 64

85 At hatching, all poults were individually weighed and placed in electrically-heated Petersime battery brooder pens. Poults were fed ad libiùim. At hatch, 1, 2, 3, and 4 days of age, five to seven poults were randomly selected and euthanized by decapitation. Yolk-fi-ee BW and small intestine weights and lengths were measured as described for Experiment 1. As in Experiment 1, a glucose tolerance assay was implemented at 4 days of age. Thirty-six poults per egg weight class were fasted for 12 hours and then injected i.m. with 2.5 mg glucose per g BW from a 50% (wt/vol) solution in 0.9% saline. Prior to injection, 30, and 60 minutes post-injection, 12 poults per time point per egg weight class were euthanized by decapitation and blood samples were collected into tubes, containing 10 pi of a 50% (wt/vol) sodium citrate solution. Plasma was separated after centrifugation and frozen. Plasma glucose concentrations were determined as described for Experiment 1. The poults were given a glucose challenge per unit BW in this experiment to account for BW differences. The glucose dosage of 2.5 mg glucose per g BW was calculated based on a predicted 100 g BW of poults at 4 days of age, therefore making the predicted dosage similar to that in Experiment 1. All results were analyzed by analysis of variance using the General Linear Models procedure of SAS [22]. The main effect tested was egg weight class. Experiment 3 Commercial turkey hens (BUTA) were reared in commercial facilities (Cuddy Farms, Inc., Danville, Ohio 43014) and photostimulated at 30 week of age. At 34 and 48 week of age, fertile eggs were collected from each of two flocks. Eggs were individually weighed and assigned (n=160 per egg weight class and hen age) to one of two egg weight classes (75 to 80 and 85 to 90 g from the 34-week-old hens and 85 to 90 g from the 48-week-old hens). 65

86 All eggs were set in a NatureFonn incubator (NatureForm, Inc., Jacksonville, Florida 32202). Upon hatching (28 days of incubation), all poults were individually weighed and placed in electrically-heated Petersime battery brooder pens. Ten poults per hen age and egg weight class were randomly selected at 27 days of incubation, hatch (0), and 1 day of age and five poults per hen age and egg weight class at 2, 3,4, 5,6, and 7 days of age. Yolk-free BW was recorded. The small intestine was excised, weighed, and measured (0, 1, 3, 5, and 7 days of age) as described for Experiment 1. As in Experiment 1 and 2, a glucose tolerance assay was implemented at 4 days of age. Thirty-six poults per hen age and egg weight class were fasted for 12 hours and then injected i.m. with 3.75 mg glucose per g BW from a 50% (wt/vol) solution in 0.9% saline. Time periods and blood collection were similar to Experiment 2. Twelve poults per time point per hen age and egg class were utilized. As in Experiment 2, the poults in Experiment 3 were given a glucose challenge per unit BW to account for BW differences. The poults in Experiment 2 were given a dosage of 2.5 mg glucose per g BW based on a predicted 100 g BW. Body weight at 4 days of age, however, averaged 75 g. Therefore, the dose response to the glucose challenge at 30 minutes post-injection was an average of 99.8 mg/dl less in Experiment 2 as compared to Experiment 1. Therefore, uncertainty remained as to whether the 2.5 mg per g BW glucose administration was sufficient to challenge metabolic regulation. The glucose dosage was subsequently increased to 3.75 mg glucose per g BW for Experiment 3. All results were analyzed by analysis of variance using the General Linear Models procedure of SAS [22]. Differences between hen production ages and egg weight classes 6 6

87 were determined using a Duncan means separation when the significance of the experimental model was < Results Experiment 1 Egg weights from each hen age and egg weight class are presented in Table 6.1. Even though eggs were stratified into weight classes, the eggs from older hens were 0.94 and 0.62 g heavier in the 80.0 to 84.9 and 85.0 to 89.9 g egg weight classes, respectively {P < 0.01). Eggs from the older hens contained less albumen (3.96 %) and more yolk relative to egg weight (3.83%, P < ). The eggs from the 44-week-old hens (80 to 89.9 g eggs) contained 3.59 g more yolk as compared to eggs from the 34-week-old hens {P < 0.05). This additional yolk deposition by the older hens consisted of 2.03g more dry matter (1.59 g more hpid; P < 0.05). Intact poult BW was consistently heavier in poults from younger hens and from similar sized egg weight classes from the younger hens at hatch, 7 and 14 days of age (P < 0.01, Table 6.1). Yolk-free BW the first day after placement (1 day of age), however, was 6.59 g lighter in poults from similar sized egg weight classes from the younger hens, but this effect was not sustained and at later ages was reversed (Table 6.2). As the residual yolk sac remaining at the older ages (7 and 14 days of age) was minimal, yolk-free BW was similarly affected by hen age (P < 0.01). Egg weight class (either within or between hen ages) did not consistently influence intestinal measurements, therefore only data from the similar egg weight classes between hen ages are reported. Poult duodenum weight was significantly heavier in poults from 34- vs 44-67

88 week-old hens in the 80 to 84.9 g egg weight class at 5 days of age (P < 0.05), but differences between hen ages within similar sized egg weight classes were not apparent at any other age (Figure 6.1). Hen age, within similar sized egg weight classes, had no affect on duodenum length. Duodenum density (g/cm) was significantly greater in poults fi"om 34- vs 44-week-old hens in the 80 to 84.9 g and 85 to 89.9 g egg weight classes at 5 days of age (P < 0.05), but differences between hen ages within similar sized egg weight classes were not apparent at any other age. Combined jejunum and ileum weight was significantly greater in poults fi"om 34- vs 44-week-old hens in the 80 to 84.9 g egg weight class at 2 and 5 days of age (P < 0.05), but différences between hen ages within similar sized egg weight classes were not apparent at 1, 3, or 7 days of age (Figure 6.2). Jejunum and ileum length was significantly greater in poults fi"om 44- vs 34-week-old hens in the 85 to 89.9 g egg weight class at 1, 2, and 5 days of age (P < 0.05), and in the 80 to 84.9 g egg weight class at 3 days of age (P < 0.05). Differences in jejunum and ileum lengths in poults due to hen age within similar egg weight classes were not apparent at 7 days of age. Jejunum and ileum density (g/cm) was significantly greater in poults from 34- vs 44-week-old hens in the 80 to 84.9 g and 85 to 89.9 g egg weight class at 2 and 5 days of age (P < 0.05), and in the 80 to 84.9 g egg weight class at 3 and 7 days of age (P < 0.05). Plasma glucose concentrations were 37.5 mg/dl greater in fasted poults fi'om 34 vs 44-week-old hens at 4 days of age prior to injection (P < 0.04, Figure 6.3). This hen age effect could largely be attributed to the poults firom the lightest egg weight class (58.1 mg/dl greater than the other egg weight classes fi'om the 34-week-old hens, P < 0.08). The poults 68

89 from the 34-week-oId hens and 75 to 79.9 g egg weight class continued to have higher glucose concentrations 60 minutes post-injection, but neither hen age or egg class significantly affected plasma glucose concentrations 30 or 60 minutes post-injection in 4-day-old, fasted poults. At 7 days of age, plasma glucose concentrations were significantly greater in poults from older hens and similar egg weight classes as compared to poults from the younger hens (P < 0.01, Figure 6.3). Poult BW at 7 days of age was significantly heavier from the younger hens (Table 6.1), thereby affecting the dose-response to the glucose tolerance assay at this age. Experiment 2 Egg weights for the 75 to 79.9 g and > 80 g egg weight classes from 32-week-old hens were ± 1.35 and ± 2.29 (mean ± SD). Intact BW at hatch was 5.34 g greater in eggs from the > 80 g weight class. Egg weight class also significantly affected yolkfree BW at hatch and 1 day of age (poults from heavier eggs were 4.22 and 7.42 g heavier at each age, respectively). Egg weight class did not significantly affect yolk-free BW at 2, 3, or 4 days of age. Duodenum or jejunum and ileum measures (weight, length, and density) were not consistently affected egg weight class during the first week after hatching. In Experiment 1, all poults were injected at 4 and 7 days of age with the same glucose dosage (0.25 g glucose). In Experiment 2, poults were injected with glucose accounting for differences in BW (2.5 mg glucose/g BW). Plasma glucose concentrations were not significantly different prior to injection, 30, or 60 minutes post-injection between 4-day-old poults from the different egg weight classes (P > 0.05). Mean glucose concentrations were 69

90 258.8 ±4.2 (mean± SEM), ± 15.3, ± 14.0 mg/dl at 0, 30, and 60 minutes after injection, respectively. Experiment 3 Egg weight means were ± 1.55, ± 1.44, and ± 1.42 g from the 75 to 80 g and 85 to 90 g egg weight class from the 34 week and 85 to 90 g egg weight class from the 48-week-old hens, respectively. Poult BW at hatch was 1.29 g heavier from the 85 to 90 g eggs from the 48- (58.15 g) vs 34-week-old hens (56.86 g,p < 0.05). Yolk-free BW, however, was not significantly affected by hen age in the 85 to 90 g egg weight class from 27 days of incubation to 7 days of age (Table 6.3). Yolk-free BW was significantly less in poults from the young hens and 75 to 80 g egg weight class as compared to the poults from the 85 to 90 egg weight class from 27 days of incubation to 1 day of age (P < 0.05). Neither hen age or egg weight class consistently affected duodenum or jejunum/ileum weight, length, or density of poults during the first week after hatch (data not presented). As in Experiment 2,4-day-old poults were injected with a glucose dosage according to BW in the glucose tolerance assay. The dosage for Experiment 3, however, was increased from 2.5 to 3.75 mg glucose/g BW. Plasma glucose concentrations were not significantly affected by hen age or egg weight class at 0 or 30 minutes post-injection (Table 6.4). The poults from the young hens continued to have higher glucose concentrations 60 minutes postinjection, however, glucose concentrations at 60 minutes post-injection were not significantly affected by the experimental model (P < 0.11). 70

91 Discussion Eggs from young turkey breeders produce lighter weight poults relative to egg weight [1, 5, 6], In addition, egg weight has been reported to transiently affect poult BW after hatching [8,11], In Experiment 1, poult BW at hatching was 1.85 g greater from similar sized eggs (80 to 90 g) from the younger vs older hens and these differences persisted through 14 days of age. Differences in yolk-free BW in that experiment, however, did not become apparent until 7 days of age in poults from similar sized eggs. Poults in Experiment 1 were from the same commercial flock and were, therefore, reared 10 weeks apart. While the heavier BW at 14 days of age in poults from the younger hens was unexpected when compared with other reports in the literature [8, 11], this difference emphasizes the importance of environmental factors after hatching on overall growth. Egg size within a hen age, however, continued to have an influence on poult BW through 14 d of age. In Experiment 3, when poults were from separate flocks but reared simultaneously, egg size had more o f an effect on yolk-free poult BW prior to or after hatching than did hen age. Body weight, however, is not necessarily an index of the relative state of metabolic homeostasis during this critical acclimation period. For example, 4-day-old poults from the 75 to 80 g egg weight classes in Experiments 1 and 3, tended to remain hyperglycemic 60 minutes following the glucose challenge. Therefore, measurements other than BW are needed to more accurately assess the dynamics of poult quality during the first week after hatching. Circulating glucose concentrations in avians are higher than in mammals and endocrine regulation of carbohydrate metabolism is also different [23,24]. Compared with mammals, the avian pancreas produces and secretes less insulin and nearly 10 times more glucagon [24]. 71

92 Newly hatched chicks, therefore, are more resistant to fasting hypoglycemia than neo-natal mammals [25]. In chicks that were fasted beginning at hatch, 2,6, 10, or 14 days of age (as compared to fed controls), Houpt [25] noted no major change in blood glucose concentrations until the second day of festing, and acute hypoglycemia did not occur until the sixth day of fasting. McNaughton et ai [26] noted a 2.4% higher mortality in chicks from 29- vs 58-weekold broiler breeder hens through 8 week of age. Much of this mortality difference was attributed to chicks from very light (47 to 54 g) eggs from the 29-week-old hens [26]. Latour etal. [13] reported wide fluctuations in serum concentrations of glucose, corticosterone, and triglyceride in chicks between 1 and 5 days of age. Whether or not fluctuations in serum constituents are reflective o f aberrations in metabolic homeostasis, they do correlate vrith peaks in early poult mortality [27]. In Experiment 1, 4-day-old poults from the 75 to 79.9 g egg weight class from the young hens had 85 to mg/dl greater plasma glucose concentrations than poults from the heavier egg weight classes at 60 minutes post-injection (250 mg glucose). When poults were then challenged with glucose on a BW basis (3.75 mg glucose/g BW) in Experiment 3, poults from the younger hens had 79 to 90 mg/dl greater plasma glucose concentrations 60 minutes after injection as compared to poults from the older hens. Assay variability, however, precluded the statistical significance of the experimental model (P > 0.05). In a similar study in which similar sized eggs were collected from 33- and 49-week-old Pekin duck hens, 3-dayold ducklings from the younger hens remained hyperglycemic 60 minutes post-glucose injection (3.75 mg glucose/g BW, [28]). 72

93 Recent evidence suggests that the yolk stalk (vitelline diverticulum) can provide a passageway for transport of yolk into the small intestine up to 72 hours after hatching [29, 30]. In mammals, the stimulatory effects of maternal colostrum and milk on intestinal development through various mitogenic factors has received considerable attention [31]. We have hypothesized that differences in the mass transfer of yolk lipid during the final week of incubation that occurs with increasing hen age [3], could influence the transfer of mitogenic factors firom the yolk into the intestine and subsequently stimulate its maturation. In the current experiments, gross weight and length measures of intestinal development were not consistently affected by either hen age or egg weight class. In a subsequent experiment the more precise measure of villus growth was determined. Distal jejunal villi of poults were determined to be 22.8 ±6.5 pm (mean ± SEM) longer at hatch from older vs younger hens [32]. Villus growth after placement, however, was unaffected by hen age or egg weight class. Therefore, differences in the transfer of the contents of the yolk sac may effect intestinal development prior to, but not after placement. fri conclusion, neither hen age or egg weight class significantly effected gross intestinal weight and length measures or BW growth during the first week after hatching. Poults from the younger hens, especially those from the 75 to 80 g egg weight class, maintained high plasma glucose concentrations 60 minutes post-injection of a test dosage of glucose. This prolonged hyperglycemia suggests these poults could not as readily adapt to this metabolic challenge as the poults from heavier eggs and older hens. 73

94 Acknowledgements The author wishes to thank Cooper Farms, Inc., Ft. Recovery, Ohio and Cuddy Farms, Inc., Danville, Ohio for their generous donation of fertile eggs used in these experiments. References 1 Applegate TJ, Lilbum MS: Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci 1996;75: Applegate TJ, Lilbum MS: Effect of hen age, body weight, and age at photostimulatiorl 2. Egg, incubation, and poult characteristics of commercial turkeys. Poultry Sci 1998;77: Applegate TJ, Lilbum MS: Effect of hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys. Poultry Sci 1998;77: Shanawany MM: Hatching weight in relation to egg weight in domestic birds. W Poultry Sci J 1987;43: Yannakopouios AL: The relationship between egg weight and shell quality on poult hatching weight. Zootech Inti 1989;3: Christensen VL, Donaldson WE, McMurtry JP: Physiological differences in late embryos ffom turkey breeders at different ages. Poultry Sci 1996;75: Wilson HR: Interrelationships of egg size, chick size, posthatching growth and hatchability. W Poultry Sci J 1991;47: Moran ET Jr: Effect of egg weight, glucose administration at hatch, and delayed access to feed and water on the poult at 2 weeks of age. Poultry Sci 1990;69: Mussehl FE, Ackerson CW: Some observations on humidity and weight loss in the incubation of turkey eggs. Neb Ag Exp St Res Bull 1934; Payne L, Siegel PB, Ortman L: Correlation of dam, egg, poult and adult weights in broad breasted bronze turkeys. Poultry Sci 1957;36:

95 11 Moran ET Jr, Reinhart BS: Breeder flock productivity and egg size efifects on broiler turkey performance and carcass quality. Poultry Sci 1981;60: Phelps PV, Edens FW, Gildersleeve RP: The posthatch physiology of the turkey poult-in. Yolk depletion and serum metabolites. Comp Biochem Physiol 1987;87A: Latour MA, Peebles ED, Boyle CR, Brake JD, Kellogg TF: Changes in serum lipid, lipoprotein and corticosterone concentrations during neonatal chick development. Biol Neo 1995;67: Nitsan Z, Ben-Avraham G, Zoref Z, Nir I: Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. Brit Poultry Sci 1991;32: Nitsan Z, Dunnington EA, Seigel PB: Organ growth and digestive enzyme levels to fifteen days of age in lines of chickens differing in body weight. Poultry Sci 1991;70: Sell JL, Angel CR, Piquer FJ, Mallarino EG, Al-Batsham HA: Developmental patterns of selected characteristics of the gastrointestinal tract o f young turkeys. Poultry Sci. 1991; 70: Konarzewski M, Kozlowski J, Ziolko M: Optimal allocation of energy to growth of the alimentary tract in birds. Funct Ecol 1989;3: Konarzewski, M, Lilja C, Kozlowski J, Lewonczuk B: On the optimal growth of the alimentary tract in avian postembryonic development. J Zool Lond 1990;222: Ricklefs RE, Starck JM, Konarzewski M: Internal constraints on growth in birds; in Starck JM, Ricklefs RE (ed): Avian Growth and Development: Evolution within the Altricial-Precocial Spectrum. New York, Oxford University Press, 1998, pp Folch J, Lees, M, Sloan-Stanley GH: A simple method for the isolation and purification of total lipids. J Biol Chem 1957;226: Turner KA: The effect of feeding high carbohydrate and fet diets following a 2 day fast upon blood metabolites and liver status in newly-hatched poults. Wooster, Ohio State University/0ARDC, SAS User s Guide: Statistics Version 5. Cary, SAS Institute,

96 23 Hazelwood RL; Endocrine control of avian carbohydrate metabolism. Poultry Sci 1971;50: Pearce J: Some differences between avian and mammalian biochemistry. Inti J Biochem 1977;8: Houpt, TR: Effects of fasting on blood sugar levels in baby chicks of varying ages. Poultry Sci 1958;37: McNaughton JL Deaton JW, Reece, FN, Haynes RL: Effect of age of parents and hatching egg weight on broiler chick mortality. Poultry Sci 1978;57: Phelps, PV, Edens FW, Christensen, VL: The posthatch physiology of the turkey poult-i. Growth and development. Comp Biochem Physiol 1987;86A: Applegate TJ, Ladwig E, Weissert L, Lilbum MS: Effect of hen age on intestinal development and glucose tolerance of the Pekin duckling. Poultry Sci 1999; manuscript submitted. 29 Esteban, S, Moreno M, Rayo JM, Tur JA: Gastrointestinal emptying in the final days o f incubation in the chick embryo. Brit Poultry Sci 1991;32: Noy Y, Uni Z, Sklan D: Routes of yolk utilization in the newly-hatched chick. Brit Poultry Sci 1996;37: Odle J, Zijlstra RT, Donavan SM: Intestinal effects of milkborae growth factors in neonates of agricultural importance. J Anim Sci 1996;74: Applegate TJ, Dibner JJ, Kitchell ML, Uni Z, Lilbum MS: Effect of turkey breeder hen age and egg size on poult development. 2. Intestinal villus growth, enterocyte migration and proliferation o f the turkey poult. Biol Neo 1999; manuscript submitted. 76

97 Days of age Hen age, week Egg weight class, g Mean egg weight (.g) ' f 56.28* 157.8* 377.5* * 60.02* 160.2* 382.4* * 54.57* 143.2* 306.9* * * 145.5* 319.1* SEM Source of variation rooaduity) Hen age Egg weight class Hen age by egg weight class ^ Means in columns are significantly different (P < 0.05) as a result of an orthogonal contrast between hen ages fi om the 80 to 84.9 g egg weight class. * Means in columns are significantly different (P < 0.05) as a result of an orthogonal contrast between hen ages from the 85 to 89.9 g egg weight class. ' n=200. Table 6.1. Egg and poult body weight from different egg weight classes and ages of turkey breeder hens. Experiment 1. 77

98 Hen age, week Egg weight class, g Days relative to hatching ^yoik-iree Doay weigni, ' Î * 164.1* 365.9* * * 374.2* -o * * 137.8* 308.4* * * 324.5* , SEM Source of variation 1 ODaDiluy ) Hen age Egg weight class Hen age by egg weight class Table 6,2

99 ^ Means in columns are significantly different {P < 0.05) as a result of an orthogonal contrast between hen ages from the 80 to 84,9 g egg weight class. ^ Means in columns are significantly different (P < 0.05) as a result of an orthogonal contrast between hen ages from the 85 to 89.9 g egg weight class. ' n=5. Table 6.2. Yolk-ffeebody weight of poults ffom different egg weight classes and ages of turkey breeder hens, Experiment 1.

100 Figure 6.1. Duodenum weight, length, and density (g/cm) of poults from different egg weight classes and ages of turkey breeder hens. Experiment 1. ^Difference between hen ages within the 80 to 84.9 g egg weight class is significant (JP < 0.05) in poults within this age. ï)ifference between hen ages within the 85 to 89.9 g egg weight class is significant (P < 0.05) in poults within this age. n=5 poults per egg weight class and hen age. Error bars indicate SEM. 8 0

101 Duodenum 3 i l Duodenum g Duodenum Pouit age (days) g g g g egg weight class, 34 wk egg weight class, 44 w k 81 Figure 6.1

102 Figure 6.2. Jejunum and ileum weight, length, and density (g/cm) of poults from different egg weight classes and ages of turkey breeder hens. Experiment 1. 'Difference between hen ages within the 80 to 84.9 g egg weight class is significant {P < 0.05) in poults within this age. ^Difference between hen ages within the 85 to 89.9 g egg weight class is significant (P < 0.05) in poults within this age. n=5 poults per egg weight class and hen age. Error bars indicate SEM. 8 2

103 Jqunum and ileum T ^ Ë È ë I NÉ NN Jejunum and ileum % Jejunum and ileum i 0.00! Poult age (days) g g egg w eight class, 34 w k g egg weight class, 44 wk g 83 Figure 6.2

104 Figure 6.3. Glucose tolerance assay of 4 (upper) and 7-day-old (lower) poults from different egg weight classes and ages of turkey breeder hens, Experiment 1. Poults from 34 and 44- week-old hens are on the left and right, respectively. Poults were injected i.m. with 0.5 ml of a 50% (wt/vol) glucose solution in 0.9% saline at time 0 and sampled 30 and 60 minutes later. Difference between hen ages within a time after injection is significant {P < 0.05). Means represent 6 to 8 poults per egg weight class and hen age for each time point. Error bars indicate SEM. 84

105 % (Ucn O 0 3 U Poults, 4 days o f age 34 wk 44 wk 1CO 700 Poults, 7 days of age 34 wk 44 w k a 300 I 200 "5, Time after injection (minutes) g VMÂ g 1 0 # g Egg weight class g 85 Figure 6.3

106 Hen age, week Egg weight class, g Days relative to hatching (.ë) "'' 47.69" 55.48" 66.74"': " " 53.41" 61.10" 72.16"'" "." " 54.18" 65.31" 76.20" " SEM \ Means in columns with different superscripts are significantly different (P < 0.05) as determined using a Duncan means separation when the significance of the experimental model (i.e. the three pre-assigned groups) was < n=10 at -1, 0, and 1 day of age. n=5 at 2 to 7 days of age. Table 6.3. Yolk-ffee body weight of poults from different egg weight classes and ages of turkey breeder hens, Experiment 3.

107 Hen age, week Egg weight class, g minutes post-injection^ (plasma glucose, mg/dl) = SEM Probability o f model effect^ O i l Poults at 30 and 60 minutes were injected i.m. with 3.75 mg glucose/g body weight. ^ n=12. ^ The experimental model determined the probability of a difference between means from the three pre-assigned groups (i.e. hen age and egg weight class). Table 6.4. Glucose tolerance assay of 4-day-old poults from different egg weight classes and ages of turkey breeders. Experiment 3. 87

108 CHAPTER? EFFECT OF TURKEY BREEDER EDEN AGE AND EGG SIZE ON POULT DEVELOPMENT. 2. INTESTINAL VILLUS GROWTH, ENTEROCYTE MIGRATION AND PROLIFERATION OF THE TURKEY POULT Abstract Villus growth, enterocyte migration and proliferation were measured in the small intestine of poults to determine if hen age and/or egg size influences these characteristics during the first week after hatching. At hatching, distal jejunal villi were 22.8 ±6.5 pm longer in poults fi-om the older (48 weeks) vs the younger (34 weeks) hens (P < 0.05). Similarly, labeled enterocytes in distal jejunal sections fi-om poults from the older hens had migrated 28 ±4.8 pm (10%) farther along the crypt-villus axis at hatching, as compared to poults fi-om the younger hens (P < 0.05). Villus growth differences and enterocyte migration were not consistently ajbfected by hen age or egg weight class in poults fi-om 1 to 7 days of age. These results suggest that even though intestinal villi may be more advanced developmentally at hatch in poults fi-om the older hens, however post-hatch growth of the intestine or the poult is not affected by hen age or egg weight class. 88

109 Introduction Turkey poult body weight (B W) at hatch can be positively affected by the age of the hen independent of changes in egg weight [1,2, 3]. Applegate and Lilbum [4, 5] partially attributed this positive effect on neo-natal poult BW to a disproportional increase in yolk deposition with advancing hen age which resulted in a greater mass transfer of major lipid subclasses from the yolk sac to the embryo during the final week of incubation. Body weight at hatch, however, is not an accurate measure of the relative physiologic state of the precocial chick or poult. Relative to other organs or tissues, growth of the gastrointestinal tract (GIT) as a proportion of BW increases after hatching and becomes maximal at 4 to 6 d of age in the poult [6,7]. The digestive and absorptive capabilities of the GIT of the chick during the first week after hatching are still immature, however, starch and lipid digestion do not limit growth in the young chick when feed intake and passage rates are accounted for [8]. Functional maturation of the small intestine involves both morphologic and physiologic changes and can be a constraint to optimal early growth of precocial birds [9, 10, 11], Physiologic maturation (in terms of digestive and absorptive fimctionality) of the GIT occurs mainly through increased production of pancreatic and intestinal enzymes [6, 7, 12, 13] and changes in nutrient transporters [14, 15, 16]. However, physical development o f the GIT, namely the increase in surface area of the small intestine, can be a more limiting factor to early growth than changes in digestive and absorptive functionality [17, 18]. The small intestine of the chick increases in length over the first 5 days after hatch, even during an imposed fast [19]. Increases in the relative weight o f the small intestine, 89

110 intestinal diameter, and villus length, however, only occur after feeding [19, 20]. Moran [21] described adjustments of villi to a critical length as a balance of nutrient needs of the body and intestinal tissue maintenance. Villus length increases over two-fold in the chick during the first 5 days after hatch [22] and villus volume (mm^ /cm^) increases 3- to 4-fold from hatch through 10 days of age [17]. Additional functional adjustments include changes in the rates of intestinal crypt cell proliferation and enterocyte turnover [23]. Enterocyte turnover rates in the small intestine were originally reported to be approximately 2 days in the chick [24]. In Japanese quail, turnover rates have been reported to vary firom 2.8 to 2.9 days in the hatchling to 8.9 to 10.6 days in the adult [25]. Slower small intestine villus growth and enterocyte migration and proliferation may contribute to reports of transient slower growth of poults from young hens during the first week after hatching [26]. To test this hypothesis, villus growth and enterocyte migration within the distal jejunum were measured in poults produced by relatively young and mature turkey breeder hens. Measurements were made between 27 days of incubation through 7 days of age. Enterocyte migration was measured after injection of a thymidine analog (bromodeoxyuridine, BrdU) and subsequent immunohistochemical staining of distal jejunum sections for BrdU at 1,2, and 3 days after incorporation. In addition, enterocyte proliferation was determined using immunohistochemical staining for enterocytes containing proliferating cell nuclear antigen (PCNA, an auxiliary protein to DNA polymerase delta which is detectable immunocytochemically only during late Gi to S phases of cell division; [27,28]). 90

111 Materials and methods Commercial turkey hens were reared in commercial facilities (British United Turkeys of America, Lewisburg, West Virginia; Cuddy Farms, Inc., Danville, Ohio) and photostimulated at 30 weeks of age. At each of 34 and 48 weeks of age, fertile eggs were collected from each of two flocks. Eggs were indiridually weighed and assigned to one of two egg weight classes (75 to 80 and 85 to 90 g from the 34-week-old hens and 85 to 90 g from the 48-week-old hens; n=160 per egg weight class and hen age). All eggs were set in a NatureForm incubator (NatureForm, Inc. Jacksonville, Florida). Poults were individually weighed upon hatching (28 days of incubation) and given ad libitian access to feed and water in an electrically-heated Petersime (Petersime, Inc., Gettysburg, Ohio) brooder battery. At 26 days of incubation, 45 eggs per hen age and egg class were injected through the air cell into the embryo (/.m.)with 0.5 mg 5-bromo-2'-deoxyuridine (BrdU, Sigma Chemical Co., St. Louis, Missouri). Excess thymidine (25 mg. Sigma Chemical Co.) was injected 180 minutes after the initial BrdU dose. Sterile water was used as the vehicle for injection of BrdU and thymidine. Ten poults per hen age and egg weight class were randomly selected at 27 days of incubation, hatch (0 day, prior to placement), and 1 day of age (1,2, and 3 days postinjection, dpi). Similarly, 1- and 4-day-old poults were injected i.p. with BrdU (10 mg/kg BW) followed with an i.p. injection of thymidine (500 mg/kg BW) 180 minutes later. The BrDU and thymidine injection amounts and times were the same as that described by Dibner et al. [29]. Five poults per hen age and egg weight class were randomly selected at 2, 3, and 4 days of age (from poults injected at I days of age) and 5, 6, and 7 days of age (from poults injected at 4 days of age) and were euthanitized by cervical dislocation. Segments of the distal 91

112 jejunum (0.5 cm proximal to the yolk stalk) were rinsed and fixed in neutral-buffered formalin, dehydrated through a graded alcohol series, cleared with xylene, and embedded in paraffin. Five-pm paraffin sections were made and subsequently stained for BrdU as previously described[30]. Briefly, slides were treated with hydrogen peroxide (3%) to remove endogenous peroxidase. Jejunal sections were digested using 0.1% trypsin (Type II-S fi-om porcine pancreas. Sigma Chemical Co.) for 30 minutes and were washed in phosphate buffered saline (PBS). A monoclonal antibody to BrdU (Becton Dickinson, San Jose, California) was diluted at 1:200 in PBS with 0.01% bovine serum albumin and slides were incubated for 2 hours at room temperature. Following incubation wdth primary antibody, slides were washed with Cadenza buffer (Shandon, Inc., Pittsburgh, Pennsylvania) and a biotinylated secondary antibody, avidin-biotin complex (Vector Elite Kit, Vector Laboratories, Burlingame, California), and chromagen indicator (Vector VIP BCit, Vector Laboratories) were used for color development. Slides were then counter-stained with methyl-green. Villus measures of distal jejunum sections were made from images obtained from an Olympus DC70 inverted microscope (Olympus America, Inc., Melville, New York), processed with a Vay Tek deconvolution imaging system (Vay Tek, Fairfield, Iowa), and measurements made using Image-Pro Plus software (Media Cybernetics, Silver Spring, Maryland). Villus height (villus tip to lamina propria) and BrdU labeled enterocyte height (lamina propria to leading edge of labeled enterocytes) were measured and the mean of 5 villi per bird was used for statistical analysis. 92

113 Additional five-^m parafibn sections from poults (27 days of incubation, hatch, 1, and 5 days of age) from the 85 to 90 g egg weight class were subsequently stained for endogenous PCNA as described by Foley et al. [31] and Uni et nr/.[32]. Slides were then counter-stained with Hematoxylin and Eosin. Numbers of PCNA stained cells per crypt (laminia propria to invagination between villi) and villi (invagination between villi to villus tip) were determined using computer-assisted image analysis (NIH Image, Bethesda, Maryland; and Adobe Photoshop 4.0, Adobe System Incorporation, Mountain View, California). Values represent the mean o f measures from 5 poults per hen age from each sampling day. All results were analyzed by analysis o f variance using the General Linear Models procedure of SAS [33]. Differences between hen production ages and egg weight classes were determined using a Duncan means separation when the significance of the experimental model was < Results Villus height (lamina propria to villus tip) of distal jejuna are presented in Table 7.1. Villi of poults from the older hens were 22.8 pm longer at hatch (0 day of age, P < 0.05). Neither hen age or egg weight class significantly affected villus length at 27 days of incubation, or after placement (1 to 7 days of age). Similarly, BrdU labeled enterocytes in distal jejunal sections from poults from the older hens had migrated 28 pm (Table 7.2) farther along the crypt-villus axis at hatch (2 dpi), as compared to poults from the younger hens (P < 0.05 ), representing 10% more of the crypt-villus axis traversed (Table 7.3, P < 0.05). Leading edge height of BrdU labeled enterocytes of poults from 34-week-old hens and g egg weight class was significantly less at 5 days o f age (1 dpi) vs the 48-week-old hens and 93

114 85-90 g egg weight class (P < 0.05). The proportion of total crypt-villus axis traversed was greater in the poults at 5 days of age (I dpi) from the older hens as compared to poults from the g egg weight class from the younger hens {P < 0.05). This proportion was also greater at 6 days of age (2 dpi) in poults from the 48- vs 34-week-old hens (regardless of egg weight class, P < 0.05). When sampling times were accounted for through calculation of the enterocyte migratory rate (pm/hour), the enterocytes in poult vidi at hatch from the 48-wk-old hens were migrating 0.55 pm/hour faster than the poults from the 34-week-old hens (Table 7.4, P < 0.05). Migratory rate of enterocytes was not significantly affected by hen age or egg weight class at any other age. The relative proportion of the crypt-villus axis migrated by BrdU-labeled enterocytes was consistently greater in poults from 27 days of incubation through 1 day of age (1,2, and 3 dpi), than in older poults (Figure 7.1, f < 0.05). In addition, the migratory rate of BrdU labeled enterocytes 1 dpi was significantly greater in poults at 27 days of incubation as compared to poults at 2 or 5 days of age (P < 0.05). Figure 7.2 contains photomicrographs of distal jejunal sections from poults at 27 days of incubation and 5 days of age. Each of these ages represent poults that had been injected with BrdU 1 day previous to collection of the tissue. The sections are stained using a monoclonal antibody for BrdU and counter-stained with methyl green. The dark stained nuclei of enterocytes containing BrdU can be found in the crypts and along the villus in both sections. Some stained cells were located in the connective tissue core of the villus at all ages. 94

115 but these were presumably either stained erythrocytes or immune tissue rather than absorptive epithelia per se. Notably, the stained enterocytes in poults before hatch cover a larger proportion of the crypt-villus axis than at the older age. The number of PCNA positive enterocytes per crypt and per villus are presented in Table 7.5. One day after feeding, poults from the older hens had significantly more proliferating enterocytes in the crypt (7.6) and in the villus (22.2) than those from the younger hens (P < 0.05). This difference, however, was not apparent at hatching or at 5 days of age. The numbers of proliferating enterocytes in the crypt region did not change considerably with age. In the villus region, however, a transient decrease was noted at hatch and 1 day of age with a subsequent increase by 5 day of age. Figure 7.3 contains photomicrographs of distal jejunal sections from poults at 1 and 5 days of age. The sections are stained using an antibody for PCNA and counter-stained with Hematoxylin and Eosin. The dark brown stained nuclei of enterocytes containing PCNA are most concentrated in the crypt region and along the length of the villus. Discussion ^plegate and Lilbum [5] reported a greater mass transfer o f lipid subclasses out of the yolk sac during the final week of incubation concomitant with differences in yolk-free embryo growth due to hen age. Differences in yolk sac uptake by embryos from different hen ages may have directly or indirectly affected villus height at hatching in the current experiment. Uni ei al. [34] noted that yolk sac ablation of chicks at hatching caused a transient decrease in villus volume and crypt depth throughout the small intestine. The reduced villus height at hatching in poults from the younger hens did not affect villus height 95

116 or enterocyte migration after placement. In addition, the reduced villus height at hatching may not have impaired subsequent nutrient utilization after placement because 7 day BW was unaffected by hen age or egg weight class [35]. Poult duodenum and jejunumæeum weight, length, and density (g/cm) were not consistently affected by hen age or egg weight class during the first week after hatch in the current experiment [35]. Changes in villus height can be caused by a variety o f factors, including; feed restriction, competition with normal GIT microflora, coccidial parasitization, fiber content of diet, changes in growth or productive needs, and plane of nutrition [21]. Transient changes in villus height, however, may not directly affect nutrient utilization. Restriction of feed intake to 75% of ad libihan intake in 6-week-old chickens, for example, substantially reduced villus height but did not significantly influence nutrient utilization [36]. Several authors have described the intestine as a relatively flexible system which can undergo morphological and ftmctional adjustments depending on demands of the organism [16, 21, 25]. For example. Uni et al. [17] noted a positive correlations of jejunal and ileal villus volumes, and enterocyte density with feed intake in chicks during the first 2 weeks of age (r=0.77, 0.67, and 0.62, respectively). Whilst measuring cytokinetic parameters of enterocyte proliferation, Starck [25] determined the duration of ^H-thymidine incorporation during the S-phase was not appreciably different between hatchlings and adults in the altrical starling (Sturtnis vulgaris) or precocial japanese quail (Coutumix cotumix japonica). Starck [25] concluded that the S- phase duration does not contribute to observable differences in growth. Regionality of cellular proliferation may, however, affect intestinal functionality. In 18- to 36-day-old chicks. Uni 96

117 et cd. [32] determined that enterocyte proliferation was not conserved to the crypt region as in mammals, rather the proportions of cells engaged in proliferation (PCNA-positive cells) were 55, 32, and 8% in the crypt, mid-villus, and upper villus, respectively, similar to the results of the current study. Also, distinct expression of maltase and sucrase within differing regions was not apparent in the chick as it is in mammals, thereby suggesting that discrete zones of proliferation and differentiation that exist in mammalian villi are not evident in avians [32]. Proliferating cell nuclear antigen (PCNA) has been used extensively as an index of cellular proliferation. Quantitatively, PCNA changes very little during the cell cycle but that fraction of PCNA which is bound to DNA is greatest during the S-phase of the cellular replication [37]. The bound form of PCNA allows for the immunoreactive visualization of the protein [37]. In the current experiment, the proportions of the vilh with BrdU positive enterocytes were significantly greater at -1, 0, and 1 day of age as compared with the older ages (2 to 7 days of age). Similarly, numbers of enterocytes engaged in proliferation (PCNA-positive cells) along the length of the villus were not appreciably different prior to hatching (27 days of incubation) and 5 day of age. These results suggest that prior to hatching, a larger proportion of the villus contained proliferative cells than during the first week after hatching. From hatching to 7 day of age, villus height increased nearly 2-fold, crypt cell proliferation remained constant, and enterocytes migrated at similar rates of approximately 2 pm/hour. Therefore, increased fimctionality of the small intestine is affected more by the net accumulation of cells than by the turnover of the mucosal epithelial surface. In neonatal mammals, slower 97

118 enterocyte migration rates, longer cell lifetimes, and a net accumulation of cells results in nutrient uptake occurring along the length of the villus rather than only at the villus tip [38]. In conclusion, hen age can significantly effect the height of distal jejunal villi and BrdU labeled enterocytes of poults at hatch. Hen age did not affect either measurement after placement and egg weight class did not significantly effect these measurements during the first week of age. These results suggest that even though intestinal villi may be more advanced developmentally at hatch in poults from the older hens, post-hatching growth of the intestinal system or the poult is not affected. References 1 Yannakopoulos AL; The relationship between egg weight and shell quality on poult hatching weight. Zootech Inti 1989;3: Applegate TJ, Lilbum MS: fridependent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci 1996;75: Christensen VL, Donaldson WE, McMurtry JP: Physiological differences in late embryos from turkey breeders at different ages. Poultry Sci 1996;75: Applegate TJ, Lilbum MS: Effect of hen age, body weight, and age at photostimulation. 1. Egg, incubation, and poult characteristics of commercial turkeys. Poultry Sci 1998;77: Applegate TJ, Lilbum MS: Effect of hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys. Poultry Sci 1998; 77: Sell JL, Angel CR, Piquer FJ, Mallarino EG, Al-Batshan HA:DevelopmentaI patterns of selected characteristics of the gastrointestinal tract of young turkeys. Poultry Sci 1991;70: Pinchasov Y, Noy Y: Early postnatal amylolysis in the gastrointestinal tract of turkey ^ovlts Meleagris gallopavo. Comp Biochem Physiol 1994; 107A:

119 8 Noy Y, Sklan D; Digestion and absorption in the young chick. Poultry Sci 1995;74: Konarzewski M, Kozlowski J, Ziolko M: Optimal allocation of energy to growth of the alimentary tract in birds. Funct Ecol 1989;3: Konarzewski M, Lilja C, Kozlowski J, Lewonczuk B: On the optimal growth of the alimentary tract in avian postembryonic development. J Zool Lond 1990;222: Ricklefs RE, Starck JM, Konarzewski M: Internal constraints on growth in birds; in Starck JM, Ricklefs RE (ed): Avian Growth and Development: Evolution within the Altricial-Precocial Spectrum. New York, Oxford University Press, 1998, pp Nitsan Z, Ben-Avraham G, Zoref Z, Nir I: Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. Brit Poultry Sci 1991;32: Nitsan Z, Dunnington EA, Seigel PB: Organ growth and digestive enzyme levels to fifteen days of age in lines of chickens differing in body weight. Poultry Sci 1991;70: Holdworth CD, Hastings-Wilson T: Development of active sugar and amino acid trasport in the yolk sac and intestine of the chicken. Amer J Physiol 1967;212: Shehata AT, Lemer J, Miller DS: Development of nutrient transporter systems in chick jejunum. Amer J Physiol 1984;246:G101-G Obst BS, Diamond J: Ontogenesis of intestinal nutrient transporters in domestic chickens (Gallus galliis) and its relation to growth. Auk 1992; 109: Uni Z, Noy Y, Sklan D: Posthatch changes in morphology and function of the small intestines in heavy-and light-strain chicks. Poultry Sci 1995; 74: Uni Z, Noy Y, Sklan D:Development of the small intestine in heavy and light strain chicks before and after hatching. Brit Poultry Sci 1996; 36: Baranyiova, E: Influence of deutectomy, food intake and fasting on the digestive tract dimesions in chickens after hatching. Acta Vet Brno 1972; 41: Baranyiova E, Holman J: Morphological changes in the intestinal wall in fed and fasted chickens in the first week after hatching. Acta Vet Bmo 1976; 45:

120 21 Moran ET Jr Digestion and absorption of carbohydrates in fowl and events through perinatal development. JN u tr 1985; 115: Dibner JJ, Kitchell ML, Atwell CA, Ivey FJ: The effect of dietary ingredients and age on the microscopic structure of the gastrointestinal tract in poultry. J Appl Poultry Res 1996;5: Starck JM: Structural variants and invariants in avian embryonic and postnatal development; in Starck JM, Ricklefs RE (ed): Avian Growth and Development: Evolution within the Altricial-Precocial Spectrum. New York, Oxford University Press, 1998, pp Imondi AR, Bird FH: The turnover of intestinal epithelium in the chick. Poultry Sci 1966;45: Starck JM: Intestinal growth in altricial European starling (Shinms vulgaris) and precocial Japanese quail (Çotumix cotumix japonica). A morphometric and cytokinetic study. Acta Anatom 1996;156: Moran ET Jr: Effect of egg weight, glucose administration at hatch, and delayed access to feed and water on the poult at 2 weeks of age. Poultry Sci 1990; 69: Sanders EJ, Varedi M, French AS: Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization. Develop 1993;118: Laskey RA, Fairman MP, Blow JJ: S phase of the cell cycle. Science 1989; 246: Dibner JJ, Atwell CA, Kitchell ML, Shermer WD, Ivey FJ: Feeding of oxidized fats to broilers and swine: effects on enterocyte turnover, hepatocyte proliferation and the gut associated lymphoid tissue. Anim Feed Sci Tech 1996;62: Kitchell ML, Dibner JJ: Rapid detection of proliferating cells using microwave fibcation and a monoclonal antibody to bromodeoxyuridine. J Histotech 1989;12: Foley J, Thai T, Maronpot R, Butterworth B, Goldsworthy G: Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats. Environ Health Perspect 1993;101: Uni Z, Platin R, Sklan D: Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. J Comp Physiol B 1998;168:

121 33 SAS User s Guide; Statistics Version 5. Cary, SAS Institute, Uni Z, Ganot S, Sklan D: Posthatch development of mucosal function in the broiler small intestine. Poultry Sci 1998;77: Applegate TJ, Lilbum MS: Effect of turkey breeder hen age and egg size on poult development. 1. Intestinal growth and glucose tolerance of the turkey poult. Biol Neo 1999; manuscript submitted. 36 Michael E, Hodges RD: IDstochemical changes in the fowl small intestine associated with enhanced absorption after feed restriction. Histochem 1973;36: Morris GF, Mathews MB: Regulation of proliferating cell nuclear antigen during the cell cycle. J Biol Chem 1989; 246: Ferraris RP, Villenas SA, Diamond J: Regulation of bmsh-border enzyme activities and enterocyte migration rates in mouse small intestine. Amer J Physiol 1992;262:G1047-G

122 Hen age, week Egg weight class, g Days relative to hatch (pm ) to ' 166.6" , to " to SEM Means in columns with different superscripts differ significantly (P < 0.05) as determined using a Duncan means separation when the significance of the experimental model (i.e. the three pre-assigned groups) was < n=10, days -1 to 1; n=5, days 2 to 7, Table 7.1. Distal jejunum villus height (lamina propria to villus tip) of poults from different egg weight classes and ages of turkey breeder hens.

123 Hen age, week Egg weight class, g Days relative to hatch Days post-injection of BrdU (pm ) to 80 76,8' 98.6" " s 85 to " " to SEM Means in columns with different superscripts differ significantly (P < 0.05) as determined using a Duncan means separation when the significance of the experimental model (i.e. the three pre-assigned groups) was < ' n=10, days -1 to 1; n=5, days 2 to 7. ^ BrdU labeled enterocyte leading edge not detected. Table 7.2. Distal jejunum bromodeoxyuridine (BrdU) labeled enterocyte height (pm) from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens.

124 Hen age, week Egg weight class, g Days relative to hatch Days post-injection of BrdU fo/ \ to ' 59.5" " 33.5" 44.3 s 85 to " '" 37.3" to SEM Means in columns with different superscripts differ significantly (P < 0,05) as determined using a Duncan means separation when the significance of the experimental model (i.e. the three pre-assigned groups) was < n=10, days - I to 1; n=5, days 2 to 7. ^ BrdU labeled enterocyte leading edge not detected. Table 7.3. Distal jejunum bromodeoxyuridine (BrdU) labeled enterocyte height (% of crypt-villus axis migrated) from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens.

125 Hen age, week Egg weight class, g Days relative to hatch Days post-injection of BrdU ^ iiu/nour^ to ' 2.14" , to " to o SEM t * Means in columns with different superscripts differ significantly {P < 0.05) as determined using a Duncan means separation when the significance of the experimental model (i.e. the three pre-assigned groups) was <0.05. n=10, days -1 to 1; n=5, days 2 to 7. ^ BrdU labeled enterocyte leading edge not detected. Table 7.4. Distal jejunum bromodeoxyuridine (BrdU) labeled enterocyte migratory rate (pm/hour) from distal jejunal villi from poults from different egg classes and ages of turkey breeder hens.

126 Figure 7.1. Bromodeoxyuridine (BrdU) labeled enterocyte height (gm), relative BrdU labeled enterocyte height (% of crypt-villus axis migrated), and BrdU labeled enterocyte migratory rate (gm/hour) of distal jejunal villi from poults from 27 days of incubation to 7 days of age. White bar, 1 day post-injection (dpi) of BrdU; black bar, 2 dpi; gray bar, 3 dpi. Means within each dpi with different superscripts are significantly different {P < 0.05) as determined using a Duncan means separation when the significance of the experimental model (age) was < Error bars indicate SEM. 106

127 160 BrdU label hei^t I e Relative BrdU label height BrdU label migratory rate i Days relative to hatch g g g g 2dpi SST " 3 dpi 107 Figure 7.1

128 mm# Figure 7.2. Distal jejunum villi stained using a monoclonal antibody for bromodeojqniridine (BrdU) and counter-stained with methyl green from poults at A) 27 days of incubation and B) 5 days o f age. Each age represents poults 1 day post-injection of BrdU. 108

129 Region^ Hen age, week Days relative to hatch " (^nunidcr oi enierocyies^ Crypt /f SEM Effect of hen age Villus SEM Effect of hen age *Crypt = lamina propria to invagination between adjacent villi. Villus = invagination between adjacent villi to villus tip. -n=5. Table 7.5. Proliferating cell nuclear antigen (PCNA) positive cells from distal jejunal villi from poults from 85 to 90 g eggs from differing ages of turkey breeder hens. 109

130 Figure 7.3. Distal jejunum villi stained using an antibody for proliferating cell nuclear antigen (PCNA) and counter-stained with Hematoxylin and Eosin from poults at 1 (A and B) and 5 days of age (C and D). Nuclei of PCNA positive cells are stained dark brown

131 CHAPTERS Effect of Hen Production Age on Egg Composition and Embryo Development in Commercial Peidn Ducks^ T. J- A PPIiG A TE * D. HARPER,^ and M. S. ULBURN*;z 'Department o f Anim al Sciences, The Ohio S ta ts Lbdoersity, Ohio Agricultural Research and Development Center, Wooster, Ohio and tm aple Leaf Farms, Inc., Franksoüle, Wtscortsin ABSTRACT A t 26,31,36, and 42 w k of age, eggs were collected from the same duck breeder flock to study the effects of hen production age on egg composition and embryo development. A t each hen age, yodc andalbumen measurements were made on a random subsample of unmcubated eggs. Embryo an d yolk sac measurements w ere made at 20,22, 24,26,27, and 28 d of incubation. Egg w eight was significantiy affected by hen production age, but most of the observed age effects occmred betw een 26 and 31 wk w ith m inim al age differences thereafter due to a quantitative feed restriction for egg we% ht. Yolk weight increased significantly {Key wards: duck, hen age, egg, embryo, yolk sac) w ith hen production age, w ith the largest increase, 6.6 g, also occurring between 26 and 31 w k of age. Yolk-free du ckling weight increased w ith hen production age at 27 d of incubation. The yolk sacs o f embryos from the 31-, 36-, and 42-wk-old hens were heavier a t 20 and 22 d, before the differentiation in embryo weight. Differences in yolk sac weight were n o t consistently affected by hen production age between 26 and 28 d of incubation. The eggs from the 42-wk-old hens w e% hed 323 g less than those from the 31-wk-old hens, b u t yolk-free duckling w eight a t 27 d w as 2.9 g heavier from the 42-wk-old hens Poultry Science 77: INTRODUCTION Over the last decade, the age at m arket w eight for commercial Pelrin ducks (currently 3.0 to 3.2 kg at 41 d) has been reduced by approximately 10 d (C. M. Turk, M aple Leaf Farms, FranksviHe, WI 53126, personal communication). Genetic selection pressure for increased grow th rate in these birds has caused flte late embryonic to early posthatch period to occupy a longer an d more significant segm ent of the growout phase. In. other domestic poultry species, increasing age of the hen during production can have significant; positive effects on embryonic growtti and subsequent hatchling BW independent of the normal, age-associated increases in egg weight. In other words, embryo a n d /o r hatcffling w eight from nrnilar w ei^tt eggs are increased when progeny from older hens are compared w ith fltose from young hens. These maternal effects have been reported for broilers (Wilson, 1991), Japanese quail (Yannakopoulos a n d T serveni-g ousi, 1987), an d tu rk e y poults Received for publicatioxi t4ovember 3, Accepted for pohlfratian June 2S. 1 9 ^ tsajazies and zrseaich support provided by state and fedeeal funds approp riated to The Ohio Agdcuiturai Research and Development Center, The Otiio State Ifniveislty. Vfanuscdpt num ber rpo whom correspondence shoiud be addressed: Idbom.1 S o su ed u (Christensen et ol, 1996; Applegate and lilbum, 1996, 1998ah). Applegate and Lilbum (1996, 1998a) reported that turkey hens will deposit proportionately more yolk frito the egg w ith increasing hen production age. They ttieorized that increased yolk deposition w ould lead to subsequent increased yolk availability for a higher grow th rate during the last w eek of incubation. Egg weight is highly correlated with, the w e i^ t of the duckling at hatch (Shanawany, 1987). The weight of the egg has been associated w ith hatchling thermoregulatory capacity in w ild M allards through 3 d of age (Rhymer, 1988) and w ith survivability of Lesser Scaup (Aythya afins} hatchlings through 2 w k of age (Dawson and Clark, 1996). Other rep orts have also suggested that the positive effects of egg size on hatchling weight carry th ro u ÿ r to market age in some strains of commercial Pekin ducks (Cerveny et al., 1988; Khizetova et ol, 1992). In most instances, increasing egg w eight is associated w ith increasing h e r age during production. The practice of feed restriction of Pekin breeder ducks to maintain egg weight uniformity w ith advancrir^ hen age and its effect on embryonic grow th has n o t been accounted for in previous studies. Therefore, the objective of the current research was to stu d y the effects erf duck breeder age on yolk and yolk sac characteristics (weight, DM, lipid) during the latter stages of incubation and corresponding developm ent of the duck embryo. Ill

132 Hen age Table 8.1 Egg, yolk, and aünzznen weights âom dodc breeder hens d anng the Ar«r 16 w k of prodncdon Albumen (wk) - ( g ) (%3) (g) {%*) 26 77S^ 28.4C c 585» * 275* 30.4b 535* 585» » 285» 322* b S.l*> b 2SJ? 335» 465C b SEM 03 0L Probability of hen age c f e t *<Hen age meaxa m w ith no mrmtnmm stxperscipt differ mgniscantiy (P $ (X05). ^Hgg weight means were haw j on 344, 400, and 4OT eggs for each sec respectively. ZYbk and albumen means were b ased o n 20 eggs p e r set. M ean egg weights for sam pled eggs from each set were 745, 9X2, 893, and 84.2 g, respectxveiy. ^ooc percentage = (yooc w ei^tt/egg w ei^it) x 100. Aibomcn. percentage = (aibtanen w ei^tt/egg w ei^it) x 100. MATERIALS AND METHODS Fertile hatching eggs were collected from the sam e commercial flock of White PeJdn^ ducks. Eggs w ere collected at 26, 31,36, and 42 wk of age. The num ber of eggs collected at each of these ages was 282, 344, 400, and 400, respectively, fri general, breeder ducks at Maple Leaf Færms are quantitatively restrict-fed starting at approxfrnately 10 w k of age and weigh between 2.8 and 3 kg at 22 w k. Feed allowrauaces are increased greafly betw een 22 an d 25 w k coincident w ith tfie onset of egg production. Peak egg production is normally reached betw een 28 and 30 w k of age and feed allowances are at^usted weekly according to egg production and egg w eight so as to maintain egg weight at or below 92 g. At each sam pling age, 20 eggs were individually w eighed an d hard<ooked in shell for albumen and yolk w eight determinations. The remaining eggs were also individually weighed and set in a Humidaire incubator.-* This incubator w as used to incubate the eggs for all four ages sampled. A t 20, 22, 24, and 26 d of incubation, 15 eggs w ere random ly selected, weighed, and the embryos w ere rem oved and euthanatized by decapitation. A t 27 and 28 d o f incubation, hatched ducklings were random ly selected from those that hatched between 26 and 27 or 27 and 28 d, respectiveiy, and euthanatized by decapitation. The yolk sacs of the embryos and hatched ducklings w ere carefully removed, weighed, and flozen. The yolk-free BW was subsequently recorded. The DM content of the yolk sac was determined after drying to a constant w e i^ t in a 60 C oven. Four yolk sacs p er hen age and embryo age were random ly selected for total lipid analysis. Total lipid was determ ined on dry yolk sacs after extraction w hh chloroform: methanol (2d.) as reported by Folch et ol (1957). 3Maple Leaf Fauns, Franksvtne, WI ^Hmxudaiie Incubator Co.. New Madison, OH All results w ere analyzed statistically by analysis of variance using the C e ia m l Linear Models procedure of SAS* (SAS institute, 1986). The main effect tested was hen production age w ithin each d ay of incubation. Differences between hen production ages were determ ined using a D uncan m eans separation when the significance of the experim ental m odel was < Régression analysis w as also conducted to determine w hether hen age h ad any effect on the rate of decline in yolk sac weight during incubation. All percentage data were subject to arc sfrie transformation prior to statistical analysis; however, the actual data are reported m the respective tables. RESULTS Over the 16-wk test period, egg w eight was significantly affected (P < ) by hen production age. Most of this effect w as associated with an 11.1 g increase between 26 and 31 w k (P 0.05, Ttible 1). Egg w e i^ t declined sligjttly betw een 36 and 42 wk. This decline was probably due to the feed restriction used to control egg size. The w eight of the albumen in unincubated eggs increased 9.2 g betw een 26 and 31 wk (P S 0.05) an d then declined a t the later ages, which was similar to w hat was observed for total egg w eight Albumen w eight as a percentage of egg we%ht decreased between 36 and 42 w k of age (P < 0.05). Yolk w eight increased 6.6 g between 26 and 31 w k o f age (P < 0.05) but did not decline over the last three production ages. Consequently, the proportion of yolk deposited increased significantly between 26 an d 42 wk (P < 0.05). The w eight of the residual yolk sac was also significantly affected (P < 0.004) by hen production age a t 20,22,24, and 27 d o f incubation (Table 2). Regression analysis revealed only a slightly more rapid decline in yolk sac w eight betw een 20 and 27 d of incubation (P = 0.16) in embryos frrom 42-wk-old hens (Y = X, rz = 0B6) compared w ith 26-wk-old hens (Y = X, rz = 074). This difierence in yolk sac weight loss betw een these ages partially explains the lack of a 1 1 2

133 Vanabie T a b le s ^ YoDc sac ivcigbt m d relative weigixi fnnn dndc breeder hfee during the f is t 16 wk a production Hen age Days of incnbation^ 20 d 22 d 24 d 26 d 2 7 d 28 d (wfc) Yoik sac wetghx ^ 1655b ILSSbe b ^ 17J2*> 1359*b » ZLSO* 20.40* 1552» UXO 6X4» 4X b 1754b 934 3J2*» 634< SEM H Probability of hen age eâêct X01 0X X Relative yoqc sac weight L54 1E2!» l.y g ISJg» US» b 1085 SEM X8 Probability of hen OJXJl 013 age effect ^ H e n age means in coiuxnns with no nmmnn superscnpt differ signifirantiy (P S. (105). Days 27 and 2S of incubaticn represent hatched diirklmgs that hatched between 26 a n d 27 d or 27 and 23 d. respectively. h e a r ts w ere based on 15 embryos o r hatched dnckiings perday of incubation unices fnhfratph, otherwise. ino duddings hatched between 27 and 23 d of incnbatior. ^Mean was based on eight hatched duckimgs. SReiative yolk sac w eiptt = (yodc sac weight/egg weight at set) x 100. significant hen production age effect on yolk sac weight a t 26 d. (the sampling age closest to the inteisection point in yolk utilization between the youngest and. oldest producfian ages). The rate a t which yolk sac weight declined was not appreciably different between any other production ages. The w eight of the yolk sac relative to egg w e i^ t at set w as significantly increased a t 24 d of incubation m eggs fiom 36-wk-<ild hens (P < 0.001) and although this trend was also observed a t 20 an d 22 d, it was not significant. The absolute w eight of the residual yolk sac was also significantly mcreased in eggs fiom 36-wk-old hens at 20, 22, 24. and. 27 d. The age of the hen had a significant effect (P S 0.05) on the total DM content of the residual yolk sac at 22, 24, 26, and 27 d of incubation. Furthermore, hen age significantly affected DM content of the yolk sac whan expressed as a percentage of residual yolk sac a t 20, 22, 26, 27, and 28 d of incubation (Table 3). The content of lipid in the residual yodc sac w as greater at 20 and 22 d of incubation in embryos fiom 31-wk-old hens tfian in embryos from tfie other hen ages (P S 0.05). W hen eggs of difiering weight but sim ilar yoqc w eight were com pared (31 vs 42 wk. Table 1), embryos fiom 42-wk-old hens had significantly lets yodc sac DM and lipid at 24 and 26 d of incubation (P 0.05; Table 3). T o tal yolk sac w eight in eggs from 31- and 42-wk-old hens was significant at 24 b u t not 26 d of incubation (P S 0.05; Table 2). Yolk-fiee embryo w eight was generally heavier throughout incubation, as hens aged such that hatchling 113 weight at 27 d increased 6 J3 g over the 16-wk production period (P < , Table 4). fiicreased differences in embryo w eight from different hen production ages w ere observed after 24 d of incubation. W hen eggs of difiering egg w eight but similar yolk w eight were compared, the relative embryo w ei^it at 26 d of incubation w as 7fi% greater fiom 42- vs 31-wk-old hens (P 0.05). The yolk-fiee duckling w eight at hatch (27 d of incubation) was also 2 3 g heavier (P < 0.05) in ducks fiom 42- vs 31-wk-old hens. DISCUSSION After studying the effetds of egg size on hatching date during the breeding season of Lesser Scaup ducklings (A. affijts), Dawson an d Clark (1996) reported that hatchlings fiom larger eggs, later in fiie breeding seascm, had a 20% greater charme of survival in the w ild through 2 w k of age. N o t cmly can egg size affect survivabili^ of wild ducddings, b u t it can also increase BW at market age in som e strains of commercial Pekin ducks (Cerveny et ol, 1988; Knizetpva et ol, 1992). After studying hatchling grow di rate in Pekin hatchlings, Khizetova et al (1988) suggested that the faster grow th rate of Pekin ducklings firam 75 vs 95 g eggs m ay be related to firster resorption of a larger yodc from a larger egg. Khizetova et ol (1988) also suggested fiiat w hen hatchling w eight was related to egg w eight a t set, newly hatched ducklings fiom 95-g eggs (612 %) were 25% heavier than hatchlings firom 75-g eggs (587 %). in data

134 Table 8^ YoOc dry rmn-ifilfarimi 3«tj <;iyqtgnt azid Ixpid atirf COOtSIlt iuizi «stbryos 6azxi duck breeder Jnm g the first 16 w k of prodnctiozl Days of mcnfaatiav V ariable H en age 20 d 22 d 24 d 26 d 27 d 28 d (w k) (%) (g) (%) (g) (%) (s) (%) (a (%) (g) (%) (g) YoQc sac DM » * 848* *" 473* 454* P *> * 818»" » " 470* 487»" 216*" 444» H9C " 846» * 4 11"" 434»" 446" 256» 489» b " 738" " " 3,40" 403" 146" * 234* SEM PxobabOitv o f hen O S ag e e S e a YoUr sac lipid P 489" " " 481" » 643* * * 513" (U 437" 493" *" » 653* " 533" " " 526»" * 120* SEM Probability of hot age effect ^ H e n age means in coltazms with no common sapgrscripts differ significantiy (P â 0.05). ^Days 27 and 28 of incnbatian represent hatched dnckiings that hatehgd between 26 and 27 d o r 27 and 28 d, respectiveiy. ^Mieans were based on 15 eaibryos or hatched dnckiings per day of mcubation. oniess indicated otherwise. ^No dnckiings hatched between 27 and 28 d o xncnbation. ^Mean was based on eight hatched dncklmgs. collected &om other spedes, the BW of the hatchling relative to egg w eight at set is 63 to 70% in poults (Payne et al., 1957; Applegate and lü b u m, 1996, 1998a), approximately 68% in the chicken (Shanawany, 1987), and approximately 71% in the goose (Dvorin et ai., 1982). In the current study, the weight of the embryo or hatched duckling (devoid of the yolk sac) was consistently heavier a t 26 and 27 d of incubation as hens aged. However, the decline in yolk sac w eight during the last week o f incubation was n o t increased in embryos horn older hens. The only differences in the slopes of the regression lines for yolk sac w e i^ t loss during the last week of incubation w as observed in eggs from the youngest (26 wk) and oldest (42 wk) production ages. Variable Yolk-free, embryo wegfa^ SBM. Probability of hen age effect Relative embryo we%ht SEM Probability of hen age effect Table 8.4 &nbryo weiÿxt (devoid of yolk sac) an d rdafivc w eight from duck breeder hmw ducxng the first 16 w k of production Days of inoibatxoti^ Hen. age 20 d 22 d 24 d 26 d 27 d 28 d (wk) 26 2S3S " 3405" 38.13" »" 3323" 4167»" " 3414P 3869»" »" » 3851» 4221» » 4552d ISO (% of egg weiglit a t s c ^ (g) »" 4329» 4859»" 5254»" »" 3833" " " 3787" 4463" 5157»" » 4410» 50.46» 5822» ^"^Hen age means in columns with no common snpcrscxpts differ significantiy (P 6 0J)5). ^Days 27 and 28 of mcubation represent hatched ducklings that hatched between 26 and 27 d o r 27 and 28 d, respectiveiy. ^^/feans were based on 15 embryos or hatched ducklings per day of mcubation indicated otherwûe. ^Nd ducklings hatched between 27 and 28 d of mcubation. "^Mean was based on hatched ducklings. ^Relative embryo weight = ^ouc-free embryo wdght/egg w d ÿ it at set) x

135 Even so, the difference between these production, ages w as small (P = 0.16). Therefore, the efedency of transfer of yolk content m ay not be appreoabiy different between hen productian ages. Nevertheless, in eggs w ith different weights but similar yoik content, em bryos horn 42-wk-old hens had significantly Tpss yolk sac DM and lipid between 22 and 26 d of incubation than those firom 31-wk-old hens. Ih addition, possible differences in ttue efficiency of lipid utilization by the developing duck embryo ÛOTn the relatively more mature hen can n o t be dism üsed. It has also been reported that the duck embryo is more efficient at utilizing egg energy than the chick embryo, ha studies w ith Bering ducks, H olub e t ol (1994) reported tiiat not only does a duck egg have m ore gross energy than eggs fiom broiler hens p rio r to incubation, b u t the transfer of ttiis gross energy to the developing duck embryo during incubaticn is also m ore efficient. In conclusion, the largest increase in egg w eight occurred between 26 and 31 wk, after which time m ean egg w eight declined slightly due to feed restriction. This slight decline in egg w e ^ t did not influence yolk deposhian, thereby resulting in a proportianai increase in yolk w eight as fiie duck hens aged. Yolk-fiee em bryo w eight w as not impaired by the decline in m ean egg w eight after 31 wk, as the weight of the yolk-ficee hatchling a t 27 d w as greater when hens were 42 w k of age. ACKNOWLEDGMENTS The authors wish to thank Maple T paf Farms, hoc., FranksviHe, WI for the generous donation of fertile eggs used in this experiment: We w ould also like to thank Rising Sun Farms, Rising Sun, OH for supplying the fertile eggs. REFERENCES Applegate, T. J., and M. S. LHbum, hidependent effects of hen age and egg size on incnbatian and poult cfiaractensdcs in commercial turkeys. Poultry So. 7SJ Applegate, T. J., and M. S. Idbum, 1998a. Effect of hen age, body weight, and age at photostimulatinn. 1. Egg, incubation, and poult characteristics of commercial turkeys. Fouitry Sd. 77: Apple^te, T. J., and M. S. Ubum. 1998b. Effect of hen age, body weight, and age at photostimulation. 2. Embryonic cfiaracteristics of commercial turkeys. Poultry %-i 77: Cerveny, J., H. Khizetova. and B. Kniz^ Effect of egg weight on growth and feed convesion of Fekm rforks and geese. Pages etc Waterfowl Producdoru Prtxsedings of die Intanational Symposium on Waterfowl Production, the SateHitp Conference for the XVŒ World's Poultry Congress, hitematianal Academv Publishers, New York. NY. Qmstensen, V. I_, W. E. Donaldson, and J. P. McMurtry, Physmlogical differences in late emlnyrw rtom turkey breeders at different ages. Poultry Sd. 75d72-17S. Dawson, R. D., and R. G. Gta-rV, Effects of variation in egg size aiui batching date on survival of Lesser Scaup Aythya i^fins ducklings. Ibis 138d Dvorin, A., Z. Nitsan, and L Nir, The utilization of goose eggs nutrients by the developing endnyo. Br. Poult. ScL 23: Fblcfa, J., M. Lees, and G. H. Sloane-Sianley, A sirrmle method for the isolation and purification of total lipids fiom animal tissues. J. BioL Chem. 226: Bfelub, A., E. Ponizilova, and E. Baranyiova, Energy losses in fowl and duck eggs during incubation. Acta Vet. Brno. 63dlS-120. Khizetova, H., J. Hyanek, and J. Cerveny, Egg size and posthatch growth of Pekin duck. Arch. Geflügelkd. 56: Khizetova, H., B. Knize, and J. Cerveny, Size of yodc sac m waterfowl and dtanges during 24 hours after hatching. Pages be Waterfowl Production: Proceedings of the International Symposium on Waterfowl Production, the Satellite Conference for the XVm Worid's Poultry Congress. Ihtematianal Academy Publishers, New York, NY. Payne, L., P. B. SiegeL and L. Ortman, Coneiatkm of dam. egg poult, and adult weights in Broad Breasted Bronze turkeys. Poultry ScL 36= Rhymer, J. M, The effect of egg size variabiiity on tftennoregulation of Mallard (Anas platyrkyndias) offepring and its implications for survrval Oecologia 75: SAS fasdtute, SAS» User's Guide: Statistics Edition. SAS kstitute fee.. Cary, NC. Shanawany, M. M., Hatching w el^ t in relation to egg weight in domestic birds. World's Poult. Sd. J. 43: Wilson, H. K., feterrelatiortships of egg size, chick size, posthatcfaing growth and hatdtâbîety. World's Poult. Sd. J Yanrtakopoulos, A. L., and A. S. Tserveni-GousL Research note: Effect of breeder quail age and egg weight on chick weight Poultry Sd. 66dS

136 CHAPTER 9 EFFECT OF HEN AGE ON INTESTINAL DEVELOPMENT AND GLUCOSE TOLERANCE OF THE PEKIN DUCKLING Abstract Two experiments were conducted to determine if hen age affects intestinal development and glucose clearance in Pekin ducklings after hatching. In Experiment 1, 85 to 92 g eggs were collected from 32- and 44-wk-old hens. The eggs from older hens contained proportionately more yolk, which resulted in a greater mass transfer of yolk sac DM and lipid from 21 to 25 d of incubation and ducklings from older hens were significantly heavier at hatching (P < ). Even though the ducklings from the older hens were heavier at hatch, BW, feed consumption, and feed efsciency were not significantly different from 7 to 35 d of age. During the first wk after hatching, the age of the hen had no consistent effect on duodenum or lower small intestinal measurements (weight, length, or density). Hen age did not significantly affect plasma glucose concentrations 0, 30, or 60 min post-injection of glucose (2.5 mg per g BW) in 3- or 6-d-old fasted ducklings. In a second experiment, 85 to 90 g eggs were collected from 33- and 48-wk-old hens to determine if differences in hen 116

137 age caused a differential response to a challenge with a higher glucose dosage at 3 d of age. No differences between hen ages were apparent in plasma glucose concentrations 0 or 30 min post-injection (3.75 mg glucose per g BW). Sixty min post-injection however, the ducklings from the younger hens had mg/dl greater plasma glucose concentrations than the ducklings from the older hens (P < 0.02). The glucose tolerance results suggest a that differences in metabolism exist between ducklings from the younger hens, even though measurable BW differences did not exist. Introduction Lilbum et al. (1997) reported that after correction for differences in egg weight, ducklings produced by older Pekin hens (35 to 50 wk of age) were 2 to 3 percent heavier at hatching than ducklings from younger hens (26 and 31 wk of age). Applegate et al. (1998) confirmed this observation and reported a disproportional change in yolk deposition with advancing hen age, ie. the yolk-to-albumen ratio increased from 0.52 to 0.61 from 31 to 42 wk of age. In that study, hen age effects on yolk-free embryo weight were not apparent until 26 d of incubation and were preceded by significant differences in the weight of the yolk sac. Several studies with chickens have reported a positive correlation between egg weight and post-hatch growth, but the correlation declines as chicks age (for review; Wilson, 1991). Differences in post-hatching growth attributable to egg weight have been reported in commercial Pekin ducks as well. In 80, 90, and 105 g eggs collected from Pekin hens in their sixth month of production, differences in BW were maintained from hatching to 42 and 35 d of age in males and females, respectively, but these differences declined after these ages, respectively (Knizetova et al., 1992). Cerveny et al. (1988) reported that egg weight class 117

138 did not influence duckling BW at 7 wk of age when eggs were collected fi'om hens in their sixth month of production, but did aflfect 7 wk BW when eggs were collected during the second month of production. In most instances, post-hatching growth differences can be attributed to increasing egg weight with advancing hen age. Hen age, however, can still exert significant effects on embryonic growth even when the practice of feed restriction of Pekin breeder ducks minimizes the normal age associated increases in egg weight (Applegate et al., 1998). Over the last decade, the age to market (currently, 3.0 to 3.2 kg at 41 d) has been reduced by 10 d (C.M. Turk, Maple Leaf Farms, Franksville, WI 53126, personal communication). Therefore, it is important to re-examine the effects of hen age on posthatching growth and d to market in modem strains of commercial Pekin ducks. In wild species, both hen age and egg size have been shown to influence survival in the wild. In wild Mallards, ducklings fi'om heavy eggs (> 56.4 g eggs) were heavier at hatch than ducklings fi'om small eggs (< 48.0 g eggs) and were able to maintain homeothermy at lower temperatures with a lower metabohc rate (Rhymer, 1988). Dawson and Clark (1996) reported that Lesser Scaup (Aythya afftns) hatchlings produced late in the breeding season fi'om larger eggs, had a 20 percent greater chance of survival in the wild through 2 wk of age. The physiological systems contributing to BW differences during early post-hatch growth of commercial Pekin ducklings fi'om young and old hens and/or egg size have not been identified. The objective of the current studies were to determine if ducklings fi'om relatively young vs older hens have slower intestinal development and tolerance to a metabolic challenge during the first wk after hatching, and subsequent growth to 35 d of age. To 118

139 minimize the influence of egg weight differences, eggs from pre-determined egg weight classes were used. Materials and methods Experiment 1 Eggs were collected from two flocks of breeder Pekin ducks at approximately 50% production (32 wk of age, n=316 eggs) and 12 wk after 50% production (44 wk of age, n=249 eggs). Eggs were individually weighed prior to setting, and only eggs from a predetermined egg size (85 to 92 g) were set (Maple Leaf Farms Research Hatchery, Warsaw, IN 46581). This egg weight range reflects the target range Maple Leaf Farms maintains after 50 % production through weekly adjustments in feed allocation. Prior to incubation, 15 and 10 eggs (from the 32 and 44-wk-old hens, respectively) were hard-cooked in shell for albumen and yolk determinations. At 21 and 25 d of incubation and at hatching, 15 eggs from the 32 wk and 10 eggs from the 44-wk-old hens were randomly selected and the embryos euthanized by decapitation. The yolk sac of the embryo was excised, weighed, placed in a plastic bag, and frozen in a dry ice/ethanol bath. The embryo, devoid of the yolk sac, was then weighed. Yolk and yolk sac DM were determined after lyophihzation and lipid determined after a chloformrmethanol (2:1) extraction (Folch et a i, 1957) of 10 samples per period per hen age. At hatch, all ducklings were individually weighed prior to transport to commercial research facilities (Maple Leaf Farms, Franksville, WI 53126) where they were placed on raised-wire floor pens and given ad libitum access to a commercial diet. 119

140 Fifteen and ten ducklings from the 32- and 44-wk-old hens, respectively were sampled at hatch (0)1,2,3,5, and 7 d of age. Ducklings were euthanized by cervical dislocation. The residual yolk sac was excised and weighed and the yolk-free BW determined. The small intestine was excised beginning at the proximal end of the duodenum extending to the ileocecal junction. The duodenum and lower small intestine (jejunum/ileum) were separated (distal end of pancreatic ducts), flushed with saline, blotted, and weighed. The lengths of the diffèrent segments of the small intestine were recorded. Segments of the distal jejunum (0.5 cm proximal to the yolk stalk) were rinsed and fixed in neutral-buffered formalin at 0, I, and 3 days of age from five ducklings per hen age. Segments of the distal jejunum were dehydrated though a graded alcohol series, cleared with xylene, and embedded in paraffin. Paraffin sections (5 pm) were made and subsequently stained with Hematoxylin and Eosin. Villus measures of distal jejunum sections were made from images obtained from an Olympus DC70 inverted microscope^ processed with a Vay Tek deconvolution imaging system^, and measurements made using Image-Pro Plus software^. Villus height (villus tip to crypt junction) and crypt depth (depth of the invagination between adjacent villi) were measured on 20 villi per bird. A glucose tolerance assay was done at 3 and 6 d of age (Turner, 1991). Within each duckling age, 36 ducklings per hen age were fasted for 12 h and then injected i.m. {Pectoralis major) with 2.5 mg glucose/g BW. The solution was 50% glucose (wt/vol) dissolved in 0.9% ^ Olympus America, Inc., Melville, N Y ^ Vay Tek, Fairfield, la ^Media Cybernetics, Silver Spring, MD

141 saline. At 0 (prior to injection), 30, and 60 min post-injection, blood samples were collected into microcentrifiige tubes after decapitation. Each tube contained 10 pi of a 50% NaCitrate solution (wt/vol) as an anti-coagulant. Plasma was collected and firozen for later glucose determination (Glucose Oxidase'*). At 3 and 6 d of age, twelve ducklings hen age and time post-injection were sampled. All remaining ducklings were individually weighed at 7 d of age, and randomly allocated to 18 pens per hen age (3 per pen). At 15 and 35 d of age, all ducklings were individually weighed and feed consumption was recorded by pen. All results were analyzed by analysis of variance using the General Linear Models procedure of SAS (SAS Institute, 1986). The main effect tested was hen age. A second model was used to determine the main effects of hen age, sex, and their interaction on BW at 7, 15, and 35 d of age. Experiment 2 Eggs were collected from each of two flocks of breeder Pekin ducks at 33 (approximately 50 % production) and 48 wk of age (n=60 eggs per flock). Eggs were individually weighed prior to setting, and only eggs from a pre-determined egg weight (85 to 90 g) were set (Maple Leaf Farms Research Hatchery, Warsaw, IN 46581). At hatch, all ducklings were individually weighed prior to transport to commercial research facilities (Maple Leaf Farms, Franksville, WI 53126) where they were placed on raised-wire floor pens and given ad libitum access to a commercial diet. *Sigma Chemical Co., St. Louis, MO

142 A glucose tolerance assay, similar to that described for Experiment 1, was done at 3 d of age. Forty-five and 42 ducklings from the 33- and 49-wk-old hens, respectively, were sampled. The only difference between this assay and that described for Experiment 1 is that 3.75 mg glucose per g BW was administered compared with. 2.5 mg in the previous study. The higher concentration was administered because the increase in plasma glucose concentrations between 0 and 30 min in Experiment 1 were much less than the previously observed 250 mg/dl increase in turkey poults reported by Turner (1991). Therefore, uncertainty remained as to whether the 2.5 mg/g BW glucose administration was sufficient to challenge metabolic regulation. All results were analyzed by analysis of variance using the General Linear Models procedure of SAS (SAS Institute, 1986). The main effect tested was hen age. Results Experiment 1 The eggs collected fi om the different ages of flocks at the beginning of the trial averaged and ±0.12 g (mean ± pooled SEM) from the 32- and 44-wk-old hens, respectively (P < ). Egg weight at transfer (23 d of incubation) relative to egg weight at set was and % ± 0.15 for eggs from the 32- and 44-wk-old hens, respectively (P < ). The eggs from the older hens had 2.2% more yolk (P < 0.002) and 1.5% less albumen (P < 0.03) than those from the younger hens (Table 9.1). At hatching, the ducklings from the older hens were 2.2 g heavier than from the younger hens {P < , Table 9.2). This BW difference at hatch, had no influence on 1 2 2

143 duckling BW at 7, 15, or 35 d of age. Feed consumption and feed efgciency were not different between hen ages from 7 to 35 d of age and averaged 5.31 kg and 0.55, respectively. The weight of the embryo devoid of the yolk sac was heavier at 21 d of incubation, (P < 0.06) and at hatching (P < ) in eggs from older hens (Table 9.3). At later ages, hen age did not have a consistent effect on yolk-free BW. The weight of the residual yolk sac was not significantly affected by hen age throughout the experiment (Table 9.4). The eggs from the older hens had more (0.7 g, P < 0.06) yolk DM prior to incubation, and more yolk sac DM (0.88 g, P < 0.04) and lipid (0.73 g, P < 0.003) at 21 d of incubation (Table 9.5). After 21 d, hen age did not significantly affect yolk sac composition. The rate of yolk sac weight loss did not differ significantly between hen ages from 21 d of incubation through hatching (R^ = 0.85, Y = X; and = 0.93, Y= X from the 32- and 44- wk-old hens, respectively, P < 0.24). Similarly, hen age did not affect the rate of DM and lipid disappearance from the yolk sac (data not shown). At 1 d of age, ducklings from younger hens had a more dense duodenum (g/cm, P < 0.05), but there were no further effects on duodenum or lower small intestine weight, length, or density the first wk after hatching (Figures 9.1 and 9.2). Villus heights, but not crypt depths, within the distal jejunum were significantly greater at 1 d of age in ducklings from 44- vs 32-wk-old hens (P < 0.05, Figure 9.3). Differences in villus heights or crypt depths were not significantly affected by hen age when ducklings were 3 d of age. The glucose tolerance assay results from 3 and 6 d of age are presented in Figure 9.4. At both ages, peak glucose concentrations at 30 min post-injection was approximately

144 mg/dl greater than non-treated birds. No statistical dififerences due to hen age were observed 0, 30, or 60 min post-injection in either 3- or 6-d-oId ducklings. Experiment 2 The eggs from the different flocks at the beginning of the trial differed by 0.66 g (P < 0.01, Table 9.6). Egg weight at transfer (22 d of incubation) relative to egg weight at set was and 89.21% ± 0.29 for eggs from the 33- and 48-wk-old hens, respectively {P < 0.01). Duckling BW was not significantly different between hen ages at hatch or 3 d of age (after ducklings had been fasted for 12 h). No statistical differences between hen ages were observed in plasma glucose concentrations prior to injection, or 30 min after administration of the higher glucose concentration (3.75 mg/g BW, Table 9.7). The ducklings from the younger hens had significantly increased plasma glucose concentrations 60 min post-injection compared with those from the older hens (33-wk-old, mg/dl; 48-wk-old, mg/dl; P < 0.02). In addition, the ducklings from the younger hens had very similar plasma glucose concentrations 60 min post-injection to what their counterparts had at 30 min post-injection, whereas the plasma concentration in ducklings from the older hens decreased 37.9 mg/dl between 30 and 60 min post-injection. Discussion At hatch, the ducklings were heavier (2.2 g, 4.01 g yolk-free BW) in similar sized eggs from 44- versus 32-wk-old hens in Experiment 1. Similar effects of hen age were presented by Applegate et al. (1998) in similar sized eggs from 31-vs 42-wk-old hens and Lilbum et al. (1997) in eggs from young (26- and 31-wk-old) vs old (35- to 50-wk-old) Pekin hens. 124

145 Applegate et al. (1998) suggested that this difference was partially attributed to a disproportional increase in yolk deposition with advancing hen age, i.e. the yolk-to-albumen ratio changed from 0.52 to 0.61 between 31 and 42 wk of age. They further related changes in embryo growth from 20 to 27 d of incubation to a greater mass transfer of DM and lipid out of the yolk sac during the final wk of incubation. In the current experiments, the yolk-to- albumen ratio increased from 0.54 to 0.60 between 32 and 44 wk of age. Similarly, subsequent differences in yolk sac mobilization between eggs from differing hen ages may have occurred after 21 d of incubation because no differences in yolk sac weights or composition were apparent at 25 d of incubation or at hatching. This proportional increase in yolk deposition and subsequent greater mass transfer of yolk during the final wk of incubation may have contributed to the difference in BW at hatching and is similar to reports in turkeys (Applegate and Lilbum, 1998b). The relative weight loss of the egg prior to transfer has not been consistently related to hen age in the turkey (Christensen and McCorkle, 1982; Leraer et a i, 1993; Applegate and Lilbum, 1996, 1998a). The results of the current study confirm this relationship in the Pekin duck. The relative weight loss of the egg in Experiment 1 (23 d of incubation) and Experiment 2 (22 d of incubation) were both significantly affected by hen age, yet were opposite in direction across each experiment. Hen age did not significantly affect post-hatching performance from 7 to 35 d of age in Experiment 1. In contrast, Cerveny et al. (1988) and Knizetova et al. (1992) reported a prolonged effect of egg weight on Pekin duckling growth. Therefore, egg weight may have more of an impact than hen age, per se, on long term growth of market ducklings. 125

146 Functional maturation of the intestine is one of the main constraints to optimal early growth of precocial birds (Konarzewski et a i, 1989, 1990; Rickelfs et a i, 1998). Intestinal growth (weight, length, density, or distal jejunum villus measurements) was not consistently affected by hen age in Experiment 1. The ducklings in Experiment 1 were considered to be in an ideal environment, as market BW was attained one wk earlier than industry averages. Therefore, we are uncertain as to the impact of hen age on intestinal development and fimctionahty in less than ideal environments. Nevertheless, the extraordinary growth o f the duckling small intestine should be emphasized. For example, turkey poults hatching from similar weight eggs to the duck eggs utilized here, have similar incubation lengths and BW at hatching to that of the Pekin duck. Yet by 7 d of age, the Pekin duckling s jejunum/ileum is 2.7 times heavier, 1.6 times longer, and 2.3 times more dense (g/cm) than that of the turkey poult (Applegate and Lilbum, unpublished data). Consequently, duckling BW is nearly double that of the poult by 7 d of age. Wineland et al. (1997) reported reduced cardiac (0.26 mg) and hepatic (3.95 mg) glycogen in Pekin embryos just prior to and at hatching in similar sized eggs from 30- vs 56- wk-old hens. The impact of this reduction in glycogen reserves during hatching on glycolytic metabolism during the first wk after hatching is uncertain. Nevertheless, when 3.75 mg glucose per g BW was administered to fasted ducklings at 3 d of age in Experiment 2, the ducklings from the 48- vs 33-wk-old had 27% lower plasma glucose concentrations 60 min post-injection. In Experiment 1, differences in plasma glucose concentrations 60 min postinjection at 3 or 6 d of age were not affected by hen age when fasted ducklings were injected with 2.5 mg glucose per g BW. The different concentrations of glucose administered between 126

147 experiments (2.5 vs 3.75) did not affect the peak plasma glucose concentration 30 min postinjection, which averaged 351 and 341 mg/dl in Experiments 1 and 2, respectively. Exact mechanism(s) affecting the improved clearance of glucose observed in Experiment 2 was not investigated. Notably, the response to glucose administration (either 2.5 or 3.75 mg/g BW) was considerably less in the duckling than in similar trials with turkey poults (Turner, 1991). The endocrine control of carbohydrate metabolism in avian species is quite different than in mammals (for review, see Hazelwood, 1971; Pearce, 1977). For example, the avian pancreas produces and secretes less insulin and nearly 10 times more glucagon than the mammalian pancreas (Pearce, 1977). In addition, avian species have much higher plasma glucose concentrations. Therefore, newly hatched chicks are more resistant to fasting hypoglycemia than neo-natal mammals (Houpt, 1958). These differences make glucose clearance a valuable tool in measuring the metabolic homeostasis in avian species. In conclusion, hen age significantly affected embryonic growth differences and hatching BW, independent of egg weight differences in Experiment 1, but did not affect hatching BW in Experiment 2. The BW differences observed in Experiment 1, however, had no effect on BW, feed consumption, or feed efficiency from 7 to 35 d of age, or on intestinal development during the first wk after hatching. Even though no BW differences were apparent at hatch or at 3 d of age in Experiment 2, the ducklings from the younger hens had a high plasma glucose concentration 60 min post-challenge. The results of the glucose tolerance assay suggest the ducklings from the younger hens have a reduced capacity to handle a glucose challenge at this age. 127

148 Acknowledgments The authors would like to thank Maple Leaf Farms, Franksville, WI for their generous donation o f eggs, time, and assistance with these experiments. References Applegate, T.J., and M.S. Lilbum, Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci. 75: Applegate, T.J., and M.S. Lilbum, 1998a. Effect of hen age, body weight, and age at photostimulation. 1. Egg, incubation, and poult characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., and M.S. Lilbum, 1998b. Effect of hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., D. Harper, and M.S. Lilbum, Effect of hen production age on egg composition and embryo development in commercial Pekin ducks. Poultry Sci. 77: Cerveny, J., H. Knizetova, and B. Knize, Effect of egg weight on growth and feed conversion of Pekin ducks and geese. Pages in\ Waterfowl Production: Proceedings of the Intemational Symposium on Waterfowl Production, the Satellite Conference for the XVm World s Poultry Congress. Intemational Academic Publishers, New York, NY. Christensen, V.L., and F.M. McCorkle, Characterization of incubational egg weight losses in three types o f turkeys. Poultry Sci. 61: Dawson, R.D., and R.G. Clark, Effects of variation in egg size and hatching date on survival of Lesser Scaup Aythya qffins ducklings. Ibis 138: Folch, J., M. Lees, and G.H. Sloan-stanley, A simple method for the isolation and purification of total lipids fi'om animal tissues. J. Biol. Chem. 226: Hazelwood, R.L., Endocrine control of avian carbohydrate metabolism. Poultry Sci. 50:9-18. Houpt, T.R., Effects of fasting on blood sugar levels in baby chicks of varying ages. Poultry Sci. 37:

149 Knizetova, H., J. Hyanek, and J. Cerveny, Egg size and post-hatch growth of Pekin duck. Archiv Geflug. 56: Konarzewski, M., J. Kozlowski, and M. Ziolko, Optimal allocation of energy to growth o f the alimentary tract in birds. Funct. Ecol. 3: Konarzewski, M., C. Lilja, J. Kozlowski, and B. Lewonczuk, On the optimal growth o f the alimentary tract in avian postembryonic development. J. Zool. 222: Lemer, S., N. French, D. McIntyre, C. Baxter-jones, Age-related changes in egg production, fertility, embryonic mortality, and hatchability in commercial turkey flocks. Poultry Sci., 72: Lilbum, M S., J. Nixon, and C.M. Turk, The effects of breeder age on incubation characteristics of commercial duck eggs. Poultry Sci. 76(Suppl. 1):91. Pearce, J., Some differences between avian and mammalian biochemistry. Intl. J. Biochem. 8: Rhymer, J.M., The effect of egg size variability on thermoregulation of Mallard (Anas platyrhynchos) offspring and its implications for survival. Oecologia 75: Ricklefs, R.E., I.M. Starck, and M. Konarzewski, 1998 Internal constraints on growth in birds. Pages in: Avian Growth and Evolution within the Altricial-Precocial Spectrum. J.M. Starck and R E. Ricklefs, edit. Oxford University Press, New York, NY. SAS Institute, SAS User s Guide: Statistics Edit. SAS Institute, Inc., Cary, NC. Turner, K.A., The effect of feeding high carbohydrate and fat diets following a 2 day fast upon blood metabolites and liver status in newly-hatched turkey poults. M. S. Thesis, The Ohio State University/0. A.R.D.C. Wilson, H R, Interrelationships of egg size, chick size, posthatching growth and hatchability. W. Poultry Sci. J. 47:5-20. Wineland, M.J., G.S. Davis, V.L. Christensen, and M. Jefferey, Effect of reduced incubator ventilation upon the duck embryo. Poultry Sci. 76(Suppl. 1):

150 Hen age, wk Egg Yolk Albumen (g) (g) (%): (g) (%)' ' SEM Probability of hen age effect ' Egg weights represent 15 and 10 eggs from the 32-and 44-wk-old hens, respectively, w ^ Yolk percentage = (yolk weight/egg weight) x 100. ^ Albumen percentage = (albumen weight/egg weight) x 100. Table 9.1. Egg weight and composition from 32- and 44-wk-old Pekin ducks, Experiment 1.

151 Hatch 7d 15d 35 d Hen age, wk Male Female Male Female Male Female (g) (kg) 32 54,1' 290% 288 1,02 1,00 3,28 3, ,3 290: 280 1,04 1,00 3,27 3,18 SEM 0, ,01 0,01 0,05 0,03 Source of Variation Hen age 0,0001 Probability 0,97 0,09 0,39 0,89 0,95 0,30 Sex 0,11 0,008 0,09 Age*Sex 0,26 0,56 0,60 Means represent 221 and 192 ducklings at hatch from 32- and 44-wk-old hens, respectively, ^ Means represent 25 male and 29 female ducklings from the 32-wk-old hens, ^ Means represent 24 male and 24 female ducklings from the 44-wk-old hens. Table 9,2, Duckling BW from 32- and 44-wk-old hens. Experiment 1,

152 Hen age, wk Days relative to hatch ' SEM Probability of hen age effect (g) U ) *Means represent 15 and 10 embryos or ducklings per sampling d from the 32- and 44-wk-old hens, respectively. Table 9.3, Embryo and duckling BW, devoid of the yolk sac, from 32- and 44-wk-old Pekin ducks. Experiment 1.

153 Days relative to hatch Hen age, wk ,75' 11,96 2,70 1,09 OAl 44 21,15 13,21 2,08 0,71 0,34 SEM 0,59 0,84 0,61 0,15 0,07 { r r \ (ë) Probability of hen age effect 0,11 0,31 0,48 0,08 0,22 Means represent 15 and 10 embryos or ducklings per sampling d from the 32- and 44-wk-old hens, respectively. u > Table 9,4, Yolk sac weight of embryos or ducklings from 32- and 44-wk-old Pekin ducks, Experiment 1,

154 Variable Hen age Egg Yolk -7-3 Days relative to hatch (wk) (%) (g) (%) (g) (%) (g) (%) (g) DM ' SEM U) Probability of hen age effect Lipid" SEM , , ,14 Probability of hen age effect ,91 Means represent 10 yolks or yolk sacs per sampling d per hen age. ^ Percentage means determined on a DM basis. Table 9.5. Yolk and yolk sac DM and lipid content of duck eggs and embryos from 32- and 44-wk-old Pekin ducks, Experiment 1.

155 Figure 9.1. Duodenum weight, length, and density (g/cm) of ducklings from 32- (black bars) and 44-wk-old (white bars) Pekin ducks. Experiment 1. Means represent 15 and 10 ducklings from the 32- and 44-wk-old hens, respectively. Error bars indicate SEM. *Difference between hen ages is significant (P < 0.05). 135

156 Duodenum weight, g r r 3 5 r Duodenum length, cm JTI ml Duodenum d< ;nsity, g/cm I.rill r r 3 5 r7 \ r Days of age 32 wk 44 wk Figure

157 Figure 9.2. Jejunum and ileum weight, length, and density (g/cm) of ducklings from 32- (black bars) and 44-wk-old (white bars) Pekin ducks. Experiment 1. Means represent 15 and 10 ducklings from the 32- and 44-wk-old hens, respectively. Error bars indicate SEM. No significant differences were observed due to hen age (JP > 0.05) 137

158 Jejunum and ileum weight, g r7 rrrfi r Jejunum and üeum length, cm r Jejunum and ileum density, g/cm Days of age 32 wk I I 44 wk r Figure

159 Figure 9.3. Villus height (gm, villus tip to crypt) and crypt depth (pm, depth of the invagination between adjacent villi) from distal jejunum sections from 32- (black bars) and 44- wk-old (white bars) Pekin ducks. Experiment 1. Positive scale represents villus height and negative scale represents crypt depth. Means represent the average of 20 villus measures from 5 birds per hen age. Error bars indicate SEM. *Difference between hen ages is significant {P < 0.05). The photomicrograph is a distal jejunal section from a 3-d-old duckling. Magnification = loox. 139

160 500 a Villus height 100 è 1 0 I rcj a > -200 Hatch 1 d 3 d 32 wk 44 wk m VO w

161 Figure 9.4. Glucose tolerance assay of 3- and 6-d-oId ducklings from 32- (black bars) and 44-wk-old (white bars) Pekin ducks. Experiment 1. Ducklings were fasted for 12 h prior to an Lm. injection of 2.5 mg glucose per g body weight. Means represent 12 ducklings per hen age and min after injection. Error bars indicate SEM. No significant differences were observed due to hen age (JP > 0.05). 141

162 3 d Plasma glucose, mg/dl 6 d Plasma glucose, mg/dl 30 min post-injection 142 Figure 9.4

163 Hen age, wk Egg at set Hatch BW 3 dbw^ (g) (g) (g) SEM Probability of hen age effect ^Means represent 60 eggs set per hen age. Hatchability of eggs set was 76 and 95% from the 33- and 48-wk-old hens, respectively. Three d BW represents ducklings which had been fasted for 12 h prior to weighing. Table 9.6. Egg weight and duckling BW at hatch and 3 d after hatching from 33- and 48-wkold Pekin ducks. Experiment

164 min post-injection Hen age, wk tpiasma glucose, mg/oij = = ^ SEM Probability of hen age effect ^ Ducklings were fasted for 12 h prior to an i.m. injection of 3.75 mg glucose per g BW. n=15 unless indicated otherwise. ^ n=l4. ^ n=13. Table 9.7. Glucose tolerance assay^ of 3-d-old ducklings from 33- and 48-wk-old Pekin ducks. Experiment

165 CHAPTER 11 SUMMARY AND CONCLUSIONS This dissertation measured how the age of the hen affects selected reproductive traits, egg composition, embryonic development, and post-hatching growth. Hatchling growth studies were conducted using the commercial turkey poult and commercial Pekin duckling as experimental systems, and focused specifically on the response to a metabolic challenge and intestinal growth. Within the turkey breeder industry, turkey hens are recommended to be photostimulated at a specific age (29 to 31 wk), rather than a specific body weight. This practice is based on field data and studies fi"om small white turkey strains in the late 1950's and early 1960's. Advances in growth rates of commercial strains since that time have made it possible for hens to achieve a desirable body weight much earlier than the recommended 29 to 31 wk of age, yet few studies report poor reproductive performance if hens are photostimulated prior to 29 to 31 wk. A cannulation experiment was designed to collect serial blood samples from turkey hens prior to and 3 d after photostimulation, in order to measure changes in the luteinizing hormone (LH) profiles from hens weighing either 11.8 or 13.1 kg. These weights reflect the range o f desirable target body weights at 145

166 photostimulation observed in commercial practice. The differences in BW were maintained across three different ages, 24 to 25, 27 to 28, and 31 to 32 wk. The youngest hens had a higher plasma baseline LH concentration prior to photostimulation and a greater increase in baseline values after photostimulation. Hen body weight within an age, or between ages did not greatly influence LH profiles. No differences were observed across ages on the weights of the Pectoralis major or on abdominal fat. The observed differences in photostimulatory response as measured by changes in baseline LH, therefore, were more dependent on chronological age than hen body weight at photostimulation. In a separate experiment, the effects of hen age on changes in hen body composition, egg components, incubation characteristics of eggs, and hatching poult characteristics were determined. These effects were determined over a 19 wk production period. As hens aged from 33 to 55 wk of age, much of the weight lost by the hen during this time could be attributed to a relative decrease in of abdominal fat, rather than to relative changes in the Pectoralis major. As has been previously reported, egg weight generally increased from 36 to 55 wk of age. In addition to increases in egg weight, relative yolk deposition increased with a corresponding decrease in relative deposition of albumen. The eggs during this production period were set in individual pedigree baskets, and incubated. Upon hatching, individual poult weights were expressed as a proportion of initial egg weights. After correcting for changes in egg weights over the entire 19 wk production period, the poults from 55-wk-old hens were 3 to 4 % heavier at hatching compared with poults from 36-wk-old hens. Poults from the older hens also had heavier yolk sacs at hatching. In addition, yolk sacs of poults from older 146

167 hens had proportionately more dry matter and lipid. Most of the differences in hatchling characteristics were associated with the poults produced by the youngest hens (36 wk of age). In a subsequent experiment, the effects of hen body weight at photostimulation (II. 8 and 12.9 kg) and age at photostimulation (29 and 31 wk) on egg composition and late embryonic growth was studied during the first 10 weeks of egg production. Three weeks after photostimulation (approximately the age of first oviposition), the BW of the hen at photostimulation had minimal effects on carcass traits. Age at photostimulation, however, did influence carcass measures, in that hens photostimulated at 31 wk had heavier ovaries, due largely to the presence of one additional large viteuogenic follicle (> 20 mm diameter) Neither hen BW or age at photostimulation had consistent affects on egg composition, or embryonic growth characteristics during the first 10 wk of production. Hen production age, however, greatly influenced egg composition and embryonic growth. As was noted in the earlier study, hens deposited proportionately more yolk into the egg with increasing hen production age. Through the first 10 wk of production, yolk sac weight at 21 d of incubation, embryo weight (devoid of the yolk sac) and liver weights at 25 d of incubation were generally increased. Yolk sacs from embryos from the oldest and youngest production ages were selected from similar weight eggs(80 to 85 g prior to incubation) and further lipid analyses were conducted. Yolk sacs of embryos from older hens had more neutral lipid (triglyceride and cholesterol ester) and phospholipid at 21 d of incubation but not at 25 d of incubation. The greater mass transfer of major lipid subclasses out of the yolk sac during this period of rapid growth was partially attributed to a proportionately larger yolk in eggs from older hens. 147

168 Ensuing experiments determined if the additional transfer of yolk and enhanced embryonic growth affected hatchling intestinal development, response to a metabolic challenge, and growth. To that extent, a glucose tolerance assay was conducted during the first wk after hatching. At 4 d of age, poults from relatively young hens, especially those from the lightest egg weight class (75 to 80 g), were unable to moderate plasma glucose concentrations and remained hyperglycemic 60 min after challenge with a super-physiologic dose of glucose. Intestinal growth was characterized initially by gross weight and length measures. A final experiment characterized post-hatching intestinal growth through viuus measures including enterocyte proliferation and migration through the incorporation and staining of distal jejunal sections for bromodeoxyuridine (BrdU, a thymidine analog). Poult intestinal weight and length measures were not consistently affected by hen age or egg weight class during the first wk after hatching. Poults from the older hens had longer villi and greater enterocyte migratory height at hatching, but these traits were unaffected by hen age or egg weight after placement. Poult growth throughout the first wk after hatching was not consistently affected by hen age. As growth was unaffected by hen age, the glucose tolerance assay proved to be a more sensitive measure of poult status or quality rather than BW per se. While studying the effects of hen age on poult enterocyte proliferation and migration, effects associated with poult age were also noted. The proportion of the villus stained with BrdU positive enterocytes was significantly greater at -1, 0, and 1 d of age as compared to older ages (2 to 7 d of age). This result suggests that when poults were injected with BrdU at 26 d of incubation, a larger proportion of the villus contained proliferative cells than during 148

169 the first wk after hatching. Daily migration rates of enterocytes were also similar (approximately 2 pm/hr) in poults from hatching to 7 d of age. The larger proportion of the villus containing proliferative cells at the earlier ages, coupled with similar migration rates, suggest that changes in function after hatching occurs due to an accumulation of cells rather than to turnover rates of the mucosal epithelium. The commercial Pekin duck provided a unique subject for studying the efîêcts of hen production age on embryonic and hatchling growth because egg weight is consistent (85 to 92 g) during an extended production period due to quantitative feed restriction of hens. The quantitative feed restriction prevents the laying of excessively large eggs and enhances the hatching flock s uniformity. As with the turkey, proportionately more yolk is incorporated into the egg as the Pekin duck aged from 26 to 42 wk of age. Duckling weight at hatch was also heavier with increasing hen age, independent of age-associated changes in egg weight. The rate of yolk sac weight loss (20 days of incubation to hatch) between embryos from different hen ages was not different, thereby suggesting that the efficiency of transfer of yolk lipid out of the yolk sac was not considerably different between hen ages. However, the absolute amounts of yolk dry matter and lipid (from similarly sized eggs) mobilized between 20 and 26 d of incubation was greater in 42- vs 31-wk-old hens, after which time differences were negligible. A subsequent experiment determined the effects of hen age on duckling growth, glucose tolerance, and intestinal development. After hatching, hen age had no consistent effects on duckling growth to 35 d of age, or small intestinal growth (weight, length, weight per unit length, or on villus measures) during the first wk after hatching. As with the turkey 149

170 poult, ducklings from the younger hens were unable to reduce plasma glucose concentrations during the course of the glucose tolerance assay. The data from poults and ducklings suggests that hatchlings from the younger hens have a lesser capacity for metabolic homeostasis at hatch, even though there are no measurable differences in BW. The turkey poult and Pekin duckling hatch from similar sized eggs, have similar lengths of incubation, and body weights at hatching. The male Pekin duckling, however, attains a mature market weight of approximately 3.2 kg in 6 wk or less, whereas the male turk^ only weighs approximately 2.3 kg at 6 wk. Similar sized eggs were collected and BW and intestinal growth data were collected at similar ages while studying the effects of hen age on post-hatch development in both the poult and duckling. Comparisons of growth and intestinal development were made between the two species during the first wk after hatching and specie comparison tables and figures are located in the Appendix. Yolk-free BW of ducklings was greater than poults beginning at 1 d after placement and were nearly twice as heavy by 7 d of age (Table A.3). Yolk sac weight was similar at 25 d of incubation, after which time the regression and utilization of the yolk sac was more rapid in the duckling than in the poult (Table A.2). Duodenum length was consistently greater in ducklings from hatch through 7 d of age (Figure A 1). Duodenum weight, however, was not heavier in the duckling until 7 d of age (Figure A. 1). Jejunum and ileum weight, length, and density (g/cm) were consistently heavier in the duck than in the turkey from hatch through 7 d of age, at which time the jejunum and ileum was 2.7 times heavier, 1.6 times longer, and 2.3 times more dense in the duck (Figure A_2). Histologic sections of the distal jejunum 150

171 revealed that the duck has more rapid villus growth from hatch to 3 d of age (Table A. 6, Figure A.3). This phenomenal difierence in intestinal growth aided the duckling in achieving an additional 143 g BW gain during this critical 7 d growth period. 151

172 BIBLIOGRAPHY Applegate, T.J., W.L. Bacon, and M S. Lilburn, Effect of age and body weight on plasma concentrations of luteinizing hormone in turkey hens before and after photostimulation. Dom. Anim. Endocrin. I4(6): Applegate, T.J., E. Ladwig, L. Weissert, and M S. Lilburn, Effect of hen age on intestinal development and glucose tolerance of the Pekin duckling. Poultry Sci. (manuscript submitted). Applegate, T.J., JJ. Dibner, M.L. Kitchell, Z. Uni, and M S. Lilburn, Effect of turkey breeder hen age and egg size on poult development. 2. Intestinal villus growth, enterocyte migration and proliferation of the turkey poult. Biol. Neo. (manuscript submitted). Applegate, T.J., and M S. Lilburn, Independent effects of hen age and egg size on incubation and poult characteristics in commercial turkeys. Poultry Sci. 75: Applegate, T.J., and M.S. Lilbum, Effect of hen age, body weight, and age at photostimulation. 1. Egg, incubation, and poult characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., and M.S. Lilbum, Effect of hen age, body weight, and age at photostimulation. 2. Embryonic characteristics of commercial turkeys. Poultry Sci. 77: Applegate, T.J., and M.S. Lilbum, Effect of turkey breeder hen age and egg size on poult development. 1. Intestinal growth and glucose tolerance of the turkey poult. Biol. Neo. (manuscript submitted). Applegate, T.J., M.S. Lilbum, and CM. Turk, Effect of hen age on egg and embryonic ch^acteristics in breeder ducks. Poultry Sci. 76(Suppl. 1):90. Applegate, T.J., J.E. Nixon, and M.S. Lilbum, Effect of turkey hen age, body weight, and age at photostimulation on egg and embryonic characteristics. Poultry Sci. 75(Suppl. 1):

173 Bacon, W.L., and D.W. Long, Changes in plasma luteinizing hormone concentration in turkey hens after switching from short-day to long-day photoperiods. Domest. Animal Endocrin. 12: Bacon, W.L., and D.W. Long, Secretion of luteinizing hormone during a forced molt in turkey hens. Poultry Sci. 75: Bacon, WJL., and K.E. Nestor, Reproductive traits of first eggs of clutches vs. other clutch positions in turkeys. Poultry Sci. 58: Bacon, W.L., and K.E. Nestor, Body weight changes during the reproductive period in four strains of turkey hens. Poultry Sci. 61: Bacon, WJL., K.L. Nestor, S.W. Long, and D.H. Ohm, Divergent selection for body weight and yolk precursor in Cotumix japonica. 2. Correlated responses in plasma lipoproteins and lipids. Poultry Sci. 61: Bacon, W.L., K.E. Nestor, and P.A. Renner, The influence of genetic increases in body weight and shank width on the abdominal fet pad and carcass composition of turkeys. Poultry Sci. 65: Baranyiova, E., Influence of deutectomy, food intake and fasting on the digestive tract dimensions in chickens after hatching. Acta Vet. Bmo 41: Baranyiova, E., and J. Holman, Morphological changes in the intestinal wall in fed and fasted chickens in the first week after hatching. Acta Vet. Bmo 45: Baranyiova, E., A. Holub, and E. Ponizilova, Changes in the mass and chemical composition of the gastrointestinal tract and liver of ducks in the first two months after hatching. Acta Vet. Bmo 52: Bayer, R.C., C.B. Chawan, F.H. Bird, and S.D. Musgrave, Characteristics of the absorptive surface of the small intestine of the chicken from 1 day to 14 weeks of age. Poultry Sci. 54: Cameron, I.L., and G. Cleffinann, Initiation of mitosis in relation to the cell cycle following feeding of starved chickens. J. Cell Biol. 21: Cerveny, J., H. Knizetova, and B. Knize, Effect of egg weight on growth and feed conversion of Pekin ducks and geese. Pages /«: Waterfowl Production: Proceedings of the International Symposium on Waterfowl Production, the Satellite Conference for the X V m World s Poultry Congress. Intl. Acad. Publ., New York, NY. 153

174 Chambers, G., and RJD. Grey, Development of the structural components of the brush border in absorptive ceils of the chick intestine. Cell Tiss. Res. 204: Chapman, D.P., W.L. Bacon, D.W. Long, K. Kurima, and W.H. Burke, Photostimulation changes the pattern of luteinizing hormone secretion in turkey hens. Gen. Comp. Endocrin. 96: Christensen, V.L., W.E. Donaldson, and J.P. McMurtry, Physiological differences in late embryos from turkey breeders at different ages. Poultry Sci. 75: Christensen, V.L., and F.M. McCorkle, Characterization of incubational egg weight losses in three types of turkeys. Poultry ScL 61: Crittenden, L.B., and B.B. Gohren, The effects of current egg production, age of the pullet, and inbreeding on hatchability and hatching time. Poultry Sci. 41: Cunningham, F., O.J. Cotterill, and E.M. Funk, The effect of season and age of bird. 1. On egg size, quality and yield. Poultry Sci. 39: Cunningham, F.E., O.J. Cotterill, and E.M. Funk, The effect of season and age of bird. 2. On the chemical composition of egg white. Poultry Sci. 39: Daly, K.R., and R.A. Peterson, The effect of age of breeder hens on residual yolk fat, and serum glucose and triglyceride concentrations of day-old broiler chicks. Poultry Sci. 69: Dawson, R.D., and R.G. Clark, Effects of variation in egg size and hatching date on survival of Lesser Scaup Aythya qffins ducklings. Ibis 138: Dibner, J.J., C.A. Atwell, M L. Kitchell, W.D. Shermer, and F.J. Ivey, Feeding of oxidized frts to broilers and swine: effects on enterocyte turnover, hepatocyte proliferation and the gut associated lymphoid tissue. Anim. Feed Sci. Tech. 62:1-13. Dibner, J.J., MJL. Kitchell, C.A. Atwell, and F.J. Ivy, The effect of dietary ingredients and age on the microscopic structure of the gastrointestinal tract in poultry. J. Appl. Poultry Res. 5: Ding, S.T., and M.S. Lilbum, Characterization of changes in yolk sac and liver lipids during embryonic and early posthatch development of turkey poults. Poultry Sci. 75: Ding, S T., KE. Nestor, and M.S. Lilbum, The concentration of different lipid classes during late embryonic development in a randombred turkey population and a subline selected for increased body weight at sixteen weeks o f age. Poultry Sci. 74:

175 Donaldson, W.E., and V.L. Christensen, Dietary carbohydrate level and glucose metabolism in turkey poults. Comp, biochem. Physiol. 98: Donaldson, W.E., and G.I. Liou, Lipogenic enzymes: parallel responses in lever glucose consumption by newly hatched chicks. Nutr. Rep. Intl. 13: Dunn, I.e., and F.J. Sharp, Photoperiodic requirements for LH release in juvenile broiler and egg-laying strains of domestic chickens fed ad libitum or restricted diets. J. Reprod. Fert. 90: Dunn, I.e., P.J. Sharp, and P.M. Hocking, Effects of interactions between photostimulation, dietary restriction and dietary maize oil dilution on plasma LH and ovarian and oviductal weights in broiler breeder females during rearing. Br. Poultry Sci. 31: Dunnington, E.A, and P.B. Siegel, Long-term divergent selection for eight-week body weight in White Plymouth Rock chickens. Poultry Sci. 75: Dvorin, A., Z. Nîtsan, and I. Nir, The utilization of goose eggs nutrients by the developing embryo. Br. Poultry Sci. 23: Esteban, S., M. Moreno, I.M. Rayo, and I. A. Tur, Gastrointestinal emptying in the final days of incubation in the chick embryo. Brit. Poultry Sci. 32: Etches, RJ., The egg. Pages in: Reproduction in Poultry. CAB International, Wallingford, Oxon, U.K. Ferket, P R, and E.T. Moran, Jr., Effect of plane of nutrition fi-om starting to and through the breeder period on reproductive performance of hen turkeys. Poultry Sci. 65: Ferraris, R.P., S.A. Villenas, and J. Diamond, Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Amer. J. Physiol. 262:G1047-G1059. Ferrer, R., I.M. Planas, and M. Moreto, Cell apical surface area in enterocytes fi-om chicken small and large intestine during development. Poultry Sci. 74: Folch, I., M. Lees, and GH. Slone-Stanley, A simple method for the isolation and purification o f total lipids fi-om animal tissues. J. Biol. Chem. 226: Foley, I., T. Thai, R. Maronpot, B. Butterworth, and G. Goldsworthy, Comparison of proliferating cell nuclear antigen to trhiated thymidine as a marker of proliferating hepatocytes in rats. Environ. Health Perspect. 101:

176 Foster, D.L, S.M. Yellon, and D.H. Olster, Internal and external determinants of the timing of puberty in the female. J. Reprod. Fert. 75: Freeman, B.M., and M.A. Vince, Development of the Avian Embryo. Chapman and Hall, London, UK. GrifBn, H.D., MM. Perry, and A.B. Gilbert, Yolk Formation. Pages in: Physiology and Biochemistry o f the Domestic Fowl. Volume 5. Edit. B.M. Freeman, Academic Press, Inc., New York, NY. Harper, J.A, Changes in body weight and conformation of broad breasted bronze turkeys during the breeding season. Poultry Sci. 29: Hazelwood, RL., Endocrine control of avian carbohydrate metabolism. Poultry Sci. 50:9-18. Hester, P.Y., RW.C. Stevens, Feed restriction of turkey hens-a review. Poultry Sci. 69: Hocking, P.M., Effects of photostimulation at 18, 24 and 30 weeks of age on the productivity of female turkeys fed ad libitum or restricted until point of lay. Br. Poultry Sci. 33: Hocking, P.M., Genetic and environmental control of ovarian fimction in turkeys at sexual maturity. Br. Poultry Sci. 33: Hocking, P.M., Relationships between egg size, body weight, and pelvic dimensions in turkeys. Anim. Prod. 56: Hocking, P.M., Gilbert, AB., C.C. Whitehead, and M.A. Walker, Effects of age and of earl y lighting on ovarian function in breeding turkeys. Br. Poultry Sci. 29: Holdsworth, C D., and T. Hastings-Wilson, Development of active sugar and amino acid transport in the yolk sac and intestine of the chicken. Amer. J. Physiol. 212: Holub, A, E. Ponizilova, and E. Baranyiova, fl994. Energy losses in fowl and duck eggs during incubation. Acta Vet. Bmo 63: Houpt, T.R, Effects of fasting on blood sugar levels in baby chicks of varying ages. Poultry Sci. 37: Imondi, AR., and F.H. Bird, The turnover of intestinal epithelium in the chick. Poultry Sci. 45:

177 Jeurissen, SU M., D. van Roozeiaar, and E.M. Janse, Absorption of carbon from the yolk into gut-associated lymphoid tissues of chicens. Devi. Comp. Immunol. 15: Kitchell, ML., and J.J. Dibner, Rapid detection of proliferating cells using microwave fixation and a monoclonal antibody to bromodeoxyuridine. J. Histotech. 12: Knizetova, H., J. Hyanek, and J. Cerveny, Egg size and posthatch growth of Pekin duck. Arch. Geflugelk. 56: Knizetova, H., B. Knize, and J. Cerveny, Size of yolk sac in waterfowl and changes during 24 hours after hatching. Pages in: Waterfowl Production: Proceedings of the International Symposium on Waterfowl Production, the Satellite Conference for the XVIII World s Poultry Congress. Intl. Acad. Pub., New York, NY. Konarzewski, M., Kozlowski, J., and M. Ziolko, Optimal allocation of energy to growth of the alimentary tract in birds. Funct. Ecol. 3: Konarzewski, M., Lilja, C., Kozlowski, J., and B. Lewonczuk, On the optimal growth of the alimentary tract in avian postembryonic development. J. Zool. Lond. 222: Krishnan, K.A., J.A. Proudman, and J.M. Bahr, Purification and partial characterization of isoforms of luteinizing hormone from the chicken pituitary gland. Comp. Biochem. Physiol. 108B: Krueger, KK., J.A. Owen, CE. Krueger, and T.M. Ferguson, Effect of feed and light regimens during the growing period on subsequent reproductive performance of Broad- Breasted White turkeys fed two protein levels. Poultry Sci. 57: Lambson, RO., An electron microscopic study of the entodermal cells of the yolk sac of the chick during incubation and after hatching. Amer. J. Anat. 129:1-20. Laskey, RA., M.P. Fairman, and J.J. Blow, S phase of the cell cycle. Science 246: Latour, M.A., E.D. Peebles, C.R. Boyle, J.D. Brake, T.F. Kellogg, Changes in serum lipid, lipoprotein and corticosterone concentrations during neonatal chick development. Biol. Neonate 67: Latour, M.A., E.D. Peebles, C.R. Boyle, S.M. Doyle, T. Pansky, and J.D. Brake, Effects of breeder hen age and dietary fat on embryonic and neonatal broiler serum lipids and glucose. Poultry Sci. 75:

178 Laskey, R.A., M.P. Fairman, and J.J. Blow, S phase of the cell cycle. Science 246: Leighton, A.T., Jr., and L.M. Potter, Reproductive performance of turkeys subjected to blackout versus brownout restricted light conditions. Poultry Sci. 48: Leighton, A.T., Jr., and R.N. ShofSier, Effect of light regime and age on reproduction of turkeys. 1. Effect of 15,24 hour, and restricted light treatment. Poultry Sci. 40: Leighton, A.T., Jr., and RN. Shofber, Effect of light regime and age on reproduction o f turkeys. 2. Restricted vs. unrestricted light. Poultry Sci. 40: Lemer, SJP., N. French, D. McIntyre, and C. Baxter-Jones, Age-related changes in egg production, fertility, embryonic mortality, and hatchability in commercial turkey flocks. Poultry Sci. 72: Lilbum, M S., Skeletal growth of commercial poultry species. Poultry Sci. 72: Lilbum, M S., and K.E. Nestor, Body weight and carcass development in different lines o f turkeys. Poultry Sci. 70: Lilbum, M S., Practical aspects of early nutrition for poultry. J. Appl. Poultry Res. (in press). Lilbum, M.S., and K.E. Nestor, The relationship between various indices of carcass growth and development and reproduction in turkey hens. Poulrty Sci. 72: Lilbum, M.S., J. Nixon, and C.M. Turk, The effects of breeder age on incubation characteristics of commercial duck eggs. Poultry Sci. 76(Suppl. 1):91. Lim, S., and F.N. Low, Scanning electron microscopy of the developing alimentary canal in the chick. Am. J. Anat. 150: Marcheselli, V.L., and N.G. Bazan, Quantitative analysis of fatty acids in phospholipids, diacyglycerols, free fatty acids and other lipids. J. Nutr. Biochem. 1: Mather, C.M., and K.F. Laughlin, Storage of hatchling eggs: the interaction between parental age and early embryonic development. Br. Poultyry Sci. 20: McCartney, M.G., D C. Borron, and H.B. Brown, Reproductive performance of turkey females as affected by growth and pre-breeder diets. Poultry Sci. 56:

179 McNaughton, J.L., J.W. Deaton, F.N. Reece, and R.C. Haynes, Effect of age of parents and hatching egg weight on broiler chick mortality. Poultry Sci. 57: Melnychuk, V i., F i. Robinson, R.A. Renema, R.T. Hardin, D.A. Emmerson, L.G., Bagley, Carcass traits and reproductive development at the onset of lay in two lines of female turkeys. Poultry Sci. 76: Merriam, G.R., and K.W. Wachter, Algorithms for the study of episodic hormone secretion. Am. J. Physiol. 243 :E Michael, E. and R.D. Hodges, Histochemical changes in the fowl small intestine associated with enhanced absorption after feed restriction. Histochemie 36: Mitchell, M.A., and MW. Smith, The effects of genetic selection for increased growth rate on mucosal and muscle weights in the different regions of the small intestine of the domestic fowl {Gallus domesticus). Comp. Biochem. Physiol. 99A: Mobbs, I.G., and DJB. McMillan, Transport across endodermal cells of the chick yolk sac during early stages of development. Amer. J. Anat. 160: Moran, E.T., Jr., Digestion and absorption of carbohydrates in fowl and events through perinatal development. J. Nutr. 115: Moran, E.T., Jr., Effect of egg weight, glucose administration at hatch, and delayed access to feed and water on the poult at 2 weeks of age. Poultry Sci. 69: Moran, E.T., Jr., and B.S. Reinhart, Poult yolk sac amount and composition upon placement: effect of breeder age, egg weight, sex and subsequent change with feeding or fasting. Poultry Sci. 59: Moran, E.T., Jr., and B.S. Reinhart, Breeder flock productivity and egg size effects on broiler turkey performance and carcass quality. Poultry Sci. 60: Morris, G_F., and RD. Hodges, Regulation of proliferating cell nuclear antigen during the cell cycle. J. Biol. Chem. 246: Mussehl, F.E., and C.W. Ackerson, Some observations on humidity and weight loss in the incubation of turkey eggs. Nebraska Agri. Exp. Sta. Res. Bull. p. 74. Nestor, K.E., Genetics o f growth and reproduction in the turkey. 9. Long-term selection for increased 16-week body weight. Poultry Sci. 63:

180 Nestor, K.E., Egg production consequences of improving growth and efisciency in turkeys. Pages /«Eoultry Genetics and Breeding. Edit. W.G. Hill, J.M. Manson, and D. Hewitt. British Poultry Science, Ltd. Nestor, K.E., and D O. Noble, Influence of selection for increased egg production, body weight, and shank width of turkeys on egg composition and the relationship of the egg traits to hatchability. Poultry Sci. 74: Nitsan, Z, G. Ben-Avraham, Z. Zoref, and I. Nir, Growth and development of the digestive organs and some enzymes in broiler chicks after hatching. Brit. Poultry Sci. 32: Nitsan, Z, E.A. Dunnington, and P.B. Seigel, Organ growth and digestive enzyme levels to fifteen days of age in lines of chickens differing in body weight. Poultry Sci. 70: Noble, D O., D A Emmerson, and K.E. Nestor, The stability of three randombred control lines of turkeys. Poultry Sci. 74: Noble, R.C., Lipid metabolism in the chick embryo: Some recent ideas. J. Exp. Zool. l(suppl.): Noble, R.C., and M Cocchi, Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29: Noble, R.C., and K. Connor, Lipid metabolism in the chick embryo of the domestic fowl {Gallus domesticus). World s Poultry Sci. J. 40: Noble, R.C., and K. Connor, The synthesis and accumulation of cholesteryl esters by the developing embryos of the domestic fowl. Poultry Sci. 63: Noble, R.C., and J.H. Moore, The partition of lipids between the yolk and yolk-sac membrane during the development of the chick embryo. Can. J. Biochem. 45: Noble, R.C., F. Lonsdale, K. Connor, and D. Brown, Changes in the lipid metabolism of the chick embryo with parental age. Poultry Sci. 65: Noble, R.C., J.H. Shand, and K. Connor, Effects of parental age on fatty acid oxidation in vitro by the liver and heart of the chick embryo. Proc. Nutr. Soc. 45:103A. Noy, Y., and D. Sklan, Digestion and absorption in the young chick. Poultry Sci. 74:

181 Noy, Y., Z. Uni, and D. Sklan, Routes of yolk utilization in the newly-hatched chick. Brit. Poultry Sci. 37: Noy, Y, and D. Sklan, Posthatch development in poultry. J. Appl. Poultry Res. 6: Obst, B.S., and J. Diamond, Ontogenesis of intestinal nutrient transporters in domestic chickens {Galltts gallus) and its relation to growth. Auk 109: Odle, J., R.T. Zijlstra, and S.M. Donavan, Intestinal effects of milkbome growth factors in neonates of agricultural importance. J. Anim. Sci. 74: O Sullivan, N.P., E.E. Dunnington, and P.B. Seigel, Relationships among age of dam, egg components, embryo lipid transfer, and hatchability of broiler breeder eggs. Poultry Sci. 70: Overton, J., and J. Shoup, Fine structures of cell surface specialization in the maturing duodenal mucosa o f the chick. J. Cell Biol. 21: de Pablo, F., L A. Scott, and J. Roth, Insulin and insulin-like growth factor I in early development: peptides, receptors and biological events. Endocrine Rev. 11: Payne, L., P.B. Siegel, and L. Ortman, Correlation of dam, egg, poult, and adult weights in Broad Breasted Bronze turkeys. Poultry Sci. 36: Pearce, J., Some differences between avian and mammalian biochemistry. Intl. J. Biochem. 8: Phelps, P.V., F.W. Edens, and R.P. Gildersleeve, The posthatch physiology of the turkey poult. I. Growth and development. Comp. Biochem. Physiol. 87A: Phelps, P.V., F.W. Edens, and R.P. Gildersleeve, The posthatch physiology of the turkey poult, m. Yolk depletion and serum metabolites. Comp. Biochem. Physiol. 87A: Pinchasov, Y, and Y. Noy, Early postnatal amylolysis in the gastrointestinal tract of \nrk&y poults Meleagris gallapavo. Comp. Biochem. Physiol. 107A: Rahn, H., V.L. Christensen, and F.W. Edens, Changes in shell conductance, pores, and physical dimensions of egg and shell during the first breeding cycle of turkey hens. Poultry Sci. 60:

182 Reidy, R.R., J.L. Atkmson,k and S. Leeson, components. Poultry Sci. 73: Strain comparisons of turkey egg Reinhart, B.S., and E.T. Moran, Jr., Incubation characteristics of eggs from older small white turkeys with emphasis on the effects due to egg weight. Poultry Sci. 58: Renema, R.A., F.E. Robinson, V.L. Melnychuk, and R.T. Hardin, The use of feed restriction for improving reproductive traits in male-line Large White turkey hens. 2. Ovary morphology and laying traits. Poultry Sci. 74: Renema, R.A., V.L. Melnchuk, F.E. Robinson, H.L. Classen, and R.D. Crawford, Reproductive organ morphology and carcass traits in unselected naturally mating female Bronze turkeys at onset of lay. Can. J. Anim. Sci. 78: Rhymer, LM., The effect of egg size variablility on thermoregulation of Mallard (Anas platyrhynchos) offspring and tis implications for survival. Oecologia 75: Ricklefs, R.E., J.M. Starck, and M. Konarzewski, Internal constraints on grovrth in birds. Pages in: Avian Growth and Development: Evolution within the Altricial- Precocial Spectrum. J.M. Starck and R.E. Ricklefs, Edit. Oxford University Press, New York, NY. Rob el, E.J., Weight class as related to body composition in agin white turkey hens. Poultry Sci. 63: Romanoff, A.L., Avian spare yolk and its assimilation. Auk 61: Romanoff, A.L., The Avian Embryo. MacMillan Co., New York, NY. Structural and Functional Development. Romanoff, A.L., Biochemistry of the Avian Embryo. Interscience Publishers, New York, NY. Roseborough, R.W., E. Geis, K. Henderson, and L.T. Frobish, Control o f glycogen metabolism in the developing turkey poult. Growth 43: Sanders, E.J., M. Varedi, and A.S. French, Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization. Develop. 118: SAS institute, SAS User s Guide: Basics. Version 5 Edition. SAS Institute, Cary, NC. 162

183 SAS Institute, SAS User s Guide: Statistics Ediition. SAS Institute Inc.,' Cary, NC. Scavo, L., J. Alemany, J. Roth, and F. de Pabio, Insulin-Iike growth factor I activity is stored in the yolk of the avian egg. Biochem. Biophys. Res. Comm. 162: Sell, J.L., Physiological limitations and potential for improvement in gastrointestinal tract function o f poultry. J. Appl. Poultry Res. 5: Sell, J.L., C.R. Angel, F.J. Piquer, E.G. Mallarino, and H.A. Al-Batshan, Developmental patterns of selected characteristics of the gastrointestinal tract of young turkeys. Poultry Sci. 70: Sexten, W.E., and M.G. McCartney, The effect of age at lighting on reproduction in the turkey hen. Poultry Sci. 52: Shanawany, M.M, Inter-relationship between egg weight, parental age and embryonic development. Br. Poultry Sci. 25: Shanawany, M.M., Hatching weight in relation to egg weight in domestic birds. World s Poultry Sci. J. 43: Sharp, P.J., Photoperiodic control of reproduction in the domestic hen. Poultry Sci. 72: Shehata, A.T., J. Lemer, and D.S. Miller, Development of nutrient transporter systems in chick jejunum. Amer. J. Physiol. 246:G101-G107. Shofi&ier, R.N., C.R. Polley, R.E., Burger, and E.L. Johnson, Light regulation in turkey management. 2. Female reproductive performance. Poultry Sci. 41: Siopes, T.D., Effects of age at lighting on reproduction of turkey hens. Poultry Sci. 71: Siopes, T.D., Turkey breeder hen performance by strain during consecutive lay periods. Poultry Sci. 74: Skewes, P.A., H R. Wilson, and F.B. Mather, Correlation among egg weight, chick weight, and yolk sac weight in Bobwhite quail {Colirms virginicanis). Florida Sci. 51: Smith, K.P., and B.B. Bohren, Age of pullet effects on hatching time, egg weight and hatchability. Poultry Sci. 54:

184 Smith, M.W., and M.A. Peacock, Comparative aspects of microvillus development in avian and mammalian enterocytes. Comp. Biochem. Physiol. 93 A: SoUer, M., Y. Eitan, and T. Brody, Effect of diet and early quantitative feed restriction on the minimum weight requirement for onset of sexual maturity in White Rock broiler breeders. Poultry Sci. 63: Starck, J.M., Intestinal growth in altricial European starling (Sturm s vulgaris) and precocial Japanese quail (Cotumix cotumix japonica). A morphometric and cytokinetic study. Acta Anatom. 156: Starck, J.M., Structural variants and invariants in avian embryonic and postnatal development. Pages in: Avian Growth and Development: Evolution within the Altricial-Precocial Spectrum. J.M. Starck and R E. Ricklefs edit. Oxford University Press, Inc., New York, NY. Turner, K.A, The effect of feeding high carbohydrate and fat diets following a 2 day fast upon blood metabolites and liver status in nevdy-hatched turkey poults. M.S. Thesis, The Ohio State University/0.A.R.D.C. Turner, K.A, The effect of reproduction phase of breeder hens, poult hatch time, and fatty acid supplementation of the breeder hen diet on livability, growth characteristics, and yolk and liver fatty acid composition in posthatch poults. Doctor of Philosophy Dissertation, Iowa State University Library, Ames, la Uni, Z., Y. Noy, and D. Sklan, Posthatch changes in morphology and function of the small intestines in heavy- and light-strain chicks. Poultry Sci. 74: Uni, Z., Y. Noy, and D. Sklan, Development of the small intestine in heavy and light strain chicks before and after hatching. Brit. Poultry Sci. 36: Uni, Z. S. Ganot, and D. Sklan, Posthatch development of mucosal function in the broiler small intestine. Poultry Sci. 77: Uni, Z., R. Platin, and D. Sklan, Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. J. Comp. Physiol. B 168: Vajda, K., J.H. Shand, S.W. West, B.K. Speake, and R.C. Noble, Cholesterol metabolism in the livers of chick embyros produced from immature birds. Biochem. Soc. Trans. 431S:22. Walzem, R.L., Lipoproteins and the laying hen: form follows function. Poultry Avian Biol. Rev. 7:

185 Wilson, H.R., Interrelationships of egg size, chick size, posthatching growth and hatchability. World s Poultry Sci. J. 47:5-20. Wilson, W.O., F.X. Ogasawara, and V S. Asmundson, Artificial control of egg production in turkeys by photoperiod. Poultry Sci. 41: Wineland, M.J., G.S. Davis, V.L. Christensen, and M. Jeffrey, Effect of reduced incubator ventilation upon the duck embryo. Poultry Sci. 76(Suppl. 1):91. Woodard, AE., H. Abplanalp, V. Stinnett, and R.L. Snyder, The effect of age at lighting on egg production and pausing in turkey hens. Poultry Sci. 53: Yaffei, N., and R.C. Noble, Further observations on the association between lipid metabolism and low embryo hatchability in eggs from young broiler birds. J. Exp. Zool. 253: Yannakopoulos, AL., The relationship between egg weigh and shell quality on poult hatching weight. Zootech. Intl. 3: Yannakopoulos, A.L., and A.S. Tserveni-Gousi, Research Note: Effect of breeder quail age and egg weight on chick weight. Poultry Sci. 66:

186 APPENDIX TURKEY AND DUCK COMPARISONS 166

187 Specie Egg' YoUe Albumen (g) (g) (%) (g) (%) Duck Turkey SEM Effect of species ^ Duck; 85 to 92 g eggs from 32- and 44-wk-old hens, n=565; Turkey: 85 to 90 g eggs from 34- and 44-wk-oId hens, n=400. Duck yolk and albumen means represent 25 eggs; Turkey yolk and albumen means represent 10 eggs. Table A. 1. Duck and turkey egg weight and egg component comparisons. 167

188 Days relative to hatch Specie Cg) Duck 20.31' Turkey SEM Effect of species ^ Duck: 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=10. ^ Duckling yolk sac weight was negligible. Table A.2. Duckling and poult yolk sac weight comparisons 168

189 Days relative to hatch Specie \ VO Duck 35, Turkey SEM 0, f n \ 18) Effect of species Duck: 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=10, Table A.3, Duckling and poult body weight (devoid of the yolk sac) comparisons.

190 Figure A. 1. Duckling (line) and poult (dashed line) duodenum weight, length, and density (g/cm) comparisons. Duck; 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=10. * Difference between species is significant (P < 0.05). Error bars indicate SEM. 170

191 4 Duodenum weight, g Duodenum length, cm Duodenum density, g/cm 0.15 O.IO I 2 3 Days relative to hatch Duck 5 7 Turkey 171 Figure A. 1

192 Days relative to hatch Specie 0 (g/100 g body weight) Duck 0.66' Turkey SEM Efrect of species Duck: 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=10. Table A.4. Duckling and poult relative duodenum weight comparisons. 172

193 r ' 5 C5 SS iiuui jz - ana 4 4 -wk-oid hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=lo. * Diflference between species is significant (P < 0.05). Error bars indicate SEM 173

194 18 15 JejumimÆeum weight, g * * * * * * T 120 Jejunum/ileum length, cm Jejunnm/ileum density, g/cm Days relative to hatch Duck Turkey 174 Figure A.2

195 Days relative to hatch Specie ikjkj g Doay weigni^ Duck 2.62' Turkey SEM Effect of species ^ Duck: 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 44-wk-old hens, n=10. Table A.5. Duckling and poult relative jegunum/ileum weight comparisons. 175

196 Days relative to hatch Variable Specie Yolk-free BW Duck Turkey SEM Effect of species Villus height (pm) Duck Turkey SEM Effect of species Crypt depth tpmj Duck Turkey SEM Effect o f species ^ Duck: 85 to 92 g eggs from 32- and 44-wk-old hens, n=25; Turkey: 85 to 90 g eggs from 34- and 48-wk-oId hens, n=10. Table A.6. Duckling and poult body weight (BW, devoid of yolk sac), distal jejunum villus height (villus tip to crypt), and crypt depth (depth of the invagination between adjacent villi) comparisons. 176

197 Figure A.3. Duckling and poult distal jejunum villi comparison at 3 d of age. Sections were 5 pm thick and were stained with Hematoxylin and Eosin. Magnification = loox 177

198 Distal Jejunum - Day 3 i à 00 ft w Turkey Duck