HIGH FIBER LOW ENERGY DIET FOR MOLT INDUCTION IN LAYING HENS: THE IMPACT OF ALFALFA ON PHYSIOLOGY, IMMUNOLOGY AND BEHAVIOR.

Transcription

1 HIGH FIBER LOW ENERGY DIET FOR MOLT INDUCTION IN LAYING HENS: THE IMPACT OF ALFALFA ON PHYSIOLOGY, IMMUNOLOGY AND BEHAVIOR A Disserttion y CLAUDIA SHARENE DUNKLEY Sumitted to the Office of Grdute Studies of Texs A&M University in prtil fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Decemer 2006 Mjor Suject: Poultry Science

2 HIGH FIBER LOW ENERGY DIET FOR MOLT INDUCTION IN LAYING HENS: THE IMPACT OF ALFALFA ON PHYSIOLOGY, IMMUNOLOGY AND BEHAVIOR A Disserttion y CLAUDIA SHARENE DUNKLEY Sumitted to the Office of Grdute Studies of Texs A&M University in prtil fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved y: Co-Chirs of Committee, Steve C. Ricke Luc R. Berghmn Committee Memers, Ted Friend Leon F. Kuen Jckson L. McReynolds Hed of Deprtment, Aln Sms Decemer 2006 Mjor Suject: Poultry Science

3 iii ABSTRACT High Fier Low Energy Diet for Molt Induction in Lying Hens: The Impct of Alflf on Physiology, Immunology nd Behvior. (Decemer 2006) Cludi Shrene Dunkley, B.S. Pririe View A&M University; M.S., Pririe View A&M University Co-Chirs of Advisory Committee: Dr. Steven C. Ricke Dr. Luc R. Berghmn Feed withdrwl is commonly used y commercil egg producers to induce molt nd stimulte multiple egg-lying cycles in their flocks. However, the prctice cn compromise the welfre of the irds y elevting stress, suppressing the immune response nd cusing norml ehvior. An lternte molt diet ws exmined using lflf diets, nd series of experiments were conducted to evlute the physiologicl, immunologicl nd ehviorl responses of lying hens fed these diets. We ssessed the chnges in the levels of lood plsm metolites fter hens were chllenged with 10 6 colony forming units of Slmonell Enteritidis (SE). Hens fed lflf diets displyed similr (P 0.05) levels of cholesterol, glucose, nd totl protein when compred to fullfed hens. Reduced levels (P 0.05) of triglycerides were oserved in the lflf fed nd feed withdrwl hens when compred to the full-fed hens. The immune responses of SE chllenged lying hens fed lflf diets showed similr (P 0.05) heterophil to lymphocyte rtios (H: L) to full-fed hens wheres the feed withdrwl hens displyed

4 iv elevted (P 0.05) H: L rtios. The feed withdrwl hens displyed reduced (P 0.05) levels of serum IgY erly in the tril when compred to the lflf nd full-fed hens. The lflf fed hens displyed lower levels of cid α glycol protein thn the feed withdrwl hens nd higher levels thn the full-fed erly during the tril ut returned to levels tht were similr (P 0.05) to the full-fed hens. The hens fed lflf diets displyed elevted non-nutritive pecking ehvior erly in the tril however, this ehvior declined to levels similr (P 0.05) to the full-fed hens y the end of the tril. No differences in ggressive ehvior were oserved etween the lflf fed hens nd the full-fed hens. Hfnium chloride did not effectively mrk the lflf diet; however, it cn e used to trck the pssge of corn-soy lyer rtion. This reserch suggests tht the ppliction of lflf s n lterntive molt diet cn e effective in reducing potentilly hrmful effects which re usully ssocited with feed withdrwl.

5 v DEDICATION I thnk God lmighty for crrying me through. I dedicte this disserttion to my sons, Kingsley Jr., Nichols, Khori nd Ricrdo. You hve ll een there with me through the highs nd the lows nd hve endured lot in order for me to complete this journey. Kingsley Sr., my husnd, we emrked on this journey together, for ll the times I felt like giving up you were lwys there to encourge me, you re my prtner in every wy. I lso dedicte this disserttion to my numerous fmily memers especilly my prents, Clrence nd Sylvi Powell, my Aunt Lucille, my rothers Dune nd Dougls nd sister in lw, Kren, my Uncle Mushe nd Aunt Crron; your constnt encourgement, pryers nd monetry support were invlule.

6 vi ACKNOWLEDGEMENTS I would like to express my sincere grtitude to Dr. Steven Ricke who took the chllenge to guide me through this progrm. I thnk Dr. Ricke for extending to me his wisdom nd expertise in reserch, for exposing me to mny res of reserch nd giving me experience tht I would not hve received elsewhere. I ws fortunte to hve hd the group of tlented scientists who served on my committee, nd I would like to thnk ech memer. My co-chir Dr. Luc Berghmn constntly encourged me nd ws lwys redy to point me in the right direction. Dr. Ted Friend ws lwys redy to help nd offer whtever resources he hd tht I needed. Dr. Leon Kuen constntly encourged me when I thought there ws no hope nd llowed me to conduct my reserch in his ls. Lst ut y no mens lest, Dr. Jckson McReynolds guided me nd hs tught me lessons I will not soon forget. I would lso like to cknowledge Dr. Victor Stnley who encourged me to strt PhD nd never left my side throughout the progrm. Thnks to Dr. Allen Byrd, Mrs. Cssie Woodwrd, Mrs. Denise Cldwell, Mr. Alert Blnks nd Mr. Erl Munson who ssisted me with experiments. Thnks to Dr. Willim Jmes nd Mr. Michel Rulerson who help me with the chrcteriztion of the rre erth metls. Specil thnks to Lynd Njongmet. She ws lwys redy to help nd encourge me; you re n inspirtion!

7 vii TABLE OF CONTENTS Pge ABSTRACT... DEDICATION... ACKNOWLEDGEMENTS... TABLE OF CONTENTS... LIST OF FIGURES... LIST OF TABLES... iii v vi vii ix xii CHAPTER I INTRODUCTION... 1 II LITERATURE REVIEW... 4 Molting in the Avin Species... 4 Control of Reproduction in Chickens... 5 Molting in the Commercil Egg Industry Animl Welfre nd Molting Immune Response during Molting The Effects of Molting on Slmonell enteric serovr Enteritidis Infection in Lying Hens Alterntive Methods of Inducing Molt High Fier Low Energy Diets Potentil of Alflf s Molting Diet III BLOOD PLASMA METABOLITE RESPONSE IN MOLTING LAYING HENS CHALLENGED WITH SALMONELLA ENTERITIDIS AND FED ALFALFA CRUMBLE DIETS Introduction Mterils nd Methods Results nd Discussion... 46

8 viii CHAPTER IV Pge IMMUNE AND STRESS PROTEIN RESPONSE IN MOLTING LAYING HENS CHALLENGED WITH SALMONELLA ENTERITIDIS AND FED ALFALFA CRUMBLE DIETS Introduction Mterils nd Methods Results nd Discussion V BEHAVIOR OF LAYING HENS ON ALFALFA CRUMBLE MOLT DIETS Introduction Mterils nd Methods Results nd Discussion VI LAYING HENS BEHAVIOR TO DIFFERENT ALFALFA-LAYER RATION COMBINATIONS FED DURING MOLTING Introduction Mterils nd Methods Results nd Discussions VII APPLICATION OF HAFNIUM CHLORIDE AS A MARKER TO ASSESS THE PASSAGE RATE OF LAYER RATION AND ALFALFA DIETS Introduction Mterils nd Methods Results nd Discussions VIII SUMMARY AND CONCLUSIONS REFERENCES VITA

9 ix LIST OF FIGURES FIGURE Pge 3.1 Effects of lflf on lood clcium levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril Effects of lflf on lood totl protein levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril Effects of lflf on lood triglycerides levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril Effects of lflf on lood cholesterol levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril Effects of lflf on lood glucose levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition tril 1 nd tril Effects of lflf on lood uric cid levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril The effects of lflf diets on heterophil: lymphocyte (H: L) rtios in Slmonell Enteritidis (SE) chllenged lying hens during molt in tril 1 nd tril The effects of lflf diets on serum ntiody production in Slmonell Enteritidis (SE) chllenged lying hens during molt in tril 1 nd tril Serum α 1 -cid glycoprotein (AGP) levels during n induced molt of Slmonell Enteritidis (SE) chllenged lying hens fed n lflf diet in tril 1 nd tril Hen dy egg production y the three tretments on dily sis from 2 wk prior molt induction nd during the 9 dy tril Percentge oservtions of hed movement ehvior of lying hens during 9 dy induced molt Percentge oservtions of wlking ehvior of lying hens during 9 dy induced molt Percentge oservtions of preening ehvior of lying hens during 9 dy induced molt

10 x FIGURE Pge 5.5 Percentge oservtions of drinking ehvior of lying hens during 9 dy induced molt Percentge oservtions of nonnutritive pecking ehvior of lying hens during 9 dy induced molt Percentge oservtions of feeder ehvior of lying hens during 9 dy induced molt Hen dy egg production y the five tretments on dily sis from 2 wk prior molt induction nd during the 9 dy tril Percentge oservtions of wlking ehvior of lying hens during 9 dy induced molt Percentge oservtions of preening ehvior of lying hens during 9 dy induced molt Percentge oservtions of hed movement ehvior of lying hens during 9 dy induced molt Percentge oservtions of drinking ehvior of lying hens during 9 dy induced molt Percentge oservtions of nonnutritive pecking ehvior of lying hens during 9 dy induced molt Percentge oservtions of feeder ehvior of lying hens during 9 dy induced molt Picture of the hens gstrointestinl trct. Ingest were removed from the Crop, Gizzrd, Ileun, Jejunum, nd Cec Plot of hfnium concentrtions in feces from the individul hens, s function of time fter dministrtion of corn-soy sed lyer rtion mrked mel Plot of hfnium concentrtions in feces from the hens, s function of time fter dministrtion of corn-soy sed lyer rtion mrked mel Concentrtion of the Hf mrker present in the lflf 100 diet in different segments of the hens digestive trct t 2 hours nd 7 hours post-dose

11 xi FIGURE Pge 7.5 Concentrtion of the Hf mrker present in the lflf 90 diet in different segments of the hens digestive trct t 2 hours nd 7 hours post-dose Concentrtion of the Hf mrker present in the lyer rtion in different segments of the hens digestive trct t 2 hours nd 7 hours post-dose

12 xii LIST OF TABLES TABLE Pge 3.1 Effects of lflf crumle diet on feed intke nd weight loss in lying hens experimentlly infected with Slmonell Enteritidis (SE) trils 1 nd The effects of lflf diet on intestinl nd ile IgA secretion in Slmonell Enteritidis (SE) chllenged lying hens during molt trils 1 nd Hfnium detection limits in smpled mtrices

13 1 CHAPTER I INTRODUCTION Birds undergo series of molts during their life spn nd will chnge t lest four different plumges from htching to their first nnul cycle (Lucs nd Stettenheim, 1972). At the end of lying cycle egg production nd qulity decline significntly, for commercil egg producers this will men period of unproductivity ecuse the molt is incomplete in commercil lying hens nd they continue to ly eggs t low rtes for prolonged period of time (Berry, 2003). Historiclly, commercil egg producers in the US will seek to otin more uniform molt within the flock nd they hve used feed removl s mens of inducing molt (Bell, 2003). Conventionl feed withdrwl progrms involves the removl of feed, wter (no more thn 3 dys) or oth from the hens nd lso reducing the photo-period in the houses to 8 h lighted period or less (Bell, 2003). After the induced molt, egg production nd qulity improve significntly compred to the pre-molt period. Commercil egg producers in the US typiclly implement induced molt progrms when lying hens re out 65 wk of ge so tht the irds cn e kept through second lying cycle. Feed withdrwl cuses hens to cese egg production nd results in the regression of the reproductive system. When the hens re stimulted to return to production, the rejuvented trct produces eggs with etter shell nd interior qulity nd t higher rte thn hd een the cse efore they were molted (Wester, 2000). This disserttion follows the style nd formt of Poultry Science.

14 2 Induced molt y feed withdrwl compromise the irds immune system mking them susceptile to infection y numer of microorgnisms (Holt, 2003; Ricke, 2003). Of prticulr interest is Slmonell enteric serovr Enteritidis (SE). USDA-Animl nd Plnt Helth Inspection Service (2000) stted tht there ws douling of environmentl SE numers in molted versus non-molted flocks. Slmonell infection in lying hens is serious food sfety concern, since infection of ird could led to the production of SE positive eggs fter molt (Humphrey et l., 1993). Among the investigted mechnisms tht re known to cuse decresed resistnce include ltertions in the intestinl microflor (Corrier et l., 1997; Holt nd Porter, 1992; Svge, 1989), intestinl physiology (Brnes et l., 1979; Corrier et l., 1997; Duke, 1986; Holt, 1993), nd host defenses (Ben-Nthn et l., 1981, 1977; Holt, 1992, 1993). The prctice of feed withdrwl hs lso een chrcterized s stressful nd frustrting (Duncn nd Wood- Gush, 1971). Dwkins (1999) stted tht the two components of niml welfre re physiologicl nd psychologicl helth. While the physiologicl helth of the irds cn e determined sed on physicl ppernce, the psychologicl helth is more difficult to ssess. Wester (2000) postulted tht ehvior of irds might give direct indiction of the well eing of hens undergoing feed withdrwl. Incresed concerns of reduced niml welfre during n induced molt y feed deprivtion hd prompted investigtions into lterntive methods. Attempts hve een mde to incorporte nutritionl imlnces, ingredients tht decrese ppetite, ddition of fillers or dministrtion of hormones to induce molts (Bell, 2003; Holt 2003; Prk et l., 2004) s mens of inducing molt. The incorportion of high fier low energy diets

15 3 to induce molt hs een suggested ecuse such diets re usully low in metolizle energy nd high fier diets will give the niml feeling of stiety resulting in reduced feed intke (Rijnen et l., 1999). Alflf hs the potentil to serve s dietry source for inducing molt, it is high in crude protein (17.5%) nd crude fier (24.1%) nd hs low level of metolizle energy of out 1200 kcl/kg when compred to lyer rtion which hs metolizle energy of 2,965 kcl/kg (NRC, 1994). It hs the slowest pssge through the gstrointestinl trct of the chicken (Sild, 1979, 1980) nd high concentrtions in the diet of lying hens decreses growth rte nd reduce egg production (Heywng, 1950). Alflf diets cn effectively induce molt resulting in second lying cycle similr to hens molted y feed deprivtion (Donlson et l., 2005) nd reduce SE coloniztion nd shedding in the gstrointestinl trct nd internl orgns of lying hens during n induced molt (Woodwrd et l., 2005; McReynolds et l., 2005, 2006). In order to complete the investigtion into the potentil of lflf s n lternte molt diet, it is necessry to exmine the physiologicl nd psychologicl effects of the diet in the lying hens during molt. The generl ojectives of these experiments were first to evlute the chnges in the physiologicl chnges in hens fed n lflf diet during n induced molt fter they were infected with SE. Second to evlute the immunologicl chnges in hens fed n lflf diet during n induced molt fter they were infected with SE. Third to ssess the ehvior of hens during n induced molt while they were fed different comintions of lflf diets. Lstly, to exmine the pssge of the lflf through the gstrointestinl trct of lying hens.

16 4 CHAPTER II LITERATURE REVIEW Molting in the Avin Species Molting in the vin species involves the periodic shedding nd replcement of fethers. It lso involves the involution of the hens reproductive system resulting in reproductive quiescence. During their nturl lifespn, irds will undergo series of different molts. From htching up to their first nnul cycle, irds will chnge t lest four different plumges including; ntl down, juvenile, lternte nd sic plumges (Lucs nd Stettenheim, 1972). Generlly speking, mny vin species will hve two molts per yer, pre-nuptil or pre-lternte molt nd post-nuptil or post-lternte molt. The post-nuptil molt is ssocited with the reproductive quiescence. It is more complete molt tht follows reeding or lying cycle nd involves the loss of fethers from the wings, til, nd ody of the ird. Molting is usully considered to e more thn simply plumge replcement since it lso involves physiologicl chnges in the ird (Stettenheim, 1972). These physiologicl chnges include vsculriztion of the fether follicles nd ppille (Stettenheim, 1972), osteoporosis (Meister, 1951; Murphy, 1996). Frctionl protein synthesis rte, not only for fether kertin synthesis rte, ut primrily for skeletl muscle synthesis (Murphy nd Truscio, 1995; Murphy, 1996) re lso chnges tht occur during molting. Others hve reported chnges in metolic rte, reduction in ody ft (Kuenzel nd Helms, 1974), nd incresed heterophil: lymphocyte rtio (Gross nd Siegel, 1983; Dvis et l., 2000) during molt.

17 5 The post-nuptil molt period is criticl period in the ird s cycle s it involves mjor remodeling of the ody nd lso the resetting of the neurl system in order to respond to photo-stimultion (Kuenzel, 2003). A vriety hormones nd neuropeptides re involved in the remodeling process. One function of vso-ctive intestinl polypeptide is to shut down the reproductive system y inititing the incution ehvior such s roodiness which is usully followed y post-nuptil molt (Kuenzel, 2003). In wild irds, the primry inititing fctor of molting is the estlishment of roodiness. After the onset of this roodiness, the hen will undergo the involution of the reproductive system, followed y cesstion of ly, nd finlly the shedding nd replcement of fethers (Sherry et l., 1980). During this time, the ird will voluntry reduce their intke of food. This is known s spontneous norexi (Mrosovsky nd Sherry, 1980). A hen cn lose up to 20% of its ody weight during spontneous norexi; one hlf of this lost weight is from the involution of the reproductive trct. After the chicks hve htched, the hens will once gin egin to consume feed nd the fethers will e replced. This process is nturl phenomenon in species tht live in environments where incution nd feeding is incomptile (Sherry et l., 1980). Control of Reproduction in Chickens Photoperiod Most irds re essentilly sesonl reeders especilly in temperte nd northern ltitudes irds reed under the influence of lengthening dys (Etches, 1996). Reproduction in irds is controlled y interctions with light schedule, feeding nd stress. Chnges in response to photoperiod re of fundmentl importnce to sesonl

18 6 cycles in vin reproductive development, they must e le to recognize the chnges in dy length in order to differentite etween long nd short dys. For this purpose, irds utilize internl circdin rhythms. The period during which ird is sensitive to light is clled the photosensitive phse nd this predicts the effects of photo-schedules. The importnce of light intensity in photoperiodic stimultion of gondotropin secretion is uncler however in studies where light intensity ws mesured the rte of egg production ws proportionl to intensity etween 0.2 nd 5 lux (Shrp et l., 1987). Short dys (SD; photophse 8 h) do not illuminte the photosensitive phse, however, long dys (LD; photophse 13 h) illumintes it nd susequently initites Luetinizing hormone (LH) secretion (Etches, 1996). In irds photo-stimultion induces n increse in plsm LH nd lso follicle-stimulting hormone (FSH) (Gooden nd Scnes, 1977; Bcon nd Long, 1995). In most vertertes, the chnging dy length is perceived nd trnsduced into ctivtion of the hypothlmo- pituitry gondotrophic (HPG) xis (Bentley et l., 2003; Follet, 1984; Nicholls et l., 1988). A stimultory photo-schedule must trnsmit two signls to the hen; first she sets her circdin clock t dwn, eleven hours fter the circdin clock strts it signls the photosensitive phse. The second signl is then trnsmitted during this phse when the exposure to light is registered y the hypothlmic photoreceptors (Etches, 1996). After period of 12 to 15 months of ly, the effects photo-periodism egins to decline nd irds cnnot mintin mximl levels of gondotropin secretions. This is referred to s photorefrctoriness nd the development of solute photorefrctoriness usully leds to the termintion of sesonl reeding in most irds. This is usully

19 7 followed y post- nuptil molt (Follett, 1984; Nicholls, 1988). Photo refrctoriness is considered to cuse molt or is t lest necessry for it s induction (Frner et l.,1980). The dissiption of photorefrctoriness, thus the re-setting of the hypothlmus cn e otined y inducing molt, therey mking the hypothlmus le to receive nd trnsmit photoperiodic signls t higher level (Etches, 1996). Photorefrctoriness, cuses the GnRH cell odies in the rin to shrink nd the fiers emnting towrds the medin eminence (ME) to decrese (Foster et l., 1987; Goldsmith et l., 1989; Boulkoud nd Goldsmith, 1991). As result, LH nd FHS relese will e reduced (Dwson nd Goldsmith, 1982, 1983) leding to gondl regression. Role of the Hypothlmus Light is perceived through photoreceptors tht trnsduce energy contined in photons into iologicl signls. For reproduction in the vin species the perception of light does not depend on the photoreceptors in the eyes ut on the photoreceptors in the hypothlmus. These photoreceptors re iologicl trnsducers tht convert the photon energy into neuron energy which re mplified y the endocrine system to control the function of the gonds which in turn influence the reproductive function, ehvior nd secondry sexul chrcteristics (Etches, 1996). A reduction in GnRH neurons, re involved in the development of photo refrctoriness (Bluhm et l., 1991; Hhn nd Bll, 1995), this results in decrese in gondotropin secretions. Gondotropic function nd synthesis re regulted y hypothlmic peptides. The relese of reproductive the hormones is controlled y relesing hormones t the hypothlmo-pituitry-xis of the rin. Short-term feed restriction hs shown effects on

20 8 the reproductive xis in lying hens. Verheyen et l. (1987) oserved tht cute feed restriction reduced plsm progesterone concentrtion. Due to feedck mechnism, the reduction of these hormones cn led to reduction in plsm concentrtion of luteinizing hormone (LH) nd n increse in follicle stimulting hormone (FSH) (Johnson nd vntienhoven, 1980; Tne et l., 1981; Decuypere et l., 1992) Prolctin (PRL) secretion is photo-induced nd reserch hs demonstrted tht it ply mjor role in molting (Juhn nd Hrris, 1958; Millm nd Hlwni, 1986; Pyne, 1972). Prolctin is nti-gondotrophic/ gondl in irds (Cmper nd Burke, 1977; Buntin et l., 1988) nd therefore the increse in plsm PLR is involved in the development of photorefrctoriness nd the induction of post-nuptil molt. Vso-ctive intestinl polypeptide (VIP) hs een identified in the vin species s PRL relesing hormone (Shrp et l., 1989; Muro et l., 1989). Immuniztion ginst VIP is known to suppress photo-induced PRL secretion in turkeys (El Hlwni et l., 1996). Dwson nd Shrpe (1998) concluded tht it is possile tht VIP cn control gondl regression, hence molt, either directly through PRL or y some other mechnisms. One of the most importnt regultory peptide in the vin hypothlmus is chicken Luteinizing Hormone- Relesing Hormone (clhrh). Two types hve een oserved, clhrh-i nd clhrh-ii. Chicken Luteinizing Hormone- Relesing Hormone I (clhrh-i) ws first isolted from the chicken hypothlmic tissue y King nd Milr (1982). The presence of clhrh-i immunorective (ir) neurons throughout the septl pre-optic re nd the nterior hypothlmus, hve een oserved in severl vin studies (Millm et l., 1993; Vn Gils et l., 1993). Most of these neurons projected to

21 9 the ME nd it is here tht they relese their peptide contents into the hypophysil portl lood system, therey stimulting the relese of LH nd FSH from the pituitry. These gondotropes further stimulte the relese of steroid hormones such s estrogen nd progesterone, from the gonds which in turn estlish positive feedck mechnism to the hypothlmus. The functionlity of the clhrh system is estrogen-dependent. The relese of clhrh in oth mmmlin nd vin species is regulted y severl neuromodultors nd neuropeptides (Lernth et l., 1988; Merchenthler et l., 1990). These neuropeptides regulte the relese of clhrh t the terminls in the ME (Contijoch et l., 1992). Arginine vsotocin (AVT) hs een considered s fctor in the regultion of clhrh-i relese in irds. Locliztion of AVT in structures throughout the centrl nervous system of chicken hs een reveled y immunochemicl studies (Tennyson et l., 1986). D Hondt nd others (2000) postulted tht AVT should e considered, s n importnt regultor of clhrh-i relese, nd hence of reproductive function since there is co-locliztion of LHRH-I nd AVT in neurons throughout the pre-optic hypothlmic re. Severl in vivo nd in vitro studies hve shown tht oth peptides re eqully potent in relesing LH nd FSH (King et l., 1988; Millr nd King, 1984; Shrp et l., 1987; Wilson et l., 1989). Shrp nd others (1987) used hens nd cockerels to compre the response in LH plsm levels fter i.v. injection with clhrh-i or clhrh-ii. They found tht in hens, clhrh-ii ws more effective in relesing LH thn clhrh-i. In lter study Shrp et l. (1989) oserved clhrh-ii in lying hens, occurred t low levels in the rin nd they lso noted tht clhrh-ii could not e detected in the ME wheres,

22 10 clhrh-i occurred in undnce. In the sme study they oserved tht immuniztion ginst clhrh-i nd not clhrh-ii, resulted in complete regression of the reproductive system nd shrp decline in the plsm LH concentrtion. These results suggested tht the secretion of gondotropins in hens ws regulted mostly, if not exclusively y clhrh-i nd not clhrh-ii. Molting in the Commercil Egg Industry Economic Fctors Domestic hens like most species of wild irds experience nturlly occurring molt. This however, is usully incomplete nd hens continue to ly eggs t low rtes for prolonged period of time (North nd Bell, 1990). This usully indictes the end of the useful life of flock, which for one-cycle (non-molted) progrm is pproximtely 80 weeks old (Bell, 2003). At the first sign of molting commercil egg producers typiclly sell these hens nd replce them with new flock. Egg producers usully mke the decision to molt hens for second lying cycle sed on the cost of eggs which is pproximtely one cent more per dozen in the second cycle nd the cost of feed nd replcement pullets (North nd Bell, 1990). Replcement pullets re the most expensive costs for lyer opertion, therefore, commercil egg producers seek to extend the productive lying life of their flock for s long s it is profitle (Prkhurst nd Mountney, 1988), therefore n induced molt is implemented s prt of progrm to optimize the use of replcement pullets on lyer frms (Bell, 2003). However, if replcement pullets re inexpensive, or if egg prices re high nd the frmer hve egg

23 11 production quots, there will then e n incentive to produce t higher prices nd molting will then not e profitle (Bell, 2003). Another economic fctor tht cn influence the decision to molt is the declining vlue of Leghorn hens crcsses nd the lso the declining numer of processors willing to uy them (Wester, 1995; McDniels nd Aske, 2000). It is estimted tht progrm tht includes molting will result in t lest 30% higher profit mrgins thn n ll pullet progrm (Berry, 2003). It hs ecome n industry wide prctice nd it contriutes significntly to the commercil industry profits. Bsed upon $0.65 per yer per hen housed in two cycle progrm compred to $0.50 in one cycle progrm the curtilment of such prctice nd return to n ll-pullet progrm would result in the requirement of 47% more chicks per yer nd the removl of 47% more spent hens ech yer (Berry, 2003). Mngement of Artificil Molt Induction Over the yers, reserchers hve developed methods of rtificil molt induction to occur t times other thn t the time of nturl molting. These methods hve resulted in the cesstion of ly nd the loss of fethers. Historiclly, induced molting involved the removl of food; wter or oth feed nd wter. It lso involved reducing the photoperiod to 10 h or less. Hens would then e fsted for pre-set period of time to cuse the complete regression of the reproductive trct (Berry, 2003). It ws in the 1950 s tht egg producers in the United Sttes first dpted the prctice of induced molting. Egg producers t tht time used multiple-molting progrms in which hens were more thn once nd the lying cycles were shortened (37 weeks). The first molt ws n verge of

24 12 60 weeks nd the second molt ws induced t pproximtely 106 weeks. Clifornin producers were the first to dopt the prctice (Bell, 2003) which spred cross the United Sttes to the most mjor egg producing regions in the mid 1970 s. In 2000, the USDA stted tht 93% of the flocks in the Southest region of the US prctice force molting intensively (USDA, 2000). Bell (2003) stted tht pproximtely 73% of the commercil lying fcilities in the US implement the prctice of induced molting to rejuvente their flocks. The prctice is done to give the frmer mximum production from hens on the commercil lyer frms y enling them to hve second or even third lying cycle. Under optiml economic conditions the useful reproductive life of lying hens cn e extended from less thn 80 weeks to more thn 110 weeks or even 140 weeks (Bell, 2003; Wester, 1995) giving the producer second nd third lying cycles. Lee (1982) stted tht molting hd eneficil effects on the individul hen s performnce nd the prctice ws lso eneficil for the overll flock mngement since the hens were synchronized for the second lying cycle. If llowed to molt nturlly, hens would egin molting t different times nd this would prolong the process through the entire flock. Molted hens in this experiment hd higher hen-dy egg production thn the non-molted control hens however; the molted hens consumed more feed post-molt thn the control hens. Bell (2003) lso oserved improved post-molt performnce in molted hens compred to their non-molted counterprts with pek of egg production during the second cycle of pproximtely 75% to 85% 13 weeks into the second cycle. Nturlly during the periods of short dy length irds in the wild will experience weight loss long with fether loss nd the regression of the reproductive system (Brke

25 13 nd Thxton, 1979; Mrosovsky nd Sherry, 1980; Etches et l., 1996), during this time they will undergo period of nturl inppetence. In the commercil lyer industry, n induced molt will usully mimic these conditions which will occur nturlly. There re three sic wys y which molt is induced; feed removl or limittion, low-nutrient rtion, nd feed dditives. Ech of these methods usully involves the ltertion of the photoperiod from long dy to short dy. Whichever method is used should e esy to pply, low cost, result in low mortlity nd should result in enhnced performnce during the second cycle (Swnson nd Bell, 1975), the method should lso void the potentil feed refusl y the irds, e redily ville, e economicl with miniml feed processing, nd provide molt induction stimulus enough to cuse sufficient reproductive trct regression during the molt (Ricke, 2003). Feed Withdrwl Commercil egg producers in the US typiclly use feed removl s their method to induce molt. This ecuse feed withdrwl is esy to implement nd will chieve ody weight loss of 25 to 35% to otin the optimum post-molt performnce in terms of egg production nd shell qulity (Wester, 2003; Bker et l., 1983). Feed withdrwl (deprivtion) involves the removl of feed for period of 5-14 dys nd the my or my not include period of rest for up to 21 dy fter the fst (Bell nd Kuney, 1992). Conventionl feed withdrwl progrms involves the removl of feed, wter (no more thn 3 dys) or oth from the hens nd lso reducing the photo-period in the houses to 8 h lighted period or less. Removl of feed will lst for period enough to cuse the complete involution of the reproductive trct (Berry, 2003).

26 14 Feed withdrwl is considered hrmful to lying hens (Wester, 2003) nd presently no scientific literture exists on the effects of feed withdrwl on domestic hens. However, work hs een done in other species of irds nd the conclusion ws drwn tht these responses were similr within the vin species. Cherel et l. (1988) concluded tht fsting (deprivtion) could e ctegorized into three phses; phse 1 is usully short (few dys) nd involves rpid decrese in ody mss loss while t the sme time there is reduction in the rte t which the ody mss is lost. Phse 2 is usully long term nd my lst severl weeks or even months in some species. During this phse most of the energy used is from the ctolism of ft (Wester, 2003). Phse 3 cn lst from dys to weeks nd protein ctolism egins (Cherel et l., 1988). Protein ctolism will result in deilittion of the ird nd eventully deth. However, the degree of fsting tht is imposed to induce molt cn e viewed s physiologicl dpttion of the hens (Wester, 2003). It hs een reported tht feed withdrwl lters the microenvironment of the crop nd cec in the intestines of the fowl (Impey nd Med, 1989; Durnt et l., 1999; Ricke, 2003). Corrier et l. (1997) stted tht though induced molting hd no pprent effect on the ph or oxidtion reduction potentil of the cec, it cused reduction in cetic cid, propionic cid nd totl voltile ftty cids (VFA) in the cec. Two yers fter Corrier s oservtions, Durnt et l. (1999) oserved incresed crop ph, nd reduced Lctocilli popultions nd VFA concentrtions in the crops of feed deprived hens.

27 15 Animl Welfre nd Molting Animl Welfre Issues There hve een incresed concerns posed y niml welfre dvoctes out the effects of induced (forced) molting on hens (Wester, 2003). It is common premise tht systems tht led to injury, disese, deformity, or other physicl signs of reduced helth cn men tht the welfre of the ird is compromised (Broom nd Johnson, 1993; Frser, 1995). There is possiility tht poor welfre cn lso result from confinement, food restriction or under-stimultion (Dwkins, 1999). Critics of forced molting hve stted tht the prctice is cruel nd in-humne since it involves the removl of food nd this mount to strvtion (Wester, 2000). Bxter (1994) reported tht hens rered in cged systems experience chronic (long term) nd cute (short term) suffering, s well s other threts to their welfre. This chronic suffering ws oserved on continuous or repeted sis for the durtion of the confinement in the cge nd cute suffering ws seen during the pre-lying period of the dy. Anderson et l. (2004) postulted tht ntgonistic ehvior such s ggression (oserved etween hens), escpe nd voidnce, nd sumission (used to determine ferlessness), nd their reltionship to stress within n niml could e potentil indictors of welfre during molting. Sherry et l. (1980) hve shown tht irds in the wild undergo period of inppetence followed y nturl molt nd Wester (1995, 2000, 2003) stted tht molting hens fred no worse thn non-molted hens. Duncn nd Petherick (1991) hve rgued tht the welfre of the niml depends on how the niml feels nd the clims tht the nimls welfre is compromised only to the extent tht the niml suffers.

28 16 There is however disgreement in how suffering cn e ssessed in irds. It is the common elief mong welfre dvocte groups tht induced molting uses the rules of the Five Freedoms for nimls recommended y the United Kingdom s Frm Animl Welfre Council (Appley et l., 2004). These lws re; freedom from hunger nd thirst, freedom from discomfort, freedom from pin, injury nd disese, freedom to express norml ehvior nd freedom from fer nd distress. Duncn (1998) stted tht symptoms of suffering were fer frustrtion nd pin nd these cn e used to dignose welfre. He further stted tht incresed ggression nd stereotypic pcing were indictive of suffering nd reduced welfre. Anderson et l. (2004) stted tht the cge environment lters norml ehvior ptterns in lying hens nd my increse the incidence of stereotyped ehviors tht hve een suggested to negtively influence the welfre of hens. Bxter (1994) concluded tht the cges filed to provide for the lying hens welfre needs since they were prevented from dust thing, forging, nesting nd roosting. Efforts hve een mde to improve the cge environment for the hens y including enrichment fcilities to improve the hens welfre (Cooper nd Alentos, 2003). Wester (2003) hve stted tht induced molting does not dversely ffect the ehvior or the welfre of hens since they did not disply high levels of displcement or norml ehviors such s stereotypy nd incresed ggression. Animl Behvior Cooper nd Alentos (2003) stted tht generlly ehviorl repertoires of wild or free rnging nimls in semi-nturl environments my e used s crude indictors of the kinds of ehviors tht might e deprived in cptive conditions. When ssessing

29 17 irds ehvior s it reltes to feed deprivtion, it hs een oserved tht hens tht previously hd ccess to resource nd re lter prevented from free ccess, will exhiit rnge of identifile though non-specific ehviors tht my e indictive of frustrtion (Cooper nd Alentos, 2003). Molting y wy of feed deprivtion rise concerns of reduced niml welfre of the lying hen since it cn e hrmful nd ehvior might give direct indiction of the well eing of hens undergoing feed withdrwl (Wester, 2003, 2000). When exmining the ehviorl profile of domesticted hens, numer of specific prmeters hve eing exmined including, feeding, drinking, comfort, socil, reproductive, nd nti-predtor ehvior (Duncn, 1970). During molting there re severl ehvior ptterns tht re considered of prticulr interest nd re potentil welfre indictors of stress, these include sumission- putting up no resistnce, ggression- ggressive ehvior towrds neighor, escpe nd voidnce- show of fer (Anderson et l., 2004), lso oserved re nonnutritive pecking- which is non-ggressive pecking t nything other thn feed, preen- mnipultion of the plumge with the eck, wlk- locomotion involving one step or more, still- complete immoility of n lert hen, feeder- ehvior directed into the feed trough nd drink- ingestion of wter from wterer (Wester, 2000). Some studies hve looked t chnges in ggressive ehvior during induced molts. The results hve een controversil. Aggrey et l. (1990) nd Hskell et l. (2000) found tht negtive interctions mong lying hens incresed during feed deprivtion in open housing systems ut not in ttery cges. Hemree et l. (1980) reported tht the ggressive ehvior of hens in colony cges during feed deprivtion did not differ from hens not

30 18 deprived of feed. Wester (1995) found no significnt differences in ggression etween cged hens on 4 dy fst nd hens tht were not deprived of feed. When Anderson et l. (1989) investigted chnges in the hens environment; they oserved tht ehviorl ptterns chnged significntly when hens were plced in cges. They concluded tht the dpttion process lowered the numer of negtive socil interctions etween hens, which in turn reduced stress nd enhnce welfre. Tonic immoility ws used y Jones (1989) to determine underlying sttes of fer. Zimmermn nd Koene (1998) reported tht clling nd incresed locomotion were indictors of frustrtion, in this cse they identified specific gkel cll (Zimmermn et l., 2000). Other reserchers oserved incresed ggression (Hskell et l., 2000), incresed locomotion (Duncn, 1970; Duncn nd Wood-Gush, 1971), incresed pecking t fether unches ws oserved y Hskell et l. (2000). While the prevention of feeding results in incresed locomotor ctivity including stereotypic pcing, cge ground nd fether pecking, ggression nd gkel clling (Duncn, 1970; Duncn nd Wood-Gush, 1971; Zimmermn nd Koene, 1998), the intensity of these responses does not, however, correlte with the intensity of the deprivtion. To dte, only limited numer of studies hve relted deprivtion of specific resources to ehviorl or physiologicl mesures of distress. Although there is no evidence tht mngement progrms involving feed withdrwl cuses deilittion when properly implemented, uncertinty is still evident round the issue of whether or not the hunger involved in feed withdrwl cuses suffering.

31 19 Immune Response during Molting A vriety of different physiologicl mechnisms contriute to the hens reduced resistnce during molting. Among the investigted mechnisms tht cuse decresed resistnce re the ltertions in the intestinl micro-flor (Corrier et l., 1997; Holt nd Porter, 1992; Svge, 1989), intestinl physiology (Brnes et l., 1979; Corrier et l., 1997; Duke, 1986; Holt, 1993), nd host defenses (Ben-Nthn et l., 1981, 1977; Holt, 1992, ; Holt nd Porter, 1992). During n induced molt, immunosuppressive effects on T-cell immunity (Holt, 1992, ) nd humorl immunity (Ben-Nthn et l., 1981, 1977) cn e seen. Even with the mssive invsion of inflmmtory cells to the site of infection, molted hens re still more susceptile to infection. Inflmmtion is sequence of complex rections designed to ttrct effector cells to the site of infection to remove nd destroy the inflmmtory stimulnts (Edwrds, 1994). The suppression of the immune system during n induced molt is evident in the reduced ntiody production, delyed type hypersensitivity, grft versus host response nd incresed circulting leukocytes with n incresed H: L rtio. Corticosterone The hens response during n induced molt is somewht relted to the nutritionl stress tht is ssocited with the withdrwl of food. Drmticlly elevted levels of corticosterone hve een seen during cute feed restriction or fsting (Freemn et l., 1981; Hrvey nd Klndorf, 1983). In mmmls, incresed levels of corticosterone decreses inflmmtion therey depressing the specific immune response. It does this y locking interleukin-1 (IL-1) trnscription (Besedovsky et l., 1986). IL-1 stimultes the

32 20 production of other cytokines including IL-2. These cytokines re responsile for ctivting the ody s poly-morpho-nucler (PMNs) cells such s mcrophges tht re the cells primrily responsile for inflmmtory response. Corticosterone levels increse in hens s the hypothlmic-pituitry-drenl (HPA) xis is ctivted y the ody s need to moilize energy. Juhn nd Hrris (1958) found tht exogenous deoxy-corticosterone ws s effective s progesterone in inducing molts. Etches et l. (1984) lso reported elevted corticosterone levels in molting hens, they found tht the levels to which corticosterone incresed depended on the type of molt induced, for exmple, methods tht induced more rpid or complete molt, hd greter increse in the corticosterone levels. Stress cuses generl deteriortion of the well-eing of nimls nd it usully involves cscde of physiologicl dptive responses (Thxton nd Puvdolpirod, 2000). Stress cuses elevtion of corticosterone levels in the lood nd this result in incresing concentrtions of glucose due to ctolism of muscle protein. The incresing levels of glucose due to gluconeogenisis results in incresing excretory uric cid which is n indiction of protein degrdtion (Brown et l., 1958; Siegel nd Vn Kmpen, 1984; Dvison et l., 1985). Physiologicl stress responses oserved in roilers s result of drenocorticotropin (ACTH) tretment includes elevtion of plsm glucose (Siegel nd Bene, 1961; Siegel, 1962), elevtion of plsm cholesterol (Siegel nd Siegel, 1966), nd immuno-suppression of humorl nd cell-medited immune responses (Thxton nd Siegel, 1970; Holt, 1992).

33 21 Circulting Leukocytes Induced molting y dietry restriction cn ffect components of the irds immune system. Brke et l. (1981, 1985) found tht dietry restriction ffected the thymus nd spleen weights, resulting in the reduced production nd mturtion of T- cells. Other reserchers found tht the totl numer of circulting leukocytes decrese significntly during molting (Ben-Nthn et l., 1977; Brke et l., 1982; Holt 1992; Holt nd Porter, 1992) therey reducing the hens ility to fight infection. Wolford nd Ringer (1962) previously found tht molting influenced the differentil white lood cell counts y cusing reduction in lymphocytes numers nd incresing heterophil numers in the peripherl lood. Lne (1987) suggested tht the vin heterophil could e used s the window to the stte of their helth nd tht the vin heterophil would respond to prolems tht re ssocited with their diet, chronic cteril infections, stress light nd trum. In the mjority of the vin species, lymphocytes re the most undnt leukocytes found in circultion (Mxwell nd Roertson, 1998). In poultry, pproximtely 59% of the totl white lood cells re lymphocytes nd 27% re heterophil. In extreme conditions the heterophil/lymphocyte rtio (H: L) cnnot lwys e used to provide n ccurte ssessment of stress, however, when heterophil ehve consistently to prticulr stressor, the H: L rtio cn e used (Mxwell et l., 1992). In light of this, H: L rtio hs ecome widely ccepted s relile nd ccurte physiologicl indictor of the stress response. Gross nd Siegel (1993) hve suggested tht some reference vlue e used s guidelines to determine the different degrees of stress. H: L rtios of out 0.20, 0.50 nd 0.80 re chrcteristic of low, optiml nd

34 22 high degrees of stress, respectively. When Mxwell (1993) investigted H: L rtio nd the role tht corticosterone plyed when feed ws withdrwn from hens, they oserved n increse in the rtio. Jones (1989) found tht fsted nd frustrted Brown Leghorn pullets hd n incresed H: L rtio. However, when Mxwell et l. (1990) investigted roilers tht were sujected to prolonged feed restriction, they found tht the H: L rtios of these irds were not significntly ltered when compred to irds tht were fed d liitum. These results confirmed results from studies tht were previously done y Brke et l., (1982), Gross nd Siegel (1989), Ktnf et l., (1988) nd Vn Niekerk et l., (1988). This suggests tht irds ecme hituted to the effects of prolonged feed restriction. During the erly phse of feed restriction, heterophil popultions responded to hormonl surge (Mxwell et l., 1991, 1992; Svory et l., 1992; Hocking et l., 1993, 1994, 1996), however, this increse cnnot e physiologiclly sustined, therefore the heterophil will no longer e recruited to the site of infection. Alodn nd Mshly (1999) postulted significntly incresed circulting leukocytes in molted hens over nonmolted control hens. An increse in H: L rtio indictes tht hens molted y wy of feed restriction re under more stress (Alodn nd Mshly, 1999). They lso elucidted tht induced molting could result in inhiiting the immune response since the totl circulting leukocytes in the erly stges of the molting period ws reduced when compred to control hens. It should not e surprising to see the presence of heterophil t the sight of invsion ecuse the vin heterophil is the primry phgocytic cell during n cute inflmmtory

35 23 response (Powell, 1987). Kogut et l. (1998) elucidted tht induced molting hs negtive effect on the functionl ctivities of the heterophil. They reported tht while heterophil responded strongly to chemotctic stimulus, in molted hens the heterophil were unle to effectively phgocytize cteri or illicit n oxidtive urst to kill the S. Enteritidis. They lso found diminished phgocytosis of opsonized S. Enteritidis during n induced molt. Recrudescence nd lyphocytic repopultion usully ccompnies reproductive quiescence in molting hens (Brke et l., 1981). Alodn nd Mshly (1999) found tht peripherl lymphocyte decresed nd lymphocyte popultion reduced during the fsting period of molt. While Holt (1992) found tht there ws reduced numer of B-cell oserved in irds on fsting induced molt, the ntiody levels ppered to e reduced. Desmidt et l. (1998) stted tht humorl immunity contriutes to eliminte S. Enteritidis from the gut of poultry. Initilly, S. Enteritidis occurs primrily occurs in the mucosl surfces nd significnt humorl immune response will generlly occur in this re. Duchet-Suchux et l. (1995) postulted tht humorl immune response is involved in the rpid elimintion of S. Enteritidis from the cec of some lines of chickens. The immunogloulin (Ig), IgA is the predominnt immunogloulin ssocited with mucosl immunity, however, IgG nd IgM responses cn lso e oserved. IgA cn e found in oth ile nd mucosl secretions (Lecq-Verheyden et l., 1972). In mmmls, IgA serves s possile opsonin for mucosl phgocytes (Kilin et l., 1988), protection ginst infection through ntiody dependent cellulr cytotoxicity (Tgliue et l., 1983), inhiiting cteril dherence (McGee et l., 1992), nd neutrlizing toxin moieties (Lnge nd Holmgren, 1978). Holt nd Porter (1992) hve oserved sustntil

36 24 mucosl immune response in experimentlly chllenged chickens ginst flgell nd or lipopolyscchrides respectively in the intestines nd ile. Seo et l., (2002) found high levels of IgA in response to S. Enteritidis infection in the crop (0.38 in infected hens nd less thn 0.10 in non-infected hens) nd ile (1.17 in infected hens nd less thn 0.10 in non-infected hens). They lso found tht chickens infected with S. Enteritidis developed n ntiody immune response s erly s nine dys in the serum nd fourteen dys in the egg. The degree of the ntiody response is therefore correlted to the degree of infesttion. Serum Acute Phse Proteins An cute phse response (APR) ws the term coined to the phenomen oserved in ptients during the cute or erly stges of n infectious illness (Fleck, 1989). The cute phse response hs een descried s the systemic, rther thn locl effects of inflmmtion nd the term ws first pplied to the precipitin rection oserved when pneumococcl extrct ws dded to the serum from ptients in the cute phse of pneumoni (Fleck, 1989). An APR is medited y serum proteins, specificlly cute phse protein (APP) is relesed into the loodstrem y vriety of stimuli including inflmmtion (Thoms nd Schrieer, 1985; Fleck, 1989; Jmieson et l., 1992), cteril infection (Morley nd Kushner 1982; Pfeffer nd Rogers, 1989) nd endotoxin exposure (Tkhshi et l., 1995). Reserchers hve found tht the presence of inflmmtory cytokines such s IL-1, IL-6 nd tumor necrosis fctor, enhnces the synthesis of APP (Klsing, 1984; Mrinkovic et l., 1989; Bumnn nd Guldie 1990, 1994). Chnges in the concentrtions of APP in the serum, though nonspecific, cn e

37 25 sensitive mens of indicting inflmmtion. Holt nd Gst (2002) found tht irds tht were experimentlly infected with S. Enteritidis nd molted or non-molted, showed significntly higher APP levels thn non-infected irds. Elevted concentrtions of serum APP ws lso oserved y Deignn et l. (2000) when they chllenged clves with S. Enteritidis. The degree of increse ws correlted to the severity of the disese. Slonen et l. (1996) nd Godson et l. (1995) sw comprle effects in cows with mstitis nd virl respirtory disese, respectively. Holt nd Gst (2002) concluded tht serum APPα 1 cid glyco protein (AGP) levels cn e effective nd rpid indictors of infection nd cn e useful in trcking the infection sttus of flock. Xie et l. (2002) exmined lipopolysccride (LPS) induced inflmmtion in chickens nd found significnt chnges in not only lood concentrtions of heterophil nd IL-6, ut lso in serum protein profiles. They found tht though the chnges were seen in heterophil nd IL-6, the serum proteins remined elevted up to 48h fter tretment. In nother study conducted y Xie nd others (2002), they concluded tht inflmmtion induced chnges in severl serum protein nd tht ovotrnsferrin is positive APP in chickens. Although not specific, the determintion of the levels of APP in the serum of molting hens cn potentilly e quick nd effective mens of determining the infection sttus of flock therey stemming potentil outrek of disese within the flock. The Effects of Molting on Slmonell enteric serovr Enteritidis (S. Enteritidis) Infection in Lying Hens In the mid-1980s, the interntionl pndemic of S. Enteritidis egn (Rodrique et l., 1990). Slmonell Enteritidis infections reched high of 3.9 per 100,000 popultion

38 26 in 1985 nd hs since declined to 1.98 per 100,000 in 1999 (Ptrick et l., 2004), however, while the totl numer of outreks declined y hlf, those in the western sttes tripled (Ptrick et l., 2004). The spred of infection to humns is consequence of the food poisoning form from chickens tht re infected. The S. Enteritidis is spred not only from contminted chicken crcsses, ut lso from eggs nd the contents of intct eggs (Anonymous, 1989). Feed withdrwl compromises the irds immune system mking them susceptile to infection y numer of microorgnisms. Of prticulr interest is S. Enteritidis. Feed withdrwl ssocited stress cuses incresed susceptiility to S. Enteritidis infection (Holt, 1993; Corrier et l., 1997; Durnt et l., 1999; Ricke, 2003; Holt, 2003) which is usully mrked y incresed intestinl shedding nd coloniztion in internl orgns such s the liver, spleen, nd ovries (Holt nd Porter, 1992; Holt, 1993; Holt et l., 1995; Thigrjn et l., 1994). Other reserchers hve shown tht emptying of the gstrointestinl trct y feed withdrwl is consistent with incresed levels of Slmonell in vriety of niml species including chickens (Humphrey et l., 1993). While feed withdrwl provides the enefits of extending the egg-lying life of the flock, it lso hs detrimentl effects on the hens immune system. In mmmls nd irds, deficient diets hve een shown to reduce humorl immunity (Ben-Nthn et l., 1977, 1981; Gross nd Newerne, 1980) nd lso cell-medited immunity (Chndr, 1990; DePsqule-Jrdieu nd Frker, 1979). Holt (1992) oserved tht cell-medited immunity ws significntly depressed, while in nother study (1992), the B-cells nd CD8+ T-cells were less infected. DePsqule-Jrdieu nd Frker (1979) detected

39 27 elevted levels of serum corticosterone, which suggested tht the hormone plyed role in the depression of cell-medited immunity. Brke nd Thxton (1979) nd Etches et l. (1984) lso noted similr response when molting hens y wy of feed withdrwl. The intestinl shed rte of S. Enteritidis is higher in irds tht re exposed to exogenous sources of the pthogen concomitntly during molt induction. These hens hd more severe infection when compred to un-molted hens (Holt nd Porter, 1992; Holt et l., 1995). These hens not only shed more orgnisms (Holt nd Porter, 1992; Holt, 1993; Holt et l., 1994, 1995) ut they lso showed significntly more inflmmtion especilly in the colon nd cecum (Holt nd Porter, 1992, Porter nd Holt, 1993; Holt et l., 1995; Mcri et l., 1997). Prolems ssocited with S. Enteritidis in the flock environment cn increse when irds re exposed to stresses such s feed deprivtion nd the spred of S. Enteritidis in lrger numers of susceptile hens in flock is of mjor importnce. Nkmur et l. (1994) oserved tht short-term feed removl cn increse horizontl trnsmission to nery hens, while Holt nd Porter (1992) oserved similr results when molting incresed horizontl trnsmission to hens in neighoring cges. The impct of ir-orne trnsmission is lso mjor concern since trnsmission to irds in cges which were distnce wy from infected irds during molt y feed withdrwl, ws oserved y Holt et l. (1998). Lying hens with S. Enteritidis infected ovries, will not only ly contminted eggs, ut infected chicks will lso e htched from these eggs. These chicks will grow to ecome pullets which will susequently ly contminted eggs (Hopper nd Mwer, 1988; Lister, 1988; O Brien, 1988). The process of molting cn cuse n increse in

40 28 infectivity of these irds (Cson et l., 1994) nd stress situtions such s feed withdrwl to induce molt cn lso cuse recurrence of previous S. Enteritidis infection (Hughes et l., 1989). Holt et l. (1995) postulted tht molting cn significntly ffect n S. Enteritidis infection t different times in the infection cycle when they oserved tht infected molted hens produced more eggs tht were S. Enteritidis contminted. They lso oserved tht molting excerted the infection s shown y the redy trnsmission of the orgnism to previously uninfected hens, n indiction tht molting mde these hens more susceptile (Holt et l., 1995). The extent to which feed withdrwl increses hens susceptiility to infection cn e seen when compring 10^3 10^4 colony forming units of S. Enteritidis necessry to infect 50% of group of un-molted hens, while fewer thn 10 orgnisms were necessry to infect molted hens (Holt, 1993; Holt et l., 1994). Gst nd Berd (1990) found S. Enteritidis in the lumin nd yolks of eggs from hens 4 weeks fter they were orlly inoculted. This ws n indiction tht internl contmintion of eggs could occur prior to the lying of the egg, when S. Enteritidis ws deposited on the shells. Keller et l. (1995) oserved tht when eggs which were not yet formed were removed from the oviduct they were contminted with S. Enteritidis much more often thn freshly lid eggs. Since induced molting y feed withdrwl is currently prcticed y commercil lyers, reserch into different methods y which to meliorte the incresed incidence of S. Enteritidis infection is necessry. The direction of interest to control this pthogen should e through intervention schemes or y using lterntive methods for inducing molt. Whichever products re used should e prt of n

41 29 lterntive method of inducing molt nd should e of economicl dvntge to the commercil frmers y successfully providing second lying cycle of high production rtes nd good qulity egg while t the sme time reducing the incidence of incresed susceptiility to pthogenic microorgnisms. Alterntive Methods of Inducing Molt Generl Concepts Due to the incresed pressure nd criticism of commercil lyer frmers for their prctice of feed deprivtion s mens of inducing or forcing molt, reserchers hve een investigting different methods to ring on molt insted of depriving hens of feed. The gols of successful molt re; for the hens to lose pproximtely 20 to 25% of their totl ody weight, for cesstion of ly long enough for the totl regression of the reproductive trct nd n cceptle nd persistent second cycle performnce (Scheideler et l., 2003). Generlly speking, egg production nd qulity deteriorte s the flock ges (Bell, 2003). After the rejuvention of the reproductive trct the hens will come into second cycle nd produce etter qulity eggs (Keshvrz nd Quimy, 2002). Bell (2003) oserved tht lying hens peked in the first cycle in wk 8 t out 90% nd in the second cycle in wk 13 t out 80%. Deteriortion of the egg qulity includes the internl nd externl trits of the egg nd improvement in egg qulity prmeters re evident fter n induced molt (Swnson nd Bell, 1975). The prmeters tht re of prticulr importnce include: lumin qulity, cndled grde, shell thickness, specific grvity, egg weight nd shell texture (Bell, 2003).

42 30 Experimentl molting techniques hve vried widely in n effort to initite molt nd cese egg production in more humne wy nd lso to llevite prolems of incresed infectivity ssocited with feed withdrwl. Attempts hve een mde to restrict feed intke therey reducing ody weight nd inducing molt. This hs een done y using nutritionl imlnces, ingredients tht decrese ppetite, ddition of fillers or dministrtion of hormones to induce molts (Bell, 2003; Holt 2003; Prk et l., 2004). Some of these methods include feeding diet tht contins less thn 0.3% clcium (Dougls et l., 1972; Mrtin et l., 1973; Gilert nd Blir, 1975; Wkeling, 1978), feeding diets with less thn 0.04% sodium (Whitehed nd Shnnon, 1974; Dillworth nd Dy, 1976; Neseth et l., 1976; Cmpos nd Bio, 1979; Ross nd Herrick, 1981), feeding diets tht contin high levels of zinc, usully in the form of zincoxide (Scott nd Creger, 1977), nd feeding diets contining iodine in the form of potssium iodine (Arrington et l., 1967). However, the results of some of these diets do not induce consistent molt in ll hens in the flock, most re either costly or mngerilly unwieldy nd cnnilistic pecking hs lso een oserved (Ricke, 2003; Prk et l., 2004; Biggs et l., 2004; Wester, 2003). High Fier Low Energy Diets A diet tht is high in fier is usully lower in metolizle energy thn diet tht hs low fier content (Wenk, 2001). Lnghns (1999) stted tht high fier diets cuse erlier stiety of n niml, while Rijnen et l. (1999) concluded tht n niml tht reches stiety oth physiclly nd nutritionlly is less stressed. With this in mind the incorportion of high fier low energy diets to induce molt hs een suggested.

43 31 High fier diets consist predominntly of plnt cell wlls, non-strch polyscchrides, nd non-crohydrte compounds including lignin, protein, ftty cids nd wx (Bch Knudsen, 2001). By definition dietry fier cnnot e digested y endogenous processes, insted the resident microorgnisms metolize it (Wenk, 2001). These diets cn lter the gstrointestinl trct y chnging its microil ctivities, the rte of pssge, metolites nd the overll effectiveness of the trct (Bch Knudsen, 2001; Wenk, 2001). A vriety of high fier diets hve een incorported in poultry feed including cotton mel (Dvis et l., 2002), jojo mel (Arnouts et l., 1993), gur mel (McGinnis et l., 1983), grpe pomce (Keshvrz nd Quimy, 2002), whet middlings (Biggs et l., 2004; Seo et l., 2001) nd lflf (Lnders et l., 2005,; Woodwrd et l., 2005; Donlson et l., 2005). Cotton seed mel is lower in energy nd protein thn soyen mel nd cn e used successfully incorported in poultry diets. Dvis et l. (2002) exmined the effects of cotton seed mel when incorported in lyer diets. They oserved tht cotton seed mels t levels of 20 to 30% of the diet resulted in reduced egg sizes nd yolk discolortion. Dvis et l. (2002) reported no significnt difference in egg production etween the control hens nd the hens fed feed contining cotton seed mel. There is however, disdvntges of using cotton seed mel in lyer diet since it cn result in the production of eggs with rown yolk discolortion. Jojo mel is y-product fter the extrction of oil from jojo seeds. It contins pproximtely 30% crude protein which mkes it potentilly useful s n ingredient for niml feed. However, when Ngou Ngoupy et l. (1982) fed jojo mel

44 32 to chickens they oserved impired ody weight gin, reduced feed intke nd impired feed efficiency. Arnouts et l. (1993) stted tht roiler chickens fed diet supplemented with 4% jojo mel exhiited reduced growth rte. Vermut et l. (1998) utilized jojo mel to induce molt in roiler reeder hens. These hens reched mximum ody weight loss 5 wks fter the strt of the molt nd stopped lying 3 wks fter molt induction. They oserved tht the oviduct regressed to the sme degree s the pir-fed hens. The oviduct re-grew completely fter the jojo mel ws removed form these hens. This result ws not oserved y Vermut et l. (1998) who fed jojo mel to growing pullets nd oserved irreversile inhiition of oviduct development resulting in no egg lying. Feeding gur mel diets to lying hens to induce molt cn e effective in cusing greter loss in ody ft nd decresed loss in ody protein when compred to molt induced y feed withdrwl (McGinnis et l., 1983). Zimmermnn et l. (1987) oserved tht lying hens stopped lying fter eing fed for 8 dys with 15% gur mel to lyer diet. They oserved tht these hens lost 31% of their ody weight within 21 d. When Keshvrz nd Quimy (2002) molted hens using grpe pomce, which is essentilly the solid remins of grpes fter the juice or oil hs een pressed out. They oserved tht the hens tht these hens stopped lying y dys 3 to 4 fter the initition of the molt similr time s the full feed withdrwl hens. They lso noticed tht the hens tht were fed grpe pomce lost similr mount of ody weight s the feed withdrwl hens (30.30 nd 30.80% respectively). No significnt differences were oserved in egg production during the first 4 wk fter the initition of molt etween the feed withdrwl

45 33 hens nd the hens tht were fed grpe pomce (7.50 nd 5% production respectively), lso no differences were oserved in specific grvity of the eggs or ny other internl egg qulities. The reproductive orgn weights (ovry nd oviduct) of the hens treted with grpe pomice were not significntly different from the hens which were deprived of feed. Although grpe pomce cn effectively induce molt nd hve post-molt performnce tht is comprle to feed withdrwl hens, ccessiility nd storge issues could potentilly e prolemtic due to its light low-density chrcteristic. Also sed on the physiologicl response of hen on the grpe pomice diet, it ppers tht this diet did not prove to e less stressful on the hens thn the conventionl method of feed withdrwl. Whet middlings re the y-products of whet flour mnufcturing. Whet rn is lso y product of whet mnufcturing nd contins higher fier thn whet middlings which is utilized s source of energy in niml feed (Bi et l., 1992). Biggs et l. (2004) evluted the efficiency of whet middling, corn gluten feed nd distillers dried grins with solules (DDGS) s lterntive methods of inducing molt. Previously, Biggs et l. (2003) showed tht diets high in whet middling or corn, were effective methods tht otined post-molt performnces tht were comprle to 10 dy feed withdrwl molt. In ltter study, Biggs et l. (2004) oserved tht ll diets (whet middling, corn gluten feed, nd distillers dried grins with solule) yielded decresed egg production nd ody weight during the molting period. However, they found tht the DDGS diet hd the highest oviduct nd ovry weight (2.50% nd 3.50% respectively of their ody weight) which suggest tht there ws limited ovrin

46 34 regression using DDGS. Biggs et l. (2004) postulted tht feeding hens with diets high in corn, whet middling, corn gluten feed or 71% whet middling 23% corn comintion could e used s effective lterntive molt progrms. They oserved no significnt differences were oserved in post-molt egg prmeters mong tretments. When Seo et l. (2001) exmined whet middlings s dietry source during molt they oserved tht the hens which were fed whet middlings stopped lying within 7 d fter the eginning of the molt. Biggs et l. (2004) oserved tht while the feed deprived hens in their study cesed egg production within 6 d, the hens tht were in the other tretments did not totlly stop producing eggs even though the hens in the whet middlings tretments reduced production t fster rte the hen in the other tretments. Post-molt feed intke nd feed efficiency of hens fed whet middlings during molt is not different from tht of hens which were molted y the conventionl feed withdrwl. The cceptnce of the feed initilly y the hens ws poor nd hens fed whet middling te two fold less feed thn other tretments. Potentil of Alflf s Molting Diet Alflf s Dietry Ingredient for Poultry As erly s the mid 1920 s scientists hve shown tht lflf lef, lflf mel, nd lflf hy cn e source of vitmin A in poultry diets (Bech, 1924; Dvis nd Bech, 1925; nd Kennrd nd Lingle, 1928). Heywng (1932) stted tht lflf lef mel cn e stisfctory source of vitmin A when fed t levels of 7.50 % to 10% of the totl feed intke. Alflf is forge crop tht is esily ccessile in most nd poultry producing regions of the United Sttes nd is currently commercilly ville in msh,

47 35 pelleted nd cue form. It contins 17.50% crude protein nd 24.10% crude fier nd hs low levels of metolizle energy, pproximtely 1200 kcl/kg when compred to lyer rtion which hs metolizle energy of 2,965 kcl/kg (NRC, 1994). Dehydrted lflf is one of severl feedstuff tht spends more thn 24 h in the chickens digestive trct (Sild, 1979) nd the longer the retention time in the cec nd the intestines, the greter the opportunity for microil degrdtion of fier nd the production of voltile ftty cids (Hungte, 1966). Alflf lef mel when fed in sufficient mounts (5% -10% of the diet) to furnish 0.2 mg of crotene per ird dily prevented the occurrence of deficiency lesions in the throts of irds (Willims et l., 1938). Heywng (1950) lso oserved tht when White Leghorn lying hens were fed diets contining 5% to 25% sun-cured lflf mel hd reduction in totl egg production occurred s the level of the lflf in the diet incresed. Drper (1948) oserved significnt decline in weight when chicks were fed lflf mel nd the rte of weight reduction incresed s the quntity of lflf in the diet incresed from 5% to 15%. Cooney et l. (1948) reported tht there ws n unidentified fctor in lflf tht resulted in reduced growth of New Hmpshire chicks when fed t 10% of the diet. Germen nd Couch (1950) lso oserved depressed growth in New Hmpshire chicks nd the depression in growth ws proportionl to the levels of lflf in the diet. Heimn nd Wilhelm (1937) oserved tht yolk pigmenttion ws greter for hens which were fed lflf thn those hens which were fed corn; this is desirle trit for consumers in some regions. Kohler (1972) hs lso stted tht lflf ws n excellent source of pigment for roiler nd lying hens. Sen et l.(1998) nd Ponte et l.(2004), found tht lflf ws well lnced

48 36 in mino cids, rich in vitmins, crotinoids nd xnthophylls tht give poultry crcss its desirle yellow color. It lso contined high levels of ioctive ntinutritive fctors such s sponins (Sen et l., 1998). Sponins re considered steroids or triterpenoid glycosides tht possess hypocholesterolemic, nti-crcinogenic, nti-inflmmtory nd ntioxidnt properties (Ro nd Gurfinkel, 2000; Ponte et l., 2004). Willims nd Bolin (1938) concluded tht when lflf ws fed in sufficient quntities to lying hens to furnish 0.2 mg of crotene per ird dily, lflf lso kept the irds in good helth nd resulted in fir egg production. Ponte et l. (2004) found tht met from roiler hens fed moderte levels of lflf ws cceptle to consumers. Fermenttion products of the microorgnisms present in the gstrointestinl trct cn limit foodorne pthogen coloniztion. McHn nd Shotts (1993) oserved 50-80% reduction in Slmonell Typhimurium popultion in the presence of short chin ftty cids. Dietry fier re preferentilly utilized y Lctocillus nd Bifidocteri species which leds to the production of lctic cid nd short chin ftty cid (Kpln nd Hutkins, 2000), this results in low ph which will mintin the norml microorgnism popultion thus preventing the estlishment of Slmonell in the gstrointestinl trct (Juven et l., 1991). Donlson et l. (2004,) oserved tht lflf cn enhnce the fermenttion properties of cecl microflor which re cple of limiting in vitro growth of Slmonell Typhimurium. Sild, (1979, 1980) oserved of the slow pssge rte of lflf through gstrointestinl trct of the chicken. This could e potentilly eneficil s it llows for etter digestion of the feed nd microil fermenttion therey mintining lnced microflor which will limit pthogenic

49 37 mircorgnisms in the gstrointestinl trct (Ricke et l., 1982; Vispo nd Krsov, 1997). Alflf Molt Diets nd Egg Production The utiliztion of lflf in lying hens nutrition hs een ttempted to improve yolk pigmenttion nd lso s protein concentrte (Burdick nd Fletcher, 1984; Miller et l., 1972). The potentil of lflf s molt diet is evident due to its slow pssge nd low energy content leving the irds with feeling of stiety (Sild, 1979, 1980). When high levels of lflf were incorported in the diets of lying hens, Whitehed et l. (1981) oserved tht the sponins contined in the lflf depressed feed consumption, ody weight, egg production liver lipid concentrtion nd plsm triglyceride concentrtions. Donlson et l. (2005) incorported lflf s molt diet nd reported tht the hens cesed production of eggs y dy 5 of the molt s opposed to dy 4 y the feed withdrwl hens. Hens fed lflf diets otined weight loss tht ws comprle to tht of the feed withdrwl hens (Woodwrd et l., 2005; Donlson et l., 2005). No significnt differences were oserved etween the feed deprived hens nd the lflf fed hens when exmining the length of time it took for the irds to return to 50% to 60% production. Donlson et l. (2005) oserved tht hens fed 100% lflf rtion returned to ly fter n verge of 14.8 dys fter molt induction, with the feed withdrwl hens chieving up to 74.29% egg production y 39 weeks post-molt. This ws not significntly different from the hens tht were fed 90% lflf diets, similr levels of egg production post-molt were lso oserved y Lnders et l. (2005). Lnders et l.

50 38 (2005) nd Donlson et l. (2005) oserved tht hens which were molted using lflf diets produced eggs with interior qulities tht were comprle to the eggs produced y the feed withdrwl hens. They lso oserved tht hens which were fed 100% lflf diets consumed the lest mount of feed nd lost weight tht ws comprle to feed withdrwl hens. Donlson et l. (2005) lso reported tht hens fed % lflf hd second cycles tht were comprle to the feed withdrwl hens they oserved tht hens which were fed 70% lflf lost the lest mount of weight nd took the longest mount of time to cese egg production. Lnders et l. (2005) conducted sensory study tht evluted the consumer ssessment of eggs produced y the hens which were treted using lflf diets. The sensory investigtion ws sed on the tste, texture nd color of the cooked egg. They found tht the eggs produced y hens molted with lflf, were not considered less desirle thn those produced y hens molted y feed withdrwl. Donlson et l. (2005) evluted the chnges in the internl orgns of the hens fed lflf molt diets compred to those hens which were molted y feed withdrwl, they oserved no significnt differences in ovry or oviduct weight mong the hens in the molting tretments (lflf or feed withdrwl), indicting tht the reproductive trct of the hens on the diet were regressed. Lnders et l. (2005,) found similrities when they compred ovry regression weights etween hens molted y feed withdrwl nd hens molted using lflf. Alflf Molt Diets nd S. Enteritidis Infection When used to induce molt in lying hens, lflf limited S. Enteritidis coloniztion nd infection during the molt (Woodwrd et l., 2005), they oserved no

51 39 significnt difference etween the lflf diets nd full fed hens in cecl enrichment positives. Woodwrd et l. (2005) oserved no differences etween the tretments in S. Enteritidis coloniztion of the crop nd postulted tht the presence of lctte within the crop could effectively inhiit Slmonell. Alflf ws efficcious in reducing S. Enteritidis coloniztion in the spleen, liver nd ovries (Woodwrd et l., 2005) nd cn lso increse voltile ftty cids such s cette, utyrte nd propionte. McReynolds et l. (2005) comined lflf with n experimentl chlorte product nd oserved results similr to tht of non-molted irds. They oserved n increse in lctic cid production, reductions in orgn coloniztion nd lso, cecl content colony forming units (cfu). When compring the effects of high fier diets on S. Enteritidis infection in molting hens, lflf diets, lflf with chlorte products (Woodwrd et l., 2005; McReynolds et l., 2005, 2006) nd whet middlings (Seo et l., 2001) fvorly reduced the levels of S. Enteritidis in the cec, ovries, livers nd spleen. The use of whet middlings lso resulted in reduced numers of S. Enteritidis in the feces. Hens fed lflf diets hd higher concentrtions of voltile ftty cids thn feed withdrwl hens.

52 40 CHAPTER III BLOOD PLASMA METABOLITE RESPONSE IN MOLTING LAYING HENS CHALLENGED WITH SALMONELLA ENTERITIDIS AND FED ALFALFA CRUMBLE DIETS Introduction Molting in domestic hens is nturlly occurring experience, however, it is usully incomplete nd hens will continue to ly eggs t low rtes for n extended periods resulting in un-profitility due to reduction of egg production nd egg qulity (Bell, 2003). To the commercil egg producers this is n indiction of the end of the useful life in flock nd t the first sign of molting commercil egg producers would either sell these hens nd replce them with new flock or induce molt. Egg producers usully mke the decision to molt hens for second lying cycle sed on the cost of eggs nd the cost of feed nd replcement pullets (North nd Bell, 1990). The cost of replcement pullets is one of the most expensive costs for lyer opertion; therefore, commercil egg producers seek to extend the productive lying life of their flock for s long s it is profitle (Prkhurst nd Mountney, 1988). Whtever method of inducing molt tht is used usully involves the chnging of the lighting period. Commercil egg producers who dpt the prctice in the US typiclly use feed removl s their method of inducing molt. Feed is removed for period of 5-14 dys nd the progrm my or my not include period of rest for up to 21 dy fter the fst to ring irds ck into mximl production (Bell nd Kuney, 1992). The prctice of feed withdrwl hs een descried s stressful (Beving nd Vonder, 1978) nd stress cuses generl

53 41 deteriortion of the well-eing of nimls nd it usully involves cscde of physiologicl dptive responses (Thxton nd Puvdolpirod, 2000). Puvdolpirod nd Thxton (2000,,c,d) hve studied physiologicl stress in chickens extensively nd stted tht it cn e determined y the evlution of lood metolites. Weer et l. (1990) reported tht feed withdrwl ws stressful to chicks since it resulted in elevted levels of plsm corticosterone. Reserchers over the yers hve developed vrious methods to induce molt such s nutritionl imlnces or the incorportion of high fier low energy diets to induce molt (Wester, 2003; Prk et l., 2004). A vriety of high fier diets hve een incorported in poultry feed including cotton mel (Dvis et l., 2002), jojo mel (Arnouts et l., 1993), gur mel (McGinnis, 1983), grpe pomce (Keshvrz nd Quimy, 2002) nd whet middlings (Biggs et l., 2004; Seo et l., 2001). Alflf hs the potentil to serve s dietry source for inducing molt. When used to induce molt in lying hens, lflf mel limited Slmonell Enteritidis (SE) coloniztion nd infection during the molt (Woodwrd et l., 2005; McReynolds et l., 2005, 2006). Lnders et l. (2005,) found tht hens molted with lflf mel or lflf pellets hd ovry regression weights tht were equivlent to tht of hens molted y feed withdrwl nd retined cceptle egg production chrcteristics in the second lying cycle. Donlson et l. (2005) oserved similr results when they exmined molting lyer hens fed with different rtios of lflf mel nd lyer rtion. One prolem ssocited with the use of lflf mel s molting diet hs een reduced feed intke nd wstge of the feed (Woodwrd et l., 2005). Lnders et l.

54 42 (2005) exmined lflf mel nd lflf pellets nd reported tht hens fed lflf pellets reentered egg production erlier thn hens fed lflf mel. Woodwrd et l. (2005) concluded tht the reduced intke oserved in hens fed lflf mel could ccount for the incomplete elimintion of SE from the cec of infected hens. It hs een shown tht using feeding pellets or crumles improved growth rte nd feed conversion in chickens (Heywng nd Morgn, 1944; Kilurn nd Edwrds, 2001). Allred et l. (1957) oserved similr results whether the feed ws pelleted or if it ws reground. They concluded tht this ws indictive of chnge in the nutritive vlue of the feed due to the pelleting process. Nir et l. (1994) explined tht the superior performnce oserved with pelleted or crumled food is ecuse they re more suitle for the chickens digestive system thn msh diet with uniform prticle size. The development of n lternte diet to feed withdrwl is essentil to llevite concerns of reduced niml welfre nd incresed infectivity. Our over-ll gol is to exmine lflf crumle diet s n lterntive molt induction diet nd compre hens physiologicl response to the physiologicl stress tht usully ccompnies feed deprivtion. Mumm et l. (2006) stted tht endocrine nd iochemicl chnges re definitive responses to stress in most species, including dult fowls. Elevted plsm levels of corticosterone, glucose, cholesterol, totl protein nd triglycerides re ssocited with stress in chickens (Siegel, 1995; Puvdolpirod nd Thxton, 2000). In this study we determined the effects of lflf crumle molt diets on lood plsm metolites.

55 43 Mterils nd Methods Experimentl Design Experiments 1 nd 2 were conducted using 250 Single Com White Leghorn hens (SCWL) over 50 weeks old, which were otined from locl commercil lying flock. Twelve hens were used in ech of 6 tretments in tril 1 nd 10 hens were used in ech of 6 tretments in tril 2. The hens were plced in wire lyer cges nd were given free ccess to wter nd un-medicted corn-soyen mel sed mshed lyer rtion tht met Ntionl the Reserch Council recommendtions for nutrients (1994). Bsed on pulished dt the lflf crumle diet ws considered to e high in crude fier (24.1%), with moderte crude protein (17.5%) nd low in metolizle energy (1200 kcl/kg; NRC, 1994). Feed nd fecl smples (1 g) were collected nd exmined for slmonelle. Smples were cultured in tetrthioninte roth nd on rillint green gr (BGA) pltes s ws previously descried y Andrews et l. (1995). All the hens nd feed used in oth trils tested negtive for Slmonell. The hens were llowed to cclimtize in the cges for period of two weeks fter which 12 (10 in the cse of tril 2) hens were rndomly ssigned to 6 tretment groups designted s followed; (1) feed withdrwl SE+ (FW+), (2) full fed SE + (FF+), (3) 100% lflf crumle SE + (ALC+), (4) feed withdrwl SE - (FW-), (5) full fed SE -, nd (6) 100% lflf crumle SE - (ALC-). The molt procedure used to induce the molt ws modifiction y Holt (1993) which ws previously descried y Brke et l. (1982). Tretment diets were pplied to ech tretment group on d 1 of the molt, t the

56 44 sme time feed ws removed from the feed withdrwl hens. Tretment diets were dministered for 12 dys, the period of time the FW hens were deprived of feed. Hens in ll the tretment groups were given wter d liitum. The SE positive hens were ll plced in the sme room, while the SE negtive hens were plced in seprte room. Foot ths were plced t the doors of ll the rooms in the fcility nd non-infected hens were cred for efore the infected hens ech dy. The hens in ll the tretment groups were exposed to 8 h light: 16 h drk photo-period one week efore chnging the diets nd removing feed from the FW hens. This light schedule continued for 12 dy period fter which the experiment ws terminted. Bcteril Strin For this experiment primry poultry isolte of S. Enteritidis (phge type 13A) from the Ntionl Veterinry Services Lortory, Ames, Iow, selected for resistnce to novoiocin nd nlidixic cid (NO nd NA) in the USDA-ARS fcility, College Sttion, TX, ws used. The medi used to culture the resistnt isolte contined 25 µg of NO nd 20 µg of NA per ml. The culture ws prepred from n overnight culture previously trnsferred three times in trypticse soy roth. The chllenge inoculum ws prepred y serilly diluting the culture in sterile phosphte uffered sline (PBS) to concentrtion of pproximtely 10 6 cfu per ml. The cfu of the chllenge inoculum ws confirmed y plting on BGA pltes. On d 4 of the molt, ll the hens in groups 1, 2 nd 3 were chllenged y crop gvge with 1ml of inocul contining pproximtely 10 6 cfu of NA nd NO resistnt SE. The chllenge dosge ws slighter higher thn the 5.6x10 4 cfu dosges reported y Holt (1993) to e the men

57 45 infectious dosge for SE in molted hens. Groups, 4, 5 nd 6 were not chllenged with SE. Blood Collection nd Chemicl Anlysis Twenty-one guge, 1.5 inch needles were used to collect pproximtely 8 ml lood from the jugulr vein in the neck of 12 irds which were rndomly selected on t the eginning of the study to otin the seline dt. Eight hens were led in similr mnner from ech tretment group on dys 2, 5, 9 nd 12 of the molt. Eight ml ws plced in 10 ml non-heprinized lood collection tues. The plsm ws seprted y centrifugtion t 2500 rpm X 15 min, the cler superntnt plsm from ech smple were collected nd plced in plstic vils nd were stored t -20 C until chemicl nlysis could e conducted. A clinicl chemistry nlyzer (Gilford Impct, 400E, Ci Corning Dinostic Corp., Oerlin, OH 44774) ws used to nlyze spectrophotometriclly, the concentrtions of the plsm metolite prmeters in ech of the smples using methods s descried y Kuen et l. (1985) nd Prk et l. (1999). Chemicl regents otined from Byer HelthCre (Byer Dignostics, Europe Limited, Chpel Lne, Swords, Co. Dulin, Irelnd) were used s outlined in the mnufcturer s mnul to determine the concentrtion of the following plsm metolites: clcium, cholesterol, totl protein, glucose, triglyceride, nd uric cid. Cuvettes were loded with 1 ml of the respective regent nd 20 µl of the smple. The cuvette were gitted to ensure proper mixing of the liquids nd then llowed to incute t room temperture for pproximtely 5 min, fter which they were red y the chemicl nlyzer.

58 46 Sttisticl Anlysis The concentrtions of lood plsm metolites were summrized in ech tretment nd mens of ech metolite over the 12 d period were nlyzed using repeted mesures design. SAS Proc GLM (SAS version 8.3, SAS Institute Inc., Cry NC, 2001) ws used with tretment, time, nd tretment y time interction nd individul hens nested within tretment s the fctors. Chicken nested within tretment ws the error term used to test for tretment effects. When significnt (P 0.05) tretment y time interctions ws found, mens were compred using Lest Significnt Difference. Results nd Discussion Feed Intke nd Weight Loss The feed intke response to the lflf crumle diet in oth experimentl trils exhiited significnt differences etween the FF groups nd the ALC groups y s much s 6 fold in oth trils (Tle 3.1). Over the 12 dy molt period for oth trils the FW+ (34.82 nd 35% in tril 1 nd 2 respectively) hens lost significntly more (P 0.05) ody weight thn the FF+ (1.67 nd 1.50% in trils 1 nd 2 respectively) nd the ALC+ (18.40 nd 20% in trils 1 nd 2 respectively) hens lost significntly more weight thn the FF+ hens.

59 47 Tle 3.1: Effects of lflf crumle diet on feed intke nd weight loss in lying hens experimentlly infected with Slmonell Enteritidis (SE) trils 1 nd 2 Tretment Tril 1 Tril 2 Feed intke %Weight loss Feed intke %Weight loss g/hen/dy /hen g/hen/dy /hen FW+ N/A ± 2.47 N/A ± 3.00 FF ± ± 2.43 d ± ± 1.30 d ALC ± ± 3.44 c ± ± 2.10 c FW- N/A ± 3.21 N/A ± 2.10 FF ± ± 4.58 d ± ± 1.90 d ALC ± ± 3.47 c ± ± 2.70 c -d Mens within columns with no common superscript differ significntly (p<0.05). FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens.

60 48 This ws consistent with results oserved y Woodwrd et l. (2005) nd Lnders et l. (2005) when they demonstrted tht lflf fed hens lost pproximtely 19% of their totl ody weight when compred to FF hens which ctully gined weight. Donlson et l. (2005) reported in molting study tht feed deprived hens lost up to 25.80% of their totl ody weight which did not differ significntly from hens fed diet with 100% lflf mel (25.10%) or diet with 90% lflf mel. The weight loss could e result of the regression of the reproductive system (Donlson et l., 2005). Bker et l. (1983) demonstrted tht pproximtely 30% reduction in ody weight is needed for the ovry to successfully regress. Berry nd Brke (1985) stted tht pproximtely 25% of the ody mss loss cn e ttriuted to reduction in liver nd reproductive orgn weights. Brke (1993) lter concluded tht ovrin weight loss occurred simultneously with ody mss loss. Donlson et l. (2005) nd Lnders et l. (2005) oserved no significnt difference etween feed withdrwl hens nd lflf molted hens in ovry weight. The ody weight loss of these hens ws significntly lower thn the non-molted hens which lso exhiited significntly higher ovrin weight. Other explntions for the weight loss seen in the ALC hens could e the reduced feed nd wter intke, low metolizle energy in lflf, nd lso these hens could e using their ft reserves nd they te much less thn the FF hen nd therefore could not mintin their ody weight. The difference in weight loss etween the hens in oth ALC groups nd the hens in oth FW could e result of the slow pssge of lflf through the gstrointestinl trct of the ALC hens mking the intestines of the trct hevier thn the FW hens. Mrosovsky nd Sherry (1980) reported tht irds hd suppressed ppetite during

61 49 nturl molt, nd Sen et l. (1988) concluded tht reduced feed intke y hens fed lflf could e s result of the low pltility. Mtsushim (1972) reported tht the sponins present in lflf could ply role in the suppression of feed intke nd growth. Erlier, Sild (1979) demonstrted the slow pssge rte of lflf (more thn 24 h) through the chickens gstrointestinl trct he concluded tht this gve the irds feeling of stiety cusing them to reduce their intke. Clcium Concentrtion Significnt tretment y dy effect ws oserved for clcium concentrtion (Figure 3.1) in oth trils 1 nd 2 (P 0.001). On d 2 of oth trils no significnt differences (P 0.05) were oserved etween the ALC+ hens nd the other tretment groups in clcium concentrtion. On d 5 in tril 1 the FF- hens exhiited significntly higher (P 0.05) concentrtions of clcium in the lood thn the other tretment groups nd in tril 2 the FF+ groups hd higher levels (P 0.05) of clcium thn the ALC+ hens nd FW+ hens which were not significntly different (P 0.05). A similr trend ws oserved on d 9 in oth trils when the FF+ hens exhiited higher concentrtions of clcium thn the FW+ nd ALC+ hens which were not significntly (P 0.05) different in tril 1. On d 12 in tril 1 the FF+ hens exhiited significntly higher (P 0.05) levels of clcium thn the FF- nd FW- hens ut not the other tretments. The ALC+ hens did not disply significnt differences (P 0.05) from the other groups. The on the finl dy of the molt in tril 2 the concentrtions of clcium were significntly higher (P 0.05) in the FF+ hens thn the FW+ nd ALC+ hens.

62 Clcium Tril 1 FW+ FF+ ALC+ FW- FF- ALC- mg/dl c d dc d d c c 5 0 Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.1: Effects of lflf on lood clcium levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

63 Clcium Tril 2 FW+ FF+ ALC+ FW- FF- ALC- mg/dl c c c Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.1: Continued

64 52 Lying hens require high levels of clcium in the diet to sustin egg shell production this ws evident in oth trils where we oserved the FF hens hd higher levels of the metolites in the lood. Clcium is moilized from the ones nd intestines nd trnsported to the reproductive trct for deposition in the shell glnd; therefore it would e expected tht the FF hens hve higher levels of clcium in the serum the FW hens nd the ALC hens ecuse they hd cesed egg production. Yosefi et l. (2003) oserved decresed levels of egg shell glnd nd the intestinl clindin during molting due to its dependency on clcium. Clndin is present in the intestines of irds efore the onset of reproduction to ccommodte high clcium demnds for shell clcifiction (Striem nd Br, 1991). Totl Protein Concentrtion A significnt tretment y dy effect ws oserved in totl protein concentrtion (Figure 3.2) in the plsm (P 0.001)in oth trils 1 nd 2. There were no significnt differences (P 0.05) in totl protein concentrtion mong the tretment groups on d 2 in tril 1 however; in tril 2 the FF+ hens exhiited significntly higher concentrtions of totl protein thn the ALC+ nd FW+ hens on d 2. On d 5 in oth trils the SE+ groups exhiited no significnt differences (P 0.05) in totl protein concentrtion, while on d 9 in oth trils, the FF+ hens exhiited higher (P 0.05) concentrtions of totl protein ( nd mg/dl in tril 1 nd 2 respectively) thn the ALC+ hens (5950 nd mg/dl in tril 1 nd 2 respectively) nd the FW+ hens (6662 nd in tril 1 nd 2 respectively). The ALC+ nd FW+ hens did not differ significntly. This

65 53 mg/dl Totl Protein Tril 1 c FW+ FF+ ALC+ FW- FF- ALC- c c c Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.2: Effects of lflf on lood totl protein levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

66 54 mg/dl FW+ FF+ ALC+ FW- FF- ALC Totl Protein Tril 2 c c Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.2: Continued

67 55 result ws repeted on d 12 of the tril 1when we oserved tht the FF+ hens gin exhiited higher (P 0.05) levels of totl protein thn the hens in the other tretments. In tril 2 on d 12 the FF+ hens exhiited higher (P 0.05) levels of totl protein thn the FW+ hens nd the ALC+ hens. The negtive tretments did not differ significntly (P 0.05) from FF+ nd the FW+ hens ut they differed (P 0.05) from the ALC+ hens. The mount of protein required for integumentl synthesis such s fether synthesis is less thn one-third of tht deposited in single egg (Murphy, 1994), however, molting involves n ccelertion of the totl protein turnover (Murphy nd Trusico, 1995) nd protein lost during n induced molt is lso eing restored (Hrms, 1983; Hoyle nd Grlish, 1987). Puvdolpirod nd Thxton (2000) reported elevted levels of totl protein in ACTH treted chickens (3960 mg/dl) when compred to nontreted chickens (3410 mg/dl). Triglyceride Concentrtion Significnt tretment y dy effects (P ) were oserved in the levels of triglycerides (Figure 3.3) found in the lood plsm in trils 1 nd 2 during the nine dy tril. On d 2 in oth trils no significnt differences were oserved mong the tretments. On d 5 in tril 1 ll FF hens exhiited significntly higher (P 0.05) levels of triglycerides in the lood thn ll FW nd ALC hens. Similr results were oserved in tril 2 on d 5 ut the FF- hens exhiited higher (P 0.05) levels thn ll the other groups (3990 mg/dl) nd the FF+ hens exhiited higher (P 0.05) levels (2665 mg/dl) thn ll FW hens nd ll ALC hens. Significnt differences (P 0.05) were not oserved mong ny of the ALC

68 Triglycerides Tril 1 FW+ FF+ ALC+ FW- FF- ALC- mg/dl Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.3: Effects of lflf on lood triglycerides levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

69 57 Triglycerides Tril 2 mg/dl FW+ FF+ ALC+ FW- FF- ALC- c c c c Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.3: Continued

70 58 or FW tretments. The trend of reducing levels of triglycerides in the lood of the FW hens nd the ALC hens ws oserved on d 9 in oth SE+ nd SE- hens in oth trils.all FF hens exhiited significntly higher (P 0.05) levels of triglycerides thn ll ALC hens nd ll FW hens. All the ALC nd FW hens exhiited similr (P 0.05) levels of triglycerides in the lood on d 9. The lowest concentrtions of triglycerides were oserved on d 12 in oth tril 1 nd 2. In generl the SE+ nd SE- hens in the FW nd ALC tretments in oth trils, exhiited significntly lower levels of triglycerides thn the FF hens. High levels of triglycerides re in circultion in lying hens, moilized from the liver for the purpose of folliculr development, during reproductive rest, the mount of triglycerides in circultion is reduced. This could e relted to the reduced ovrin weight oserved y Lnders et l. (2005) when they molted hens with lflf mel (5.1 g), pellet (7.0 g) feed withdrwl (6.2 g) nd compred the results to non-molted hens (35.5 g). Puvdolpirod nd Thxton (2000-d) stted tht n elevtion in triglyceride levels could e used s stress indictor, this elevtion in triglycerides results from the trnsporttion of the triglycerides from the liver to tissues such s the muscles nd dipose tissues (Assmnn nd Jerzy-Roch, 2003). Apprently this ws not the cse in the hens which were molted in the current study since these hens did not disply incresed levels of triglycerides in the lood. Anthony et l. (1999) lso oserved reduced plsm concentrtions of triglycerides in fsted hens which were chllenged with Psteurell multocid nd Mumm et l. (2006) oserved decresed plsm levels of triglycerides in

71 59 lying hens during dreno- corticotrophin hormone induced stress while they oserved n increse in roilers. Cholesterol Concentrtion There ws no tretment y dy effects (P > 0.85) in cholesterol concentrtion (Figure 3.4) in oth trils 1 nd 2, we however oserved tretment effects. No significnt differences (P > 0.05) were oserved etween the ALC+ hens nd the other tretment groups in levels of cholesterol in the plsm on d 2 of oth trils nd 2 however; in tril 2 the FF+ hens hd significntly higher levels of lood plsm cholesterol thn the FW+ hens. Significntly elevted (P 0.05) cholesterol concentrtions were oserved in the FW+ hens on d 5 in oth trils 1 nd 2. On d 5 the ALC+ hens exhiited levels similr (P > 0.05) to the FF+ hens in tril 1 nd 2. No significnt differences (P > 0.05) were oserved in tril 1 on d 9 nd 12 mong the tretment groups in cholesterol concentrtion In tril 2 on d 9 the FW hens hd significntly higher (P 0.05) levels of cholesterol thn the FF+ hens, the ALC+ hens were not significntly different from the FF+ hens. On d 12 in tril 2 the FW- hens hd significntly higher levels of cholesterol thn ll ALC hens. Throughout the molt the FW nd ALC hens displyed incresing levels of serum cholesterol. This increse ws not lwys significntly different t P 0.05 from the FF hens which t times exhiited lower levels of cholesterol in the plsm. Wlzem et l. (1994) postulted tht this trend ws expected in molted hens, s the resorption of tretic follicles during the process of molt should increse the mount of circulting

72 Cholesterol Tril 1 FW+ FF+ ALC+ FW- FF- ALC- 250 mg/dl Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.4: Effects of lflf on lood cholesterol levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

73 61 mg/dl FW+ FF+ ALC+ FW- FF- ALC Cholesterol Tril 2 c c c c c c c 50 0 Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.4: Continued

74 62 cholesterol in the lood, lso, filure to ovulte could cuse n increse in insolule frction of cholesterol, resulting in higher concentrtions of cholesterol. Mumm et l. (2006) stted tht plsm cholesterol levels re incresed during stress nd Siegel (1995) stted tht elevted levels of plsm cholesterol were ssocited with stress in chickens. Puvdolpirod nd Thxton (2000) stted tht stressed irds exhiited elevted levels of plsm cholesterol. The reduced levels of cholesterol oserved in the ALC hens in this study could e result of lflf sponins which cn reduce the levels of serum cholesterol in the lood. It is sid tht lflf sponins form insolule complexes with cholesterol in the gut lumen (Coulson nd Evns, 1960). This is consistent with the reduction in lood plsm cholesterol when sponins were fed to chicks nd dult roosters oserved y Griminger nd Fisher (1958). Glucose Concentrtion No tretment y dy effect (P > 0.2) ws oserved in glucose concentrtion (Figure 3.5) in the plsm however, there were tretment effects in oth tril 1 nd 2. In tril 1 ll the FF hens exhiited higher levels (P 0.05) of glucose in the lood thn the ALC+ hens, the FW+ hens ws not significntly different (P > 0.05) from the FF+ hens or the ALC+ hens. On d 2 in tril 1 the ALC+ hens did not disply significntly different levels of glucose thn the FW+ hen or the FF+ hens, however, the FF+ hens exhiited higher levels of lood glucose thn the FW hens. On d 5 of the molt in tril 1 the FF+ hens hd significntly higher (P 0.05) glucose concentrtions when compred to the FW+ nd the ALC+ hens which were not significntly different. In tril 2 on d 5, the FF+ hens were not significntly different (P > 0.05) from ny of the other tretment

75 63 mg/dl FW+ FF+ ALC+ FW- FF- ALC Glucose Tril 1 d c c cd Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.5: Effects of lflf on lood glucose levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

76 FW+ FF+ ALC+ FW- FF- ALC Glucose Tril 2 c c mg/dl Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.5: Continued

77 65 groups, the ALC+ nd FW+ hens were not significntly different (P > 0.05). In tril 2 on d 5, the FF+ hens were not significntly different (P > 0.05) from ny of the other tretment groups, the ALC+ nd FW+ hens were not significntly different (P > 0.05). The results of glucose concentrtion on d 9 of the tril 1 showed the FW+ nd the ALChens exhiited the lowest (P 0.05) concentrtions of glucose nd there were no differences etween the other tretment groups. No differences (P > 0.05) were oserved mong the tretments in tril 2. On d 12 of tril 1 the FW+ hens exhiited lower (P 0.05) levels of glucose in the lood thn the FF+ hens nd the ALC+ hens. This ws not the sme in tril 2, the ALC- hens exhiited the lowest levels of plsm glucose nd ws significntly different(p 0.05) from the other tretment groups except for the ALC+ hens. The FW+ nd FW- hens were deprived of feed for totl of 12 d while the ALC+ nd ALC- hens were given low clorie lflf diet yet oth the FW hens nd the ALC hens exhiited glucose levels tht were t times similr to the FF+ nd FF- hens. Plsm glucose concentrtions in chickens will generlly decline t the eginning of feed restriction (Ktnf et l., 1989; Edwrds et l., 1999) however, Scnes et l. (1980) did not oserve reductions in glucose concentrtions in feed restricted dult irds. Anthony et l. (1999) lso oserved depressed glucose concentrtions during feed deprivtion. Puvdolpirod nd Thxton (2000) reported tht glucose levels incresed in ACTH stressed induced chickens. Cherel et l. (1988) investigted molting fsts in king penguins nd oserved tht glucose did not chnge sustntilly during molting. Ngr

78 66 nd Meyer (1963) stted tht incresed circulting glucocorticoids induce gluconeogenesis which leds to elevtion in plsm glucose. Uric Acid Concentrtion Significnt tretment y dy effect (P < ) ws oserved in uric cid concentrtion (Figure 3.6). On d 2 uric cid concentrtion of the FW+ (2.04 nd 2.03 mg/dl for trils 1 nd 2 respectively) nd the ALC+ (1.75 nd 2.25 for trils 1 nd 2 respectively) hens were significntly reduced (P 0.05) when compred with the FF+ hens (5.14 nd 5.83 in trils 1 nd 2 respectively). On d 5 of the molt in tril 1 the FF+ hens hd significntly higher (P 0.05) uric cid (4.90) concentrtion when compred to the FW+ (1.86) nd the ALC+ hens (2.61), which did not significntly differ. The FF+ group hd higher levels of uric cid (6.15) thn the ALC+ hens (3.12) nd FW hens (2.67) groups in tril 2. The ALC+ nd FW+ hens did not differ significntly. On d 9 of the molt the FW+ hens exhiited lower concentrtions of uric cid (3.32 in oth tril 1 nd 2) thn the FF+ hens (5.95 nd 5.72 for trils 1 nd 2 respectively) nd the ALC+ hens (5.05 nd 5.72 for trils 1 nd 2 respectively), the ALC+ nd FF+ did not differ significntly. On the finl dy of tril 1, the ALC hens exhiited higher levels of uric cid thn the FW hens. No significnt differences were oserved mong the tretments on d 12 in tril 2. At the eginning of the tril reduction in the uric cid concentrtion ws oserved in oth the FW+ nd FW- hens nd the ALC+ nd ALC- hens. For the FW hens this could hve een the result of the lck of protein source which the hens would usully get from the diet. The ALC hens initilly did not redily ccept the lflf diet t

79 Uric Acid Tril 1 c FW+ FF+ ALC+ FW- FF- ALC- mg/dl cd c d cd c c cd c d 1 0 Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.6: Effects of lflf on lood uric cid levels in molting lying hens on dys 2, 5, 9, nd 12 fter molt initition in tril 1 nd tril 2. Hens were infected on d 4 of the tril therefore there were no SE positive hens on d 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments within individul dys tht hve no common superscript re significntly different (p 0.05).

80 Uric Acid Tril 2 FW+ FF+ ALC+ FW- FF- ALC- 5 mg/dl Dy 2 Dy 5 Dy 9 Dy 12 Dy of Tril Figure 3.6: Continued

81 69 first s feed source which could ccount for the reduction in uric cid concentrtion oserved t the eginning of the tril. Puvdolpirod nd Thxton (2000) oserved incresed levels of uric cid in stressed irds. Cherel et l. (1988) found tht plsm concentrtion of uric cid ws highly vrile, incresing fter the thirteenth dy of the fst, which ws n indiction of the utiliztion of endogenous proteins. Anthony et l. (1999) oserved low uric cid concentrtion erly fter feed deprivtion in slow growing turkeys nd reported tht the rte of protein degrdtion incresed with the durtion of feed withdrwl nd protein degrdtion ws indicted y plsm uric cid levels. The levels of uric cid in the molting groups stedily incresed towrds the end of the molt period, the ALC+ hens exhiited higher levels of uric cid thn the FW+ hens, which could e result of incresed microil ctivity within the gstrointestinl trct of the ALC hens when compred to the FW hens. Thxton nd Puvdolpirod (2000) dvised cution when ttempting to dignose stress using physiologicl chnges since it consisted of series of non-specific doptive response tht fcilittes the return to homeostsis, however, lood plsm metolites cn e used s indictors of incresed stress, sed on work done y other reserchers. In generl, the non-chllenged hens exhiited levels of lood plsm metolites tht were similr to their SE chllenged counterprt, therefore the presence of n infective orgnism in the hens did not lter the results even though this would men the SE+ hens were exposed to n dditionl stressor. It hs een estlished tht induced stress results in the elevtion of the lood plsm metolites triglycerides, glucose, cholesterol, totl protein nd uric cid. These elevtions were generlly not oserved in the lflf fed

82 70 hens s they displyed reducing levels of triglycerides which could e ssocited with the regression of the reproductive trct. The lflf fed hens displyed glucose levels tht were comprle with the full-fed hens, while the SE+ FW hens exhiited mens glucose levels for the molt period tht ws higher thn the fed hens, whether, lflf fed or full-fed. The totl protein levels exhiited y the ALC hens were t times lower or similr to the FF hens for the tril nd could e result of reduced feed intke during the period. At the eginning of the tril the ALC hens exhiited reducing levels of uric cid, this however chnged nd y the end of the tril these levels were similr to tht of the FF hens. This cn e interpreted s the result of protein degrdtion nd since lflf is good source of protein we cn conclude tht the diet ws eing metolized. Bsed on the results from this study we conclude tht lflf crumle diets did not result in elevtion of the metolic stress mrkers exmined. Further studies hve een performed to evlute the immune response of molting hens fed n lflf diet.

83 71 CHAPTER IV IMMUNE AND STRESS PROTEIN RESPONSE IN MOLTING LAYING HENS CHALLENGED WITH SALMONELLA ENTERITIDIS AND FED ALFALFA CRUMBLE DIETS Introduction Molting occurs nturlly in the vin species usully t the end of reeding seson. For commercil egg producers this will men period of unproductivity ecuse the molt is incomplete in commercil lying hens nd they continue to ly eggs t low rtes for prolonged period of time (Berry, 2003). In order to otin more uniform molt within the flock commercil egg producers in the US historiclly hve used feed removl to induce molt. Conventionl feed withdrwl progrms involves the removl of feed, wter (no more thn 3 dys) or oth from the hens nd lso reducing the photoperiod in the houses to 8 h lighted period or less. The removl of feed should lst for period which is long enough to cuse the complete involution of the reproductive trct (Berry, 2003). Induced molting y feed withdrwl increses the susceptiility of hens to Slmonell Enteritidis (SE) infection (Holt, 2003; Ricke 2003). Slmonell infection in lying hens is serious food sfety concern, since infection of ird could led to the production of SE positive eggs fter molt (Humphrey et l., 1993). While poultry is not the only source of SE to humns, it is y nd lrge the most common when the source of the infection cn e trced, nd eggs re the primry source (Ptrick et l., 2004). A vriety of different physiologicl mechnisms contriute to the hens reduced resistnce

84 72 to pthogens during molting. Among the investigted mechnisms tht re known to cuse decresed resistnce include ltertions in the intestinl microflor (Corrier et l., 1997; Holt nd Porter, 1992; Svge, 1989), intestinl physiology (Brnes et l., 1979; Corrier et l., 1997; Duke, 1986; Holt, 1993), nd host defenses (Ben-Nthn et l., 1981, 1977; Holt, 1992, 1993). During n induced molt, immunosuppressive effects on T-cell immunity (Holt, 1992, 1993) nd humorl immunity (Ben-Nthn et l., 1981, 1977) hve een reported nd even with the mssive invsion of inflmmtory cells to the site of infection, molted hens re still more susceptile to infection. Wolford nd Ringer (1962) oserved tht molting influenced the differentil white lood cell counts y cusing reduction in lymphocytes numers nd incresing heterophil numers in the peripherl lood. An cute phse response is medited erly during inflmmtion, nd is mnifested y n increse in the production of serum proteins from the liver (Godson et l., 1995). These serum proteins, specificlly cute phse protein (APP) re relesed into the loodstrem y vriety of stimuli including inflmmtion (Thoms nd Schrieer, 1985; Fleck, 1989; Jmieson et l., 1992), cteril infection (Morley nd Kushner 1982; Pfeffer nd Rogers, 1989) nd endotoxin exposure (Tkhshi et l., 1995). Holt nd Gst (2002) oserved tht irds tht were experimentlly infected with SE nd were molted or not molted, exhiited sustntilly higher APP levels thn non-infected irds. They concluded tht serum APP; specificlly α 1 cid glyco-protein (AGP) levels cn e effective nd rpid indictors of infection nd cn e useful in trcking the infection sttus of flock.

85 73 Animl welfre concerns of reduced welfre of hens tht re force molted y feed deprivtion hs led to investigtions into lterntive methods of inducing molt. Different methods hve een exmined for their potentil to induce molt insted of using feed deprivtion nd the methods usully incorporte some form of nutritionl imlnce to induce molts (Bell, 2003; Prk et l., 2004). Low energy high fier diets such s whet middlings, cotton seed mel nd jojo mel hve een shown to successfully induce molts (Biggs et l., 2004; Dvis et l., 2002; Arnouts et l., 1993) nd limit SE coloniztion (Seo et l., 2001). Our previous reserch hs evluted the potentil of lflf to serve s the primry dietry component for molt induction nd retention of egg production during the second egg lying cycle (Donlson et l., 2005; Lnders et l., 2005,; Woodwrd et l., 2005). When used to induce molt in lying hens, lflf limited SE coloniztion nd infection during the molt (Woodwrd et l., 2005; McReynolds et l., 2005; 2006). Due to the concerns of immunosupression during n induced molt, the present study ws undertken to ssess the cpility of lflf to reduce immunosupression during n induced molt. The specific ojectives were to evlute the following immune responses to SE infection; serum ntiody, intestinl mucosl ntiody, ile ntiody, chnges in the H: L rtios nd lso to evlute the levels of AGP concentrtion in the serum of SE infected hens on n lflf crumle diet fed during n induced molt.

86 74 Mterils nd Methods Experimentl Design Experiments 1 nd 2 were conducted using 250 Single Com White Leghorn hens (SCWL) over 50 weeks old, which were otined from locl commercil lying flock. Twelve hens were used in ech of 6 tretments in tril 1 nd 10 hens were used in ech of 6 tretments in tril 2. The hens were plced in wire lyer cges nd were given free ccess to wter nd un-medicted corn-soyen mel sed mshed lyer rtion tht met Ntionl the Reserch Council recommendtions for nutrients (1994). The lflf crumle diet ws considered to e high in crude fier (24. 1%), with moderte crude protein (17. 5%) nd low in metolizle energy (1200 kcl/kg; NRC, 1994). Feed nd fecl smples (1 g) were collected nd exmined for slmonelle. Smples were cultured in tetrthioninte roth nd on rillint green gr 1 (BGA) pltes s ws previously descried y Andrews et l. (1995). All the hens nd feed used in oth trils tested negtive for Slmonell. The hens were llowed to cclimtize in the cges for period of two weeks fter which 12 (10 in the cse of tril 2) hens were rndomly ssigned to 6 tretment groups designted s followed; (1) feed withdrwl SE+ (FW+), (2) full fed SE + (FF+), (3) 100% lflf crumle SE + (ALC+), (4) feed withdrwl SE - (FW-), (5) full fed SE -, nd (6) 100% lflf crumle SE - (ALC-). The molt procedure used to induce the molt ws modifiction y Holt (1993) which ws previously descried y Brke et l. (1982). Tretment diets were pplied to ech tretment group on d 1 of the molt, t the

87 75 sme time feed ws removed from the feed withdrwl hens. Tretment diets were dministered for 12 dys, the period of time the FW hens were deprived of feed. Hens in ll the tretment groups were given wter d liitum. The SE positive hens were ll plced in the sme room, while the SE negtive hens were plced in seprte room. Foot ths were plced t the doors of ll the rooms in the fcility nd non-infected hens were cred for efore the infected hens ech dy. The hens in ll the tretment groups were exposed to 8 h light: 16 h drk photo-period one week efore chnging the diets nd removing feed from the FW hens. This light schedule continued for 12 dy period fter which the experiment ws terminted. Bcteril Strin For this experiment primry poultry isolte of S. Enteritidis (phge type 13A) from the Ntionl Veterinry Services Lortory, Ames, Iow, selected for resistnce to novoiocin nd nlidixic cid (NO nd NA) in the USDA-ARS fcility, College Sttion, TX, ws used. The medi used to culture the resistnt isolte contined 25 µg of NO nd 20 µg of NA per ml. The culture ws prepred from n overnight culture previously trnsferred three times in trypticse soy roth. The chllenge inoculum ws prepred y serilly diluting the culture in sterile phosphte uffered sline (PBS) to concentrtion of pproximtely 10 6 cfu per ml. The cfu of the chllenge inoculum ws confirmed y plting on BGA pltes. On d 4 of the molt, ll the hens in groups 1, 2 nd 3 were chllenged y crop gvge with 1ml of inocul contining pproximtely 10 6 cfu of NA nd NO resistnt SE. The chllenge dosge ws slighter higher thn the 5.6x10 4 cfu dosges reported y Holt (1993) to e the men

88 76 infectious dosge for SE in molted hens. Groups, 4, 5 nd 6 were not chllenged with SE. Blood Collection Twenty-one guge, 1.5 inch needles were used to collect pproximtely 8 ml lood from the jugulr vein in the neck of 12 irds which were rndomly selected on t the eginning of the study to otin the seline dt. Eight hens were led in similr mnner from ech tretment group on dys 2, 5, 9 nd 12 of the molt. Seven ml ws plced in 10 ml non-heprinized lood collection tues nd the remining 1 ml ws plced in EDTA tues to e used for smering microscope slides to count for heterophil to lymphocyte rtio. The plsm ws seprted y centrifugtion t 2500 rpm X 15 min, the cler superntnt plsm from ech smple were collected nd plced in plstic vils nd were stored t -20 C until chemicl nlysis could e conducted. One drop of lood ws plced on microscope slides to e counted for heterophil to lymphocyte rtio (H: L). The serum ws plced into liquots of 200 μl nd stored t -20 C until it ws used for ELISA ssys. Serum smples were diluted in sterile PBS to 1: 320. Intestinl Smple Collection nd Preprtion Following euthnsi, smples were collected from the ileum nd the cec. The smples were immersed in ice cold Hnk s Blnced Slt Solution (HBSS) contining 500 IU/ml of penicillin nd 500 μl of streptomycin (Zigtermn, et l., 1993). The gut contents were removed y gently flushing ech tissue section with HBSS. Ech tissue smple ws cut into pieces weighing 0.3 g nd susequently cut in three smller pieces nd finlly wshed with HBSS contining 500 IU/ml penicillin nd 500 μl streptomycin.

89 77 The tissue smples were suspended in 6 ml of RPMI 1640, supplemented with 100 IU/Ml of gentmicin nd 40 mm HEPES uffer. The suspensions were susequently centrifuged for 5 mins (300 x g). Smples were incuted t 41 C, 5% CO 2, nd 95% ir in 12-well culture pltes for 16 h. After incution, the smples were plced in 200 μl liquots nd stored t -20 C until they were used for ELISA ssys to mesure ntigen specific IgA. The intestinl medi smples were diluted in PBS (ph 7.4) 1: 2. Bile Smpling During necropsy, pproximtely 1 ml of ile ws spirted from the gll ldder of ech hen using 22 inch guge X 0.5 inch needle with 2 ml syringe. The smples were plced into micro centrifuge tues nd spun t 15,000 rpm for 30 min t 4 C to seprte the lrger proteins. The smples were stored t -20 C until needed nd t tht time smples (100 μl) were diluted to 1: 250 in PBS (ph 7.4) for ELISA. Indirect ELISA An indirect ELISA ws performed on the serum, intestinl nd ile smples tht were otined from the irds. Lipopolyscchride (LPS) t concentrtion of 50 µg: 10 mg ws used to cot the 96-well pltes with 190 µl of the solution pipetted in ech well on the pltes. The pltes were susequently llowed to incute overnight nd the following dy they were rinsed four times with PBS Tween (ph 7.4, 0.5% Tween 20). The smples from the serum, intestinl medi nd ile were dded (190 µl of ech) to the wells using the dilutions mentioned previously. After incution nd rinsing, horse rdish peroxidse-conjugted got nti-chicken IgG (for the serum smples) nd IgA (for the ile nd intestinl smples) were dded to ech well. The pltes were gin incuted

90 78 for 1h nd rinsed efore 150 μl of 1-step Turo TMB ws dded to ech well. After 20 mins, the rection ws stopped y dding 50 μl of 1 M sulfuric cid. The pltes were red for sornce y multi-well plte reder (Spectr Mx, Microplte Spectrophotometer, Sunnyvle, CA) t wvelength of 450 nm. AGP Assy Serum levels of AGP were evluted using commercilly ville rdil immunodiffusion try (Crditech Services Inc. Louisville, KY). The grose trys were impregnted with nti-ser specific for chicken AGP. Ech try hd ten wells cut into the grose for smple ppliction. Ech kit contined two stndrd smples; low of 250 μg/ml nd high of 1000 μg/ml of AGP. Of these stndrds, 5 μl were plced into individul wells on the try s the results would lter e used to provide dt for generting stndrd curve. The other 8 wells were filled with 5 μl of the test smples, plced in moisture chmer, nd incuted overnight t 25 C. The interction of the migrting AGP from the stndrds nd the serum smples through the gr with the nti- AGP tht ws present in the mtrix ws indicted y the formtion of ring. The concentrtions of AGP in the smples nd stndrds were directly correlted with the dimeter of the rings, ssuming the higher the concentrtion of AGP in the smple, the lrger the dimeter of the ring tht would e formed. The dimeters of the two stndrds concentrtions vlues were mesured using mesuring guge provided in the kit. The vlues were plotted on semi-logrithmic pper provided in the kits with the concentrtion of AGP on the horizontl xis nd the dimeter on the verticl xis. A line

91 79 ws drwn etween the two points nd served s the reference curve for the concentrtions of the remining smples on the try. Sttisticl Anlysis Intestinl nd ile IgA dt were determined y nlysis of vrince using the generl liner models procedures. Significnt differences (P 0. 05) were seprted using Duncn s multiple rnge tests nd sttisticl nlysis softwre. The H: L, APP nd serum ntiody dt were summrized in ech tretment nd mens of ech prmeter over the 12 d period were nlyzed using repeted mesures design. Proc GLM (SAS version 8.3, SAS Institute Inc., USA, 2001) ws used with tretment, time, nd tretment y time interction nd individul hens nested within tretment s the fctors. Chicken nested within tretment ws the error term used to test for tretment effects. When significnt (P 0.05) tretment y time interctions ws found, mens were compred using Lest Significnt Difference. Results nd Discussion Heterophil: Lymphocyte (H: L) Rtio Significnt (P < 0.001) tretment y time effect ws oserved in H: L rtios in oth trils 1 nd 2 (Figure 4.1). In tril 1 ll the tretment groups exhiited similr rtios up to d 2 of the molt (P 0. 05). In tril 2, the ALC hens exhiited n increse in H: L rtio on d 2 fter which n increse in the H: L rtios ws oserved for oth FW groups, this increse ws oserved in oth trils nd the response ws not oserved in the ALC hens or the FF hens. On d 4 SE ws dministered to groups 1, 2, 3, the dy fter n increse in the H: L rtio ws oserved in the FW- nd FW+ hens (0.32

92 80 Rtio Heterophil: Lymphocyte Tril 1 FW+ FF+ ALC+ FW- FF- ALC Dy of Tril c c Figure 4.1: The effects of lflf diets on heterophil: lymphocyte (H: L) rtios in Slmonell Enteritidis (SE) chllenged lying hens during molt in tril 1 nd tril 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments with the sme letter re not significntly different t p 0.05.

93 Heterophil: Lymphocyte Tril 2 FW+ FF+ ALC+ FW- FF- ALC- Rtio Dy of Tril Figure 4.1: Continued

94 82 nd 0.55 for tril 1 nd 0.30 nd 0.29 for tril 2 respectively), the increse oserved on d 5 ws significntly higher thn the rtios for the other groups (P 0.05). The ALC hens exhiited rtio of 0.06 for the ALC+ hens nd 0.13 for the ALC- hens in tril 1 nd 0.16 for the ALC+ hens nd 0.14 for the ALC- hens in tril 2, these results were not significntly different (P 0.05) from the FF+ hens (0.09 nd 0.13) nd the FF- hens (0.24 nd 0.17) in trils 1 nd 2. On d 9 pek in the H: L rtio ws oserved in oth trils 1 nd 2 for oth FW groups. The H: L rtio for the FW+ hens in the trils were 1.24 in tril 1 nd 0.54 in tril 2, while the FW- hens exhiited rtios of 1.74 in tril 1 nd 0.42 in tril 2. These rtios were significntly different from the other tretment These rtios were significntly different from the other tretment groups (P 0.05). Agin the mount of heterophil tht oth ALC groups produced ws not sttisticlly different (P 0.05) from the FF groups in oth trils. On d 12, drmtic reduction in the H: L rtios were oserved in the FW hens in oth trils, however, the rtios were still significntly higher (P 0.05) thn the other tretment groups in tril 1 ut not significntly different from the ALC- group in tril 2. It hs een reported tht increses in the H: L rtio in hens is n indictor of chronic stress sed on increses in corticosterone levels (Gross nd Siegel, 1983; Gross, 1989; Mxwell, 1993; Mxwell et l., 1992; Siegel, 1995) nd Lne (1987) suggested tht chnges in the levels of the vin heterophil could e used s the window to the stte of irds helth. The findings in the current study were consistent with reports y other reserchers (Dvis et l. (2000) who reported incresed H: L rtios nd incresed corticosterone levels when hens experienced restricted food ccess. Kogut et l. (1999) lso oserved significnt rise in

95 83 the numer of circulting heterophils within two dys of feed withdrwl which remined throughout the feed withdrwl period. They lso reported tht the highest rise in heterophil occurred during the first week of the molt with drmtic reduction y the end of the molt. The results from these trils re lso consistent with studies reported y Alodn nd Mshlly (1999) who oserved significntly higher circulting heterophil on d 5 of molt, when they compred FW hens with non-molted control hens. They concluded from their results tht the FW hens were under more stress thn the control hens. In the mjority of the vin species, lymphocytes re the most undnt leukocytes found in circultion (Mxwell nd Roertson, 1998). In poultry, pproximtely 59% of the totl white lood cells re lymphocytes nd pproximtely 27% re heterophil. Brke et l. (1981) reported tht recrudescence nd lymphocytic repopultion usully ccompnies reproductive quiescence in molting hens nd Alodn nd Mshly (1999) found tht peripherl lymphocytes decresed nd lymphocyte popultions chnged during the fsting period of molt. Jones (1989) found tht fsted nd frustrted Brown Leghorn pullets exhiited incresed H: L rtios. Contrry to wht ws oserved in the current study, Mxwell nd others (1990) investigted roilers tht were sujected to prolonged feed restriction, they found tht the H: L rtios of these irds were not significntly ltered when compred to irds tht were fed d liitum, they concluded tht irds ecme hituted to the effects of prolonged feed restriction. During the erly phse of feed restriction, the vin heterophil responds to hormonl surge of corticosterone (Mxwell et l., 1991, 1992, ; Svory et l., 1992; Hocking et l., 1993, 1994, 1996), however, this increse is not physiologiclly sustined; therefore the

96 84 heterophils re no longer recruited to the site of infection nd resulting in reduction of the rtio. Serum Antiody Production Significnt tretment y dy effect ws oserved for serum IgG levels in oth tril 1 (P < 0.03) nd tril 2 (P < 0.002). The results (Figure 4.2) reveled tht on the dy fter the SE ws dministered to the hens (d 5), the FW+ hens produced less (P 0.05) IgY thn the ll the other tretment groups, this ws oserved in oth tril 1 nd 2. The ntiody sornce levels for the FW+ hens ws pproximtely 0.69, while ll the other groups exhiited ntiody sornce levels of 0.81 or greter. There were no significnt differences (P 0. 05) etween the ALC+ hens nd the FF+ hens. In tril 1 on d 9 reduction in ntiody sornce levels ws oserved in ll the tretment groups. In tril 2 the FW+ hens they produced significntly less (P 0.05) IgY (0.65) on d 9 thn the ALC+ hens (0.79) nd the FF+ (0.75). No significnt differences (P 0.05) were oserved etween the ALC+ hens nd the other tretment groups on d 9 in tril 2. In oth trils 1 nd 2 on d 12, n increse ws oserved in mount of IgY produced y the FW+ nd ALC+ hens. The FW+ hens exhiited significntly higher (P 0.05) ntiody sornce levels in tril 1thn ll the other tretments with sornce levels of 0.91 while the FF+, nd ALC+, hd sornce levels of 0.64 nd 0.80 respectively, these groups were not significntly different (P 0.05), no significnt differences were oserved mong tretment groups on d 12 in tril 2.

97 85 A t 450nm Serum Antiody Tril 1 FW+ FF+ ALC+ FW- FF- ALC c c Dy of Tril Figure 4.2: The effects of lflf diets on serum ntiody production in Slmonell Enteritidis (SE) chllenged lying hens during molt in tril 1 nd tril 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments with the sme letter re not significntly different t p 0.05.

98 86 A t 450 nm FW+ FF+ ALC+ FW- FF- ALC- Serum Antiody Tril Dy of Tril Figure 4.2: Continued

99 87 Holt (1992) oserved significnt reduction in ntiody response to Brucell ortus in molting hens on d 5 ut no other effects were oserved. They concluded tht molting exerted miniml effects on the humorl system. Ben-Nthn et l. (1980) previously reported tht irds chllenged with sheep red lood cell or Stphylococcus ureus nd deprived of food for 48 h significntly reduced ntiody titers when compred to chllenge fed irds. In this study the FW+ hens exhiited reduced levels of circulting IgY erly in the molt ut lter incresed towrds the end of the 12 dy fst, this ws not oserved in the FF+ or the ALC+ hens indicting tht feed deprivtion did hve negtive effect on humorl response erly in the fst. Holt (1992) oserved reduced numer of B-cell oserved in irds on fsting induced molt while Desmidt et l. (1998) stted tht humorl immunity contriuted to the elimintion of SE from the gut of poultry. Intestinl nd Bile Antiody Response The FW+ hens exhiited significntly higher levels (P 0.05) of ileum IgA ntiody sornce levels thn the other tretment groups in tril 1 ut no significnt differences (P 0.05) were oserved etween the ALC+ hens nd the FF+ hens. In tril 2 no significnt difference (P 0.05) in the ileum IgA ntiody sornce levels mong the FW tretment groups nd the ALC tretment groups (Tle 4.1). The FW+ nd FWhens yielded IgA sornce levels of 1.43 nd 1.35 respectively while the ALC+ nd ALC- hens exhiited IgA sornce levels of 1.29 nd 1.09 respectively. There were no significnt differences (P 0.05) etween the two FF groups nd the ALC- group ws not significntly different (P 0.05) from the FF- group. However, the cecl IgA

100 88 production for the FW+ group ws significntly higher (P 0.05) IgA sornce levels thn the other tretment groups in tril 1 while in tril 2 the FW+ hens yielded significntly higher levels of IgA in the cec thn the FF hens nd the ALC hens. Mesurements of the ile ntiody sornce levels production in tril 1 indicted tht there were no differences mong the tretment groups. In tril 2 ll SE + groups hd higher IgA production with sornce levels of 1.50 (FW+ hens), 1.51 (FF+ hens), nd 1.51 (ALC+ hens). This ws significntly different (P 0.05) from the FW- nd FFhens (1.42 ech), ut not (P 0.05) from the ALC- hens (1.49). It ws reported y Holt (1992) tht induced molting hd significnt effects on circulting T lymphocytes in hens erly during fst, this in turn could hve significnt impct on ny immune response tht requires T cell supopultions. Nencione et l. (1983) nd Lillehoj (1991) stted tht intestinl intrepithelil lymphocytes cn potentilly protect ginst intestinl pthogens. Brito et l. (1993) lso oserved significnt increses in ntiody titer in the ile, intestinl mucos nd the serum of young chicks tht were infected with S. Typhimurium. Our ile ntiody production results were lso consistent with results oserved y Seo et l. (2002) who oserved opticl density vlues from SE positive ile tht were significntly different (P 0.05) from those of negtive smples. Zigtermn et l. (1993) lso oserved no difference in the cystic ile of chicken infected with Eimer tenell. They concluded tht this could hve een the result of reltively low concentrtions of specific IgA in the presence of high concentrtions of non- specific IgA. Mockett nd Rose (1986) lso were unle to show significnt responses in ile due to high ckground. The incresed levels of IgA oserved in the ALC- hens could

101 89 Tle 4.1: The effects of lflf diet on intestinl nd ile IgA secretion in Slmonell Enteritidis (SE) chllenged lying hens during molt in trils 1 nd 2 Tretment 1 Ileum Cec Bile Tril 1 FW+ 0.19± ± ±0.06 FF+ 0.11±0.03 c 0.06± ±0.03 ALC+ 0.12±0.05 c 0.06± ±0.02 FW- 0.18± ± ±0.05 FF- 0.12±0.04 c 0.05± ±0.03 ALC- 0.14± ± ±0.02 Tril 2 FW+ 1.43± ± ±0.03 FF+ 0.52±0.14 c 0.37±0.10 c 1.51±0.003 ALC+ 1.29± ±0.08 c 1.51±0.02 FW- 1.35± ± ±0.01 c FF- 0.72±0.09 c 0.25±0.06 c 1.42±0.04 c ALC- 1.09± ±0.02 c 1.49±0.01 -c Mens within columns with no common superscript differ significntly (p<0.05). Mens within columns represents ntiody titers in different segments of the intestine of hens in the tretments groups. 1 FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens.

102 90 e the due to the presence of sponins in the lflf. Sponins re steroids glycosides tht possess nti-inflmmtory nd ntioxidnt properties (Ro nd Gurfinkel, 2000). AGP Response Significnt (P < 0.006) tretment y dy effect ws oserved in the study (Figure 4.3). In tril 1 on d 2 we oserved no significnt difference (P 0.05) 2 etween the ALC tretment nd the other tretment groups, however, there were significnt differences (P 0.05) etween the FW hens nd the FF hens. On d 5 of the molt the FW hens nd the ALC hens produced similr (P 0.05) quntities of AGP nd were significntly higher (P 0.05) thn the FF hens. On d 9 in tril 1 ll tretments produced significntly different (P 0.05) mounts of AGP with the FW hens producing more thn the other two groups nd the ALC hens producing more thn the FF hens. On d 12 of the molt in tril 1 the FW hens produced significntly more (P 0.05) AGP thn the ALC nd the FF hens which were not significntly different (P 0.05). In tril 2 long with the infected groups, non-infected groups were included. There were no significnt differences (P 0.05) mong the tretment groups on d 2 of the molt. On d 5 in tril 2 we oserved no significnt differences (P 0.05) etween the molted infected nd the FW+ hens. The FF+ nd FF- hens were not significntly (P 0.05) different from ech other, or significntly different (P 0.05) from the two lflf groups. The non-molted groups were however significntly different (P 0.05) from the FW+ nd the FW- hens. The ALC+ nd the ALC- hens were not significntly different from the FF+ hens ut they were different from the FW- hens.

103 AGP Tril 1 FW+ FF+ ALC μg/ml c Dy of Tril Figure 4.3: Serum α 1 -cid glycoprotein (AGP) levels during n induced molt of Slmonell Enteritidis (SE) chllenged lying hens fed n lflf diet in tril 1 nd tril 2. FW+= Feed withdrwl SE positive hens, FF+= Full fed SE positive hens, ALC+= Alflf crumle diet SE positive hens. FW-= Feed withdrwl SE negtive hens, FF-= Full fed SE negtive hens, ALC- =Alflf crumle diet SE negtive hens. Tretments with the sme letter re not significntly different t p 0.05.

104 AGP Tril 2 FW+ FF+ ALC+ FW- FF- ALC μg/ml c c c c c c Dy of Tril Figure 4.3: Continued

105 93 Although Holt nd Gst (2002) oserved no significnt differences etween molted infected nd non-molted infected hens in two of four trils, they did oserve numericl differences with the molted infected hens producing higher concentrtions of AGP thn the non-molted infected hens they lso oserved no significnt differences mong the tretments on d 1 nd 3 post chllenge ut sw n elevtion in the AGP serum levels of the molted infected hens on d 8 nd d 10 post chllenge. These results were similr to wht we oserved on d 9 of the molt when the FW+ hens hd significntly higher titers of AGP thn the other tretments. Similrly on d 12 of the molt oth the FW+ hens nd the FW- hen hd significntly higher AGP levels thn the other tretment groups, while the ALC hens did not differ from the FF hens. In conclusion the infectious stte of lying hens during n induced molt is fctor in their immune response, generlly, the immune response of the SE- hens during this tril were typiclly lower thn the SE+ hens. Our findings suggest tht sed immunologicl indictors such s H: L rtio, n lflf crumle diet cn limit the increse in heterophili during the molt tht is occurs in hens molted y feed deprivtion, this ws true whether or not hens were infected with SE. Incresed heterophili cn cuse reduced function of the heterophil (Kogut et l., 1999) resulting in immunosuppression nd reduced resistnce to infections such s slmonellosis which cn e trnsmitted to humns vi poultry met nd eggs. The reduced serum ntiody response tht ws oserved in the FW hens t the eginning of the molt ws not oserved when hens were fed lflf crumle during n induced molt however, it ppers tht the infectious stte of the hens plyed role since the SE- hens did not disply reduced

106 94 ntiody levels. Likewise the ALC hens did not exhiit the incresed ntiody titers in the cec tht were oserved in the FW hens. In the current study we oserved tht lflf molt diets reduced the level of AGP in the serum to levels similr to non-molted full fed hens. This would indicte tht the induction of molt using lflf molt diets cn reduce inflmmtion from n SE outrek furthermore even in the sence of n infectious gent such s SE the lflf diet effectively reduced the levels of AGP in the serum during n induced molt. This corresponds with our previous work tht lflf sed diets effectively induce molt with post-molt performnce comprle to tht of feed deprived hens nd limit SE infection (Woodwrd et l., 2005; McReynolds et l., 2005, 2006). However, it is pertinent to note tht other fctors such s endotoxin exposure nd cteril infection cn cuse rise in the levels of serum AGP (Thoms nd Schreier, 1985; Pfeffer nd Rogers, 1989; Tkhshi et l., 1995). The use of n lflf diet during n induced molt could hve significnt impct on food sfety y reducing immunosuppression nd lleviting niml welfre concerns of reduced resistnce to disese due to strvtion.

107 95 CHAPTER V BEHAVIOR OF LAYING HENS ON ALFALFA CRUMBLE MOLT DIETS Introduction Commercil lying hens, like most species of wild irds, experience nturlly occurring molts (Swnson nd Bell, 1975). In the vin species molting usully involves the periodic shedding nd replcement of fethers, ut is usully incomplete in commercil lying hens nd they continue to ly eggs t low rtes for prolonged period of time (Swnson nd Bell, 1975). An incomplete molt mens period of unprofitility due to reduction of egg production nd the end of the useful life of flock (Berry, 2003). One of the lrgest incurred expenses in the commercil lyer industry is tht of replcement pullets (Bell, 2003). Commercil egg frmers typiclly extend the productive lying life of their flock from less thn 80 wk to 110 wk or even 140 wk through the use of induced molting (Bell, 2003). After the induced molt, egg production, nd egg qulity re improved significntly compred to the pre-molt period (eg. Wester, 2003; Bell, 2003; Swnson nd Bell, 1975). Molting my e induced y feed withdrwl for up to 10 dys (e.g. Christms et l., 1985), nd (or) wter withdrwl for two dys (North nd Bell, 1990), long with reduction of dy-length (e.g. Hemree et l., 1980). Feed nd wter withdrwl is controversil nd hs een outlwed in Europe ut feed withdrwl hs een used for molt induction in the U.S. (Appley et l., Prk et l., 2004). Feed withdrwl is lso prolemtic ecuse it hs een shown in experimentl models to increse Slmonell Enteritidis coloniztion nd infectivity in the gstrointestinl trct of lying hens (Holt, 2003; Ricke, 2003).

108 96 The prctice of feed withdrwl hs een chrcterized s stressful (Beuving nd Vonder, 1978) cusing generl deteriortion of the well-eing of nimls nd usully involves cscde of physiologicl dptive responses (Thxton nd Puvdolpirod, 2000). Duncn nd Wood-Gush, (1971) stted tht feed deprivtion ws frustrting nd negtively ffected the welfre of the ird due to the initil period of fsting to ring out the cesstion of ly. Wester (2000) postulted tht ehvior might e direct indictor of the well eing of hens undergoing feed withdrwl, however, studies tht quntified ggressive ehvior during induced molts hve een contrdictory. Aggrey et l. (1990) nd Hskell et l. (2000) reported incresed ggression in feed deprived hens while Hemree et l. (1980) did not oserve this incresed ggression. Wester (1995) found no significnt differences in ggression etween cged hens on 4 dy fst nd hens tht were not deprived of feed. Wester (2003) stted tht lthough there is no evidence tht mngement progrms involving feed withdrwl cuse deilittion when properly implemented, there is dete whether or not the hunger involved in feed withdrwl cuses suffering. In light of this, vrious methods of nutrient restriction tht would void long term feed withdrwl nd the use of dietry dditives hve een investigted (Berry, 2003, Wester, 2003, Prk et l., 2004). Alterntive methods for molt induction include feeding low clcium (Breeding et l.,1992) or low sodium diets nd high dietry zinc (Berry nd Brke, 1985), or high fier, low energy diets (Donlson et l.,2005; Lnders et l., 2005, ; Woodwrd et l., 2005; Biggs et l., 2003, 2004; Seo et l., 2001). Ech method is typiclly employed in comintion with chnge in the photoperiod, nd

109 97 usully leds to ody weight loss, the cesstion of egg production, regression of the reproductive system nd induction of molt (Shippee et l., 1979; Bell, 2003; Prk et l., 2004). Among the high fier low energy molt diets, lflf molt diets hve een shown to effectively regress the reproductive system nd ring out rpid return to egg ly t rte similr to feed withdrwl hens (Donlson et l, 2005; Lnders et l, 2005,, Woodwrd et l., 2005). Alflf diets lso pper to reduce S. Enteritidis coloniztion in the orgns nd reduce intestinl shedding of the pthogen during molting when compred to feed withdrwl hens (Woodwrd et l, 2005, McReynolds et l., 2005, 2006). However, hen ehvior during induced molting using lflf molt diets hs not een investigted. The ojective of this study ws to evlute the ehvior of hens nd compre these ehviorl ptterns to tht of feed withdrwl hens over 9 dy induced molt. Mterils nd Methods Experimentl Design Ninety Single Com White Leghorn hens pproximtely 60 weeks old were otined from locl commercil lyer frm. The hens were divided into two groups nd plced in floor pens with five roiler irds in ech pen, for period of 2 wks. This ws done to trin the hens to use the nipple wterers in the ttery cges. The hens in ech tretment were plced in identicl djoining temperture controlled rooms to prevent the influence of the ehvior of one tretment group on nother.

110 98 The hens were rndomly plced on oth tiers of two- tier ttery cges, with three hens per cge nd n llownce of 1100 cm 2 sq ft per ird. Twenty four hens were plced in ech room for two week cclimtiztion period, during which they were fed lnced un-medicted corn-soy en mel sed msh lyer rtion tht met the Ntionl Reserch Council requirements for nutrients (NRC, 1994) nd wter d liitum. The full-fed (FF) group served s the control group nd remined on the cclimtiztion rtion. Alflf typiclly contins 17 to 18% crude protein nd 24 to 25% crude fier nd hs low metolizle energy (1200 kcl/kg) when compred to lyer rtion with 2,965 kcl/kg (NRC, 1994). Hens in the ALC tretment group were fed lflf crumle d liitum for the 9 d tril. The lflf crumle ws otined y pssing 2 cm long lflf pellets through crumler tht reduced the pellets to pproximtely cm. Feed ws withdrwn (FW) from the third group for the 9 d durtion of the tril. One week prior to the eginning of the tril, the lighting schedule in the three rooms ws chnged from 16 h light, 8 h drk to 8 h light, 16 h drk. On dy 1, only the lflf diet ws offered to the ALC hens nd feed ws withdrwn from the FW hens. Eight cges (three hens in ech cge) on oth the upper nd lower tiers in ech room were video recorded. Two cmers were mounted pproximtely 1.2 m wy nd 30 ove the cges in ech of the three rooms, with one cmer focused on four cges. The video were recorded to digitl multiplexer (Kltel DVMRe Triplex ez Digitl Video Multiplexer Recorder, Corvllis, OR). Recordings egn t 1200 h ech dy nd ended t 1400 h nd two 10 min intervls were nlyzed ech dy from d 0 to d 9 of the tril.

111 99 One ten min intervl recorded t 1230 h nd second 10 minute oservtion period strted t 1330 h. Behvior Prmeters The 24 hens were the sujects for the ehvior nlysis in ech tretment. Seven different ehviors were dpted from Wester (2000) to ssess the irds welfre during the tril. Hed movement ws considered to e the rpid individul hed movement of n lert ird. This is suggestive of visul surveillnce y ird of its environment. Feeder ctivity involved ny pecking ehvior tht ws directed towrds the feeding trough while the focl ird ws close enough to the trough to et or when the ird hd her hed in the trough. Preening involved the mnipultion of the plumge with the ek. Drinking ws considered to e the pprent ingestion of wter y pecking from the nipple wterers. Nonnutritive pecking ehvior ws recorded when hens were pecking t nything other thn the feed, which included pecking t the cge floor, sides nd their own feet. Wlking ws defined s the locomotion of the hen involving t lest one step. Aggression ws recorded s ny ggressive pecking ehvior directed to nother hen either in the sme cge or in neighoring cge. Aggressive ehvior ws oserved s 0/1 occurrence. The dt were in the form of counts t one minute intervls tht were then summrized s the dily men percentge of oservtions in which the suject ws performing prticulr ehvior. Sttisticl Anlysis The verge of ech ctivity of the three hens in ech cge ws summrized nd mens of ech ehvior on ech of the nine dy period were nlyzed s cge verges

112 100 using repeted mesures design. Proc GLM (SAS version 8.3, SAS Institute Inc., 2001) ws used with tretment, time, nd tretment y time interction nd individul hens nested within tretment s the fctors. When significnt (P 0.05) tretment y time interctions ws found, mens were compred using Lest Significnt Difference. Results nd Discussion Aggressive Behvior nd Egg Production The study ws uneventful nd proceeded s plnned. There were no mortlities in the ny of the three tretments throughout the nine-dy molt. Aggressive ehvior ws not oserved in the FF or the ALC hens throughout the 9 d tril. One incidence of ggression etween hens in seprte cges ws oserved mong the FW hens on d 8 (dt not shown). Aggressive ehvior ws oserved y Duncn nd Wood-Gush (1971) nd Hskell et l. (2000) in frustrted hens. Wester (2000) nd Anderson et l. (2004) oserved tht ggression in food deprived hens declined s the molt progressed. McCown et l. (2006) oserved tht hens molted y feed deprivtion nd hens molted while on low clorie diet displyed incresed ggressiveness during the fst period. The ALC hens stopped lying on d 6 of the tril the sme time s the FW hens (Figure 5.1). The FF hens did not stop lying throughout the tril. This ws consistent with Biggs et l. (2004) who oserved tht hens molted on whet middlings diet stopped lying y d 6 of the tril. Previously Seo et l. (2001) oserved hens molted with whet middlings stopped lying on d 7. When Keshvrz nd Quimy (2002) induced molt in lying hens using grpe pomce, hens stopped lying y d 4 fter the

113 101 Egg Production Hen-dy egg production _15 _13 _11 _9 _7 _5 _3 _ Dy of Tril Figure 5.1: Hen dy egg production y the three tretments on dily sis from 2 wk prior molt induction nd during the 9 dy tril. The negtive numers represents the pre-tril dys. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24hens per group.

114 102 initition of the molt. Hed Movement No significnt tretment y dy effects (P 0.40) ws oserved mong the three tretment groups in hed movements (Figure 5.2) throughout the 9 d tril. These results suggest tht the ALC nd FW hens were no less ttentive or lert thn the FF hens. This ws similr to oservtions y McCown et l. (2006). However, Wester (2000) oserved incresed levels of ttentiveness mong fsted hens, s indicted y incresed hed movement n indiction of their lertness during the molt. Since the hens in the current study were seprted sed on tretment, the ctivities of one group would not ffect the other. Wester (2000) utilized prtitions to seprte tretments ut they were not isolted from the vocliztions of the control hens. Wlking Activity Significnt tretment y dy effects ws oserved in wlking ctivity (P 0.001). On d 1 of the tril the ALC hens spent more time wlking (Figure 5.3) thn the FW nd FF hens (P 0.03). On d 2 the FF hens did not differ significntly (P 0.10) from the other tretments, however the ALC hens spent more time (P 0.001) wlking thn the FW hens. On d 3 the ALC hens spent more time (P 0.05) thn the FW nd FF hens wlking round the cges. On d 4 the ALC hens nd the FF hens spent more time (P 0.03) thn the FW hens wlking. The results of d 5 were similr to wht ws oserved on d 3, the FF hens nd the FW hens spent less time thn the ALC hens wlking. While on d 6 wlking ehvior ws similr to d 4, the ALC nd FF hens spent more time wlking thn the FW hens.

115 Hed Movement 10 % Oservtion Dy of Tril Figure 5.2: Percentge oservtions of hed movement ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. No significnt differences were recorded t p 0.05.

116 Wlking 25 % Oservtion c Dy of Tril Figure 5.3: Percentge oservtions of wlking ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. Tretments within the sme dy with different letters re significntly different t p 0.05.

117 105 On d 7 the FF hens spent more time thn the other tretments involved in wlking ehvior, the ALC hens spent more time thn the FW hens, which spent the lest mount of time wlking. On d 8 the FW hens spent more time wlking thn the other tretments ut there were no differences etween the ALC hens nd the FF hens. There were no significnt differences mong ny of the tretments on d 9. Reduced wlking ctivity ws oserved in the FW hens s the molt period progressed up to d 7 when they exhiited the most wlking ctivity, fter this, they returned to level tht ws similr to the other tretments. This ehvior could e expected ecuse these hens were not eing fed nd would exist on energy reserves from the liver nd dipose tissues (Cherel et l., 1988). The level of reduction oserved in the FW ws not seen in the ALC hens. During molting most irds, whether in cptivity or under nturl conditions, decrese ll locomotive ctivities (Murphy, 1996). The significnt reductions in locomotor ctivity tht re common during the periodic molt of irds nd result in energy svings tht cn lmost compenste for energetic cost due to integumentl loss nd replcement during nturl molt in cptive hens (Beckerton nd Middleton, 1983; Lindström et l., 1993). Preening Activity Preening ctivities through the durtion of the tril pproched significnt tretment y dy effects (P 0.06). The ALC hens exhiited significntly more (P 0.05) preening (Figure 5.4) thn the FF nd FW tretment groups on d 1 of the molt with mens of 8.0, 2.4 nd 3.5% respectively. There were no significnt differences (P 0.05) mong the three groups in preening on d 2 nd d 3 of the tril.

118 106 Preening % Oservtion Dy of Tril Figure 5.4: Percentge oservtions of preening ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. Tretments within the sme dy with different letters re significntly different t p 0.05.

119 107 On d 4 of the tril the ALC hens did not disply significnt differences (P 0.50) from the other tretment groups however, the FW hens spent significntly more (P 0.04) time preening thn the FF hens. The differences etween the three tretments on d 5 ws not significnt (P 0.05) however, on d 6 the FF hens spent significntly more (P 0.04) time preening thn the FF hens ut the ALC hens were not significntly different (P 0.05) from either FW hens or FF hens. On d 7 the FF hens spent less (P 0.05) time preening thn the other groups ut there ws no differences etween the ALC hens nd the FW hens. For the remining two dys of the tril there were no significnt differences mong ny of the tretments. The FW nd ALC hens in the present study were oserved spending n incresing mount of time preening up to d 7 of the tril. The FF hens in this tril could lso e seen performing preening ctivity ut they spent less time involved in the ctivity thn the FW nd ALC hens. The preening ehvior of these two groups my e relted to loss of fethers during the molt period. Shedding of the fethers ws sujectively determined y the first signs of the fethers under the cges on d 8 in the FW tretment nd d 9 in the ALC tretment. Preening ehvior cn e stimulted y integumentry irrittions which cn e n indiction of fether push out (Wester, 2000), s displcement ction in situtions of conflict or frustrtion (Duncn nd Wood-Gush, 1972) or s comfort ehvior (Nicol, 1989). This ehvior is generlly suppressed when hens hve less time (Budier, 1996). Wester (2000) oserved the FW hens performed more preening ehvior thn the FF hens on dys 8 to 10 ut not t other times.

120 108 Drinking Significnt tretment y dy effects (P 0.002) were oserved in drinking ehvior. The FF hens spent the most time (P 0.05) drinking throughout the molt period, followed y the ALC hens nd the FW hens (Figure 5.5) during the 9-dy tril. The FF hens were drinking significntly more (P ) thn irds in other tretments on d 1 while the ALC nd FW were not different (P 0.05) on this dy. Similr results were oserved on d 2, 4, 5, 8 nd 9. On d 3 nd 6 the FW hens spent significntly less (P 0.05) time thn the other tretments drinking nd the ALC hens spent less time (P 0.05) thn the FF hens drinking ehvior on these sme dys. There were no significnt differences (P 0.05) etween the ALC hens nd the other two tretments on d 7 however, the FF hens spent more time drinking (P 0.05) thn the FW hens. Drinking ehvior of the FW nd ALC hens declined from the eginning of the tril to levels tht were lower thn the FF hens. These reduced levels oserved in the FW hens did not return to the levels oserved in the FF hens, while the ALC hens exhiited similr mount of drinking s the FF hens on d 7. The reduced drinking ehvior oserved specificlly in the ALC nd FW hens could e due to oservtions mde in previous reports tht lflf fed hens do not et s much of their diet s the FF hens nd no diet ws ville for the FW hens to consume (Donlson et l., 2005). This is supported y Woodwrd et l. (2005) who oserved tht lflf fed hens et significntly less feed thn full fed hens. They lso reported tht full fed hens drnk more wter thn lflf fed hens nd feed deprived hens. Wester (2000) exmined the ehvior of hens molted y feed

121 Drinking % Oservtion c c Dy of Tril Figure 5.5: Percentge oservtions of drinking ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. Tretments within the sme dy with different letters re significntly different t p 0.05.

122 110 deprivtion nd oserved tht the drinking ehvior of these hens declined fter the first few dys of feed withdrwl. Nonnutritive Pecking A significnt dy y tretment effect ws oserved for nonnutritive pecking. On the first dy (Figure 5.6) of the tril (d 1), there ws significnt difference (P 0.001) etween the FW hens nd the ALC nd FF hens with mens of 50.90, 22.10, nd 29.80% respectively. There were no differences etween the ALC nd FF hens on this dy. On d 2 nd 4, the ALC hens exhiited intermedite ehvior with the FW hens spending more time in nonnutritive pecking nd the FF hens spent the lest mount of time. On d 3 no significnt differences (P 0.10) were oserved etween the FW nd the ALC hens, ut these two tretments spent significntly more (P ) time involved in this ctivity thn the FF hens. The FW hens spent significntly more (P 0.002) time on d 4 in nonnutritive pecking thn the other groups while the ALC spent significntly more (P 0.01) time thn the FF hens. A similr pttern ws oserved d 5 nd d 6 with no differences (P 0.05) oserved etween the FW nd the ALC hens. On d 7 the FW hens spent significntly more time (P 0.005) thn the other tretments displying nonnutritive pecking ehvior while the ALC hens spent more time (P 0.05) thn the FF hens expressing this ehvior. On the finl two dys of the molt there ws no difference (P 0.05) etween the FF (42.45 nd 37.79% for d 8 nd 9 respectively) nd the ALC hens (48.01 nd 33.73% for d 8 nd 9 respectively), while the FW hens (69.75,

123 111 % Oservtion c Non-Nutritive Pecking c c Dy of Tril Figure 5.6: Percentge oservtions of nonnutritive pecking ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. Tretments within the sme dy with different letters re significntly different t p 0.05.

124 112 nd 79.73% for d 8 nd 9 respectively) spent significntly more (P 0.05) time in nonnutritive pecking ctivities. The incresed nonnutritive pecking oserved in the ALC hens could hve een ecuse of the chnge of feed since the ehvior susequently declined in these hens fter the third dy on the diet. The FW hens however, continued incresing levels of nonnutritive pecking ehvior up to the end of the 9 d tril. When prevented from performing specific ctivity such s eting, hens hve tendency to sustitute or redirect one ctivity with nother ction (Cooper nd Alentos, 2003). Wester (1995) found n pprent rousl in ehvior directed to forging nd feeding nd elieved tht nonnutritive pecking ws typicl response of chickens to feed deprivtion. Svory nd Fisher (1992), nd Svory et l. (1992) lso oserved nonnutritive pecking ehvior in egg-type pullets nd growing roiler reeders when they were deprived of feed. Svory nd Fisher (1992) concluded tht nonnutritive pecking ws redirection of consummtory response from nutritive stimulus to nonnutritive stimulus. Recently McCown et l. (2006) exmined the ehvior of molting hens on low-cloric diet nd oserved tht the feed deprived hens exhiited incresed levels of cge pecking, the ehvior lso incresed in the non-fst induced tretment from the levels tht ws oserved pre-molt while the non-molted hens did not disply incresed level of cge pecking. Appley nd Hughes (1991) reported tht even in the presence of ville food, irds spent most of their dy forging, this could ccount for the nonnutritive ehvior oserved in the FF hens.

125 113 Feeder Activity From this study it ws not possile to tell if the hens were ctully eting the feed or merely pecking t the feeding trough. Becuse of this prdox rose where the FW hens were lso scored for the mount of time they spent feeding. We interpreted this score not s the mount of time they spent feeding ut s the mount of time they went to check the feeding trough. Significnt tretment y dy effects (P ) were oserved for feeder relted ctivity for the 9 dy tril, however, on the first dy no significnt differences (P 0.50) were oserved in feeder relted ctivities (Figure 5.7) etween the FF hens nd the ALC hens with mens of 27.5 nd 31.6% respectively. The FW hens spent 12.5% of their time on d 1 visiting the feeder, which ws significntly different (P 0.01) from the other tretment groups. On d 2 the three tretment groups exhiited significntly different (P 0.05) responses, the FF hens spent significntly more (P 0.02) time t the feeder thn the other groups nd the ALC hens spent significntly more (P 0.05) time thn the FW hens. On d 3 there were no significnt differences etween the FF hens nd the ALC hens while the FW hens spent less (P 0.05) time t the feeder. With the exception of d 5, the FW hens spent less (P 0.05) time t the feeder thn the other groups for the reminder of the tril. The ALC hens spent significntly less (P 0.05) time t the feeder thn the FF hens from d 4 to d 8, ut y d 9 there were no significnt (P 0.40) differences etween the ALC hens nd the FF hens.

126 114 Feeder Activity % Oservtion c c c c c Dy of Tril Figure 5.7: Percentge oservtions of feeder ehvior of lying hens during 9 dy induced molt. = hens fed n lflf crumle diet (ALC); = hens molted y feed withdrwl (FW); = non-molted control hens (FF). N= 24 hens per group. Tretments within the sme dy with different letters re significntly different t p 0.05.

127 115 It is pprent tht rpid decline in feeder ctivity occurs in hens experiencing complete removl of feed. Similr ehvior response ws oserved y Wester (1995, 2000) who reported tht hens tht were molted y feed withdrwl spent progressively less time visiting the feeder s the molt period progressed. It ws lso oserved in this study tht s hens spent less time t the feeder, they spent more time involved in nonnutritive pecking ctivities; which ws consistent with Wester (2000) who oserved tht reduction in feeder relted ctivities y FW hens ws ccompnied y n increse in nonnutritive pecking. Duncn nd Wood-Gush (1972) elieved tht hens needed less thn n hour per dy to et sufficient food when complete rtion ws provided. It ppered tht some djustment to lflf s feed source occurred s feeder ctivity initilly declined, then incresed towrds the end of the molt period. However, feeder ctivity from d 1 nd onwrds ws lwys greter thn FW indicting tht the irds were responding to the presence of feed. The reduced feeding ehvior in ALC irds compred to FF irds could e the result of reduced intke due to unpltility of sponins present in lflf (Mtsushim, 1972; Sen et l., 1998) nd/or slow pssge rtes. This ws demonstrted y Sild (1979), who reported tht lflf exhiited the slowest pssge rte in chickens requiring more thn 24 h to e clered from the chickens gstrointestinl trct. He concluded tht this gve the irds feeling of stiety cusing them to reduce their intke. When Donlson et l. (2005) evluted the utiliztion of different rtios of lflf nd lyer rtion for molt induction, they oserved tht hens fed diet contining 100% lflf mel te significntly less (82 g/hen) for 9 d tril when compred to hens fed diet contining 90% lflf plus 10% lyer rtion

128 116 ( g/hen) nd hens fed diet contining 70% lflf plus 30% lyer rtion ( g/hen). In conclusion, concerns of reduced welfre hve een n issue of induced molting y feed deprivtion. Behviorl priorities during n induced molt cn e n effective wy of ssessing hens welfre while they re fsting (Wester, 2000). In this study, the welfre of the hens tht were molted using lflf ws intermedite to the FF hens nd the FW hens sed on their ehvior. The ALC hens did not exhiit displcement ehvior, stereotypy or ggression tht is indictive of frustrtion nd these hens were given the opportunity to et even though initilly they did not ccept the diet. Bsed on nonnutritive pecking nd feeder ctivity it would pper tht once hens djusted to the lflf diet their ehvior pproximted hens fed non-molt diet. However, in ddition to ehvior oservtions, metolic nd immune indictors need to e monitored to determine whether high fier diets cn decrese physiologicl stress in hens while they re undergoing molt nd if long-term effects eyond the molt period occur.

129 117 CHAPTER VI LAYING HENS BEHAVIOR TO DIFFERENT ALFALFA-LAYER RATION COMBINATIONS FED DURING MOLTING Introduction In the vin species molting usully involves the periodic shedding nd replcement of fethers (Lucs nd Stettenhein, 1979). Birds undergo series of molts during their life spn. They chnge t lest four different plumges from htching to their first nnul cycle. This includes ntl down, juvenile, lternte, nd sic plumges (Lucs nd Stettenheim, 1972). Molting lso involves the regression of the hens reproductive system resulting in reproductive quiescence (Berry, 2003). Domestic hens like most species of wild irds experience nturlly occurring molt however, this is usully incomplete nd hens continue to ly eggs t low rtes for prolonged period of time (Swnson nd Bell, 1975). This would men period of un-profitility due to reduction of egg production nd the end of the useful life of flock (Berry, 2003). The productive lying life of flocks cn e extended from less thn 80 wk to 110 wk or even 140 wk through the use of induced molting (Bell, 2003). At the end of lying cycle egg production nd qulity decline significntly, n induced molt usully improves the hens performnce. After the induced molt, egg production nd qulity re improved significntly compred to the pre-molt period (Wester, 2003; Swnson nd Bell, 1975). Over the yers, numer of different pproches to rtificilly induce molt in commercil lying hens hve een developed. These include feed withdrwl for up to 10 dys (Christms et l., 1985), wter withdrwl for two dys (North nd Bell, 1990)

130 118 nd photoperiodic reduction (Hemree et l., 1980). The method of feed nd wter withdrwl is mtter of grve concern to niml dvoctes nd the prctice hs een outlwed in Europe (Appley et l., 2004). In light of the niml welfre controversies ssocited with feed withdrwl molting nd the dditionl prolem of incresed Slmonell Enteritidis infection lterntive mens to induce molt tht might e considered more humne hve een sought in the U.S. (Holt, 2003, Berry, 2003, Ricke, 2003; Prk et l., 2004). These lterntives fll in two ctegories: vrious methods of nutrient restriction tht would void long term feed withdrwl nd the use of dietry dditives (Wester, 2003). Although egg production prmeters hve een extensively exmined, in mny of these lterntive pproches, none hve exmined the ehviorl effect of these diets on the hens. Some of these lterntive methods hve included feeding low clcium (Breeding et l., 1992), low sodium diets nd high dietry zinc (Berry nd Brke, 1985). Some studies hve looked t chnges in ggressive ehvior during induced molts nd the results hve een contrdictory. Aggrey et l. (1990) found tht negtive interctions mong lying hens incresed during feed deprivtion in open housing systems ut not in ttery cges. Hemree et l. (1980) reported tht the ggressive ehvior of hens in colony cges during feed deprivtion did not differ from hens not deprived of feed. Wester (1995) found no significnt differences in ggression etween cged hens on 4 dy fst nd hens tht were not deprived of feed while Hskell et l. (2000) found incresed ggression in frustrted hens. Alflf-lyer rtion comintions hve een shown to e effective for molt induction, reduction of SE infection nd retention of

131 119 optiml egg production in the second egg lying cycle (Donlson et l, 2005; Lnders et l, 2005,, Woodwrd et l., 2005, McReynolds et l., 2005, 2006). The ojective of this tril ws to evlute the ehviorl ptterns of hens fed different comintions of lflf nd lyer rtion nd compre these ehviorl ptterns to tht of feed withdrwl hens in nine dy induced molt. Mterils nd Methods Experimentl Design A totl of 250 lying hens t pproximtely 53 weeks old were otined from ner-y lyer unit nd used for this tril. Hens were rndomly put in five tretment groups nd were plced in individul cges (1100 cm 2 sq/ird) in three tril rooms for two week cclimtiztion period. During this time they were fed lnced unmedicted corn-soy en mel sed msh lyer rtion tht met the Ntionl Reserch Council requirements for nutrients (NRC, 1994) nd wter d liitum. The lflf tretment groups were plced in one room, while the FF tretment nd the FW tretment were ech plced in seprte room. A totl of 39 hens were plced in the five tretment groups nd 6 hens in ech tretment were oserved for ehvior ptterns. The tretments consisted of three lflf rtions; A90 which consisted of 90% lflf mel nd 10% lyer rtion, A80 which consisted of 80% lflf mel nd 20% lyer rtion nd A70 which consisted of 70% lflf mel nd 30% lyer rtion. Alflf typicllly contins 17 to 18% crude protein nd 24 to 25% crude fier nd hs low metolizle energy (1200 kcl/kg) when compred to lyer rtion with 2,965 kcl/kg (NRC, 1994). Hens in the A90, A80 nd A70 tretment groups were fed their respective comintion of

132 120 lflf mel nd lyer rtion d liitum for the 9 d tril. The FF tretment remined on the pre-tril diet nd the FW hens were not fed during the 9 d of the tril. One week efore the dministrtion of the diets, the lighting schedule in ech of the three rooms ws chnged from 16h light/8h drk to 8h light/16h drk. Behvior ws recorded using 10 cmer Digitl Video Recorder Multiplexer system (Kltel DVMRe Triplex ez Digitl Video Multiplexer Recorder, Corvllis, OR). Two cmers were mounted pproximtely 1.2 m wy nd 30 ove the cges of ech tretment in ech of the three experimentl rooms. Two 10 min intervls were nlyzed ech dy from d 0 to d 9 of the tril. Recordings egn t 1200 h ech dy nd ended t 1400 h. One ten min intervl ws tken t 1230 h nd second 10 minute oservtion ws tken t 1330 h for detiled nlysis. Behvior Prmeters Eight hens in individul cges were the sujects for the ehvior nlysis in ech tretment. Seven different ehviors were dpted from Wester (2000) to ssess the irds welfre during the tril. Hed movement ws the rpid individul hed movement of n lert ird. This is suggestive of visul surveillnce y ird of its environment. Feeder ctivity involved ny pecking ehvior tht ws directed towrds the feeding trough while the focl ird ws close enough to the trough to et or when the ird hd her hed in the trough. Preening involved the mnipultion of the plumge with the ek. Drinking ws the pprent ingestion of wter y pecking from the nipple wterers. Nonnutritive pecking ehvior ws recorded when hens were pecking t nything other thn the feed, which included pecking t the cge floor, sides nd their own feet.

133 121 Wlking ws defined s the locomotion of the hen involving t lest one step. Aggression ws recorded s ny ggressive pecking ehvior directed to nother hen either in the sme cge or in neighoring cge. Aggressive ehvior ws oserved s 0/1 occurrence, tht is, it either hppened or did not hppen during the oservtion time. The dt were in the form of counts t one minute intervls tht were then summrized s the dily men percentge of oservtions in which the suject ws performing prticulr ehvior. Sttisticl Anlysis The verge of ech ctivity of hens in ech tretment ws summrized nd mens of ech ehvior over the nine dy period were nlyzed using repeted mesures design. Proc GLM (SAS version 8.3, SAS Institute Inc., Cry, NC, 2001) ws used with tretment, time, nd tretment y time interction nd individul hens nested within tretment s the fctors. Chicken nested within tretment ws the error term used to test for tretment effects. When significnt (P 0.05) tretment y time interctions ws found, mens were compred using Lest Significnt Difference. Results nd Discussion Aggression nd Egg Production Aggressive pecking t neighors ws not significnt in this tril. One incidence ws oserved etween cges on d 1 in the A90 nd A70 hens nd nother incidence ws oserved etween cges in the FF hens nd the FW hens (dt not shown). When Wester (2000) nd Anderson et l. (2004) induced molt y feed withdrwl they oserved ggression erly during the molt induction. The ggression declined s the

134 122 period progressed. This reduction in ggressiveness ws not oserved y McCown et l. (2006) when they induced molt y feed deprivtion nd lso y incorporting low clorie diet. They oserved tht ggression incresed s the period progressed. The low incidence of ggression oserved in this tril could result from the hens eing housed in individul cges, therey minimizing the ird to ird interction usully seen in multihen or colony cges. Egg production dt ws collected nd the A80 hens stopped lying on d 5 of the tril while the FW nd A90 hens cesed production on d 6 (Figure 6.1). The A70 hens did not stop egg production up to d 9 of the tril. Donlson et l. (2005) evluted molt induction nd ird performnce with different rtios of lflf diets, they oserved tht the A70 hens took n verge of 5.75 d to cese egg production while the A90 hens stopped lying 4.92 d nd the FW hens stopped 4.42 d (they did not exmine A80 hens). The hens in the current study took longer time to stop lying, however, the trend pper to e the sme ecuse the A90 hens stopped lying t the sme time or in the cse of the A80 hens efore the FW hens while the A70 hens did not stop until d 9. This could e due to n incomplete molt ecuse of the higher level of high clorie lyer rtion tht ws present in the A70 feed. Donlson lso noted tht the A70 hens exhiited significntly lower post-molt egg production tht ws similr to FF hens, while the A90 hens hd post-molt egg production similr to the FW hens.

135 123 Egg Production FF FW Hen-Dy Egg Production _15 _13 _11 _9 _7 _5 _3 _1 Dy of Tril A90 A80 A70 Figure 6.1: Hen dy egg production y the five tretments on dily sis from 2 wk prior molt induction nd during the 9 dy tril. The negtive numers represents the pre-tril dys.

136 124 Wlking Activity No significnt (P > 0.4) tretment y dy effect ws oserved in wlking (Figure 6.2) in this tril, however significnt (P < 0.001) tretment effects were oserved during the 9 d tril. Over this period the FW, A70 nd A80 hens spent significntly more time thn the thn the FF nd the A90 hens wlking. The FF hens spent more time thn the A90 hens wlking. In this tril the FW hens (18.64%) hd n overll men tht ws higher thn the FF hens (12.69%) nd the A90 hens (5.87%), indicting tht they wlked more throughout the period thn these two groups. The A70 nd A80 hens hd mens of nd 17.28% respectively which were not significntly different (P 0.05) from the FW hens ut were significntly different (P 0.05) from the A90 nd FF hens (dt not shown). The wlking ehvior oserved in this tril ws not consistent with wht ws oserved y McCown et l. (2006) who reported no significnt differences mong the tretments throughout the molt period. Wester (2000) oserved tht hens undergoing feed withdrwl spent 40% of their time resting during n induced molt. It would e expected tht hens tht were feed deprived would reduce their ctivities in n effort to conserve energy, Murphy (1996) stted tht during molt irds reduce ll their locomotive ctivities.

137 125 % Oservtion Wlking Dy of Tril A70 A80 A90 FW FF Figure 6.2: Percentge oservtions of wlking ehvior of lying hens during 9 induced dy molt. N= 6 hens per group. No significnt differences were recorded t P 0.05.

138 126 Preening Activity Preening ctivity during the 9 d induced molt did not disply significnt (P < 0.2) tretment y dy effects. Significnt tretment effects were oserved on d 5 nd 6 (Figure 6.3). On d 5 the A90 hens spent significntly (P < 0.007) more time thn the FF hens preening, ut were not significntly different (P > 0.05) from the other tretments. No significnt differences were oserved mong the other tretment groups on d 5. On d 6 the A90 hens displyed significntly more time thn the FF nd the A70 hens preening while they did not differ from the A90 hens nd the A80 nd FW hens. On d 6 no differences were oserved etween the A80, A70, FW nd FF hens. The highest incidence of preening oserved during this tril ws on d 5, the ltter prt of the induced molt. For the durtion of the tril the A90, A80 nd FW hens spent more time preening thn the A70 nd FF hens, this could hve een result of folliculr irrittion due to fether push-out ecuse through sujective oservtions we oserved n increse in fethers under the cges of the FW hens on d 8 nd under the cges of the A80 nd A90 hens on d 9 (dt not shown). Shedding ws not oserved in the A70 hens until d 12 (two dys fter the tretment diet ws removed from the hens). Preening my e performed s displcement ction in situtions of conflict or frustrtion (Duncn nd Wood-Gush, 1972). However, Wester (2000) reported tht the mount of time tht ws spent preening followed qudrtic trend tht peked towrds the end of molt induction; he postulted tht rther thn ttriuting the incresed preening to frustrtion, it could e ttriuted to response to fether push-out.

139 127 % Oservtion Preening Dy of Tril A70 A80 A90 FW FF Figure 6.3: Percentge oservtions of preening ehvior of lying hens during 9 dy induced molt. N= 6 hens per group. Tretments within the sme dy with different letters re significntly different t P 0.05.

140 128 Hed Movement Significnt tretment y dy effects (P < ) ws oserved in hed movement (Figure 6.4) in this tril. On d 1 of the molt the A80 hens spent significntly more (P 0.01) time thn the other tretments in hed movement. There were no significnt differences etween the A70, FF nd FW hens in hed movement nd the A90 hens spent significntly less (P 0.002) time thn the other tretments in hed movements on d 1. No significnt differences (P > 0.4)were oserved on d 2 mong the tretments, however, on d 3, 4 nd 9 the A90 hens spent more (P 0.03) time performing hed movements thn the FW hens. The A80, A70, nd FF hens did not disply significnt differences from ny other group. No significnt differences were oserved in hed movement mong tretments on d 5, 6, 7 nd 8. Most of the hed movements oserved during the tril were performed during the eginning of the tril. The FW hens spent the lest mount of time involved in this ehvior for the 9 d period (dt not shown). Similr results were oserved y Wester (2000) who lso noted tht the highest frequency of hed movement occurred during the first 3 d of feed withdrwl nd oserved no differences etween the FW nd non-molted hens during the lst dys of feed withdrwl. McCown et l. (2006) evluted ehvior of fst-molted lying hens, low-clorie molted lying hens nd nonmolted lying hens, they reported no significnt differences mong tretments in hed movements. The lflf fed hens were no less ttentive during the induced molt ecuse they displyed overll mens tht were similr to the FF hens.

141 129 % Oservtion Hed Movement c Dy of Tril A70 A80 A90 FW FF Figure 6.4: Percentge oservtions of hed movement ehvior of lying hens during 9 dy induced molt. N= 6 hens per group. Tretments within the sme dy with different letters re significntly different t P 0.05.

142 130 Drinking Activity Significnt (P < 0.001) tretment y dy effects ws oserved in drinking ctivity (Figure 6.5). On d 1 of the tril FF hens spent significntly more (P 0.006) time thn the A80, A70 nd the FW hens drinking. The A90 hens spent significntly more (P 0.004) time thn the FW hens ut did not they differ from the A80, A70, nd FF hens. On d 2 the FF hens spent significntly more (P 0.01) time thn the other groups drinking, there were no significnt (P > 0.05) difference mong the other tretments. No significnt differences were oserved mong the tretments on d 3 nd 4, however, on d 5, 6, nd 8 the FF hens spent more (P < ) time drinking thn the other tretments while there were no differences (P 0.05) mong the other tretment groups. On d 7 nd 9 no differences (P > 0.05) were oserved mong the groups. The FF hens spent more time thn the other groups drinking while the FW hens spent the lest mount of time. This ehvior is somewht expected ecuse the FW hens were not eting it would men they would drink less nd since the FF hens which were provided dry rtion, they drnk more wter. This explntion could e true lso for hens on the lflf diets ecuse even though we did not evlute feed intke it ppered tht these hens did not et s much s the FF hens so therefore they would potentilly drink less. For the durtion of the tril the A80 hens exhiited drinking ehvior tht ws closest to the FF hens. The A90 hens drnk less thn the A80 hens ut they were not significntly different. The A70 hens were oserved visiting the wterers the lest mong the lflf fed hens nd they were not different from the FW hens.

143 Drinking A70 A80 A90 FW FF % Oservtion c c Dy of Tril Figure 6.5: Percentge oservtions of drinking ehvior of lying hens during 9 dy induced molt. N= 6 hens per group. Tretments within the sme dy with different letters re significntly different t P 0.05.

144 132 Woodwrd et l. (2005) evluted wter intke of hens on n lflf molt diet nd oserved tht these hens drnk 2 fold less wter during the molt when compred to the FF hens. The FF hens lso drnk 2 folds more thn the FW hens. Wester (2000) oserved tht drinking ehvior declined in feed deprived hens fter the first few dys of feed withdrwl. Nonnutritive Pecking Significnt tretment y dy effect did not (P < 0.10) occur in nonnutritive pecking ehvior (Figure 6.6) however significnt (P < 0.001) tretment effects were oserved in this tril. On d 1 of the tril the FW hens spent significntly more (P 0.009) time thn the other groups in nonnutritive pecking. On the sme dy the A70 hens displyed significntly higher (P 0.05) levels of nonnutritive pecking thn the FF hens ut A70 ws not higher thn the A90 nd A80 hens. The A90 nd A80 hens did not differ (P > 0.05) from the FF hens. On d 2, 3 nd 4 the FW hens spent more (P 0.04) time performing nonnutritive pecking thn the A80 nd the FF hens. The A80 nd the FF hens were not significntly different (P > 0.05) from the A70 nd the A90 hens which were not significntly different (P > 0.05) from ny of the tretments. On d 5 no significnt differences (P > 0.05) were oserved mong the groups, however, on d 6 the FW hens spent significntly more (P 0.03) time thn the FF hens in nonnutritive pecking ehvior. The lflf fed tretments were not different from ny of the other tretments on d 6. On d 7 the A70 hens were not significntly different (P > 0.05) from the other tretments, however, the FW hens displyed significntly higher (P 0.001)

145 133 % Oservtion c c Non-Nutritive Pecking Dy of Tril A70 A80 A90 FW FF Figure 6.6: Percentge oservtions of nonnutritive pecking ehvior of lying hens during 9 dy induced molt. N= 6 hens per group. Tretments within the sme dy with different letters re significntly different t P 0.05.

146 134 nonnutritive pecking ehvior from the A90, A80, nd FF hens. On d 8 nd 9 the FW hens spent significntly more (P ) time thn the other tretments in nonnutritive pecking ehvior. Over the nine dy period of feed withdrwl the FW hens spent significntly more time performing nonnutritive pecking thn ll the other tretment groups while the lflf tretment groups compred fvorly to the FF hens. Among the lflf tretments the A90 hens spent most time performing nonnutritive pecking ehvior, they were not significntly different from the A70 hens. Of the lflf tretments, the A80 hens spent the lest mount of time performing nonnutritive pecking nd were similr to the FF hens, while the A90 nd A70 hens spent more time thn the FF nd the A80 hens. The incresing occurrence of nonnutritive pecking oserved erly in the tril in the lflf fed hens lter susided to occurrences similr to the FF hens, this could e ecuse they did not dpt immeditely to the chnge of diet. Nonnutritive pecking is thought to e redirected forging ehvior tht is seen in irds in the wild nd evidence shows tht even when hens re provided with dequte food they re still motivted to forge (Duncn nd Hughes, 1972). Petherick nd Rutter (1990) stted tht hens will ct in predictle mnner to feed deprivtion y working hrder for food the longer they hve een without it. Other reports in studies where hens were deprived noted tht feed restricted chickens hve exhiited incresed nonnutritive pecking (Svory nd Fisher, 1992; Svory et l., 1992; Wester, 2000; McCown et l., 2006).

147 135 Feeder Activity Significnt (P < ) tretment y dy effect ws oserved in feeder ehvior (Figure 6.7) during this tril. On d 1 of the tril the A70 nd A90 hens spent significntly more (P 0.006) time t the feeder thn the other tretments nd they were not different from ech other (P 0.8). The A80, FF nd FW hens did not differ (P 0.3) significntly on d 1. No significnt differences were oserved mong tretments on d 2. On d 3 the FF hens spent significntly more (P 0.03) time t the feeder thn the A80 nd the FW hens. The A90 nd A70 hens were not significntly different (P 0.05) from ny of the other tretments on d 3. On d 4 the A80 hens spent significntly more (P 0.001) time t the feeder thn the FW hens, no other significnt differences were oserved on this dy. No significnt differences were oserved on d 5, however, on d 6, 7, nd 9 the FW hens spent significntly less (P ) time thn the other tretments t the feeder, while the other tretments were not significntly different. On d 8 the A90, A80 nd A70 hens spent significntly more (P ) time thn the FW hens t the feeder nd they were not significntly (P 0.05) different from the FF hens. The FF hens nd FW hens did not disply significnt difference (P ) t the feeder. As ws nticipted, the hens which hd feed spent more time t the feeder thn the FW hens. Since we could not tell whether or not ird ws eting, we scored ech time the ird visited the feeding troughs nd ecuse of this the FW hens were sometimes given score for feeding. At the eginning of the tril the A90 nd A70 hens visited the feeder more often thn ny other group, however, from d 2 up to d 5 the

148 136 % Oservtion Feeder Dy of Tril A70 A80 A90 FW FF Figure 6.7: Percentge oservtions of feeder ehvior of lying hens during 9 dy induced molt. N= 6 hens per group. Tretments within the sme dy with different letters re significntly different t P 0.05.

149 137 numer of visits declined, fter d 5, the A90, A80, nd A70 hens ll spent incresing mounts of time visiting the feeder nd were not significntly different from ech other for the durtion of the tril. For the durtion of the tril the A90 hens exhiited feeder ehvior tht ws closest to tht displyed y the FF hens. Of the lflf fed hens A70 hens displyed the lest mount of time visiting the feeder nd the A80 ws intermedite. The reduction in visits to the feeder t the initil stges of the tril could hve een n djustment period for these hens to the chnge of diet. The reduced intke of the lflf diet erly in the tril could lso e due to the unpltility of lflf ecuse of the presence of sponins (Sen et l., 1998) or the slow pssge of the lflf through the gstrointestinl trct of the chicken (Sild, 1979, 1980). It hs een demonstrted tht hens fed lflf diets te significntly less thn hens fed lyer rtion (Woodwrd et l., 2005; Donlson et l., 2005). When Donlson et l. (2005) compred different rtios of lflf diets they oserved the A70 hens te more thn the A90 hens. We lso oserved wht ppered to e reltionship etween feeder ctivity nd nonnutritive pecking ecuse s nonnutritive pecking declined in frequency, feeder ctivity incresed ut the reduction in frequency ws not dependent on the tretment. Wester (1995, 2000) reported similr reltionship when molting hens y feed deprivtion, they lso stted tht hens which were molted using feed withdrwl spent progressively less time visiting the feeder s the molt period progressed. It hs een implied tht feed deprivtion s mens of inducing molt cn result in reduced niml welfre, however, Wester (1995, 2000, 2003) hs stted tht there is

150 138 no evidence tht hens deprived of feed for period to induce molt ctully suffered during the molt induction or tht they suffer long term effects from the induced molt. Little ehvior work hs een done investigting the effects of lterntive molt diets on lying hens. The results from this tril indicted tht the lflf molt diets did not result in incresed ggressiveness in the molting hens. The A90 nd A80 hens stopped lying t time tht ws comprle to the FW hens. The hens fed lflf- lyer rtion diets remined ctive nd lert throughout the tril s indicted y their hed movement nd wlking ctivities during the tril. The lflf fed hens spent less time drinking thn the FF hens ut this could e ecuse they te less thn the FF hens. When compring the lflf diets, the A70 hens spent the lest mount of time drinking nd the lest mount of time t the feeder, on the other hnd the A90 hens spent the most mount of time eting ut the A80 hens drnk more thn them. At the eginning of the tril the hens fed lflf diets spent similr mount of time performing nonnutritive pecking s the FW hens, however, this declined towrds the end of the tril. Reduced nonnutritive pecking ehvior ws not oserved in the FW hens nd insted the ehvior incresed in these hens towrds the end of the tril. With the reduction in nonnutritive pecking ehvior, n increse in feeder relted ctivity ws oserved, this indicted tht the hens required some time to ecome ccustomed to the new diet. The A90 hens compred lest fvorly with the FF hens in nonnutritive pecking ehvior. The A80 hens ppered to e the closest to the FF hens nd the A70 hens were lso similr to the FF hen in nonnutritive pecking ehvior. The use of different comintions of lflf diets to induce molt hve een explored (Donlson et l., 2005) nd the A70 ws the lest

151 139 fvorle due the incomplete regression of the reproductive trct, in this tril the A70 hens spent less time preening nd egn shedding t lter dte thn the other two lflf diets, lso, they did not stop completely lying up to d 9. No dverse effects to the hens welfre surfced in the A90, A80, or A70 hens during this tril sed on the ehvior prmeters we exmined; in fct the diets effectively reduced some of the norml ehviors such s incresed nonnutritive pecking which hs een oserved in feed deprived hens. Additionl studies re necessry to exmine physiologicl nd immunologicl chnges ssocited with stress in hens tht re fed these diets to induce molt.

152 140 CHAPTER VII APPLICATION OF HAFNIUM CHLORIDE AS A MARKER TO ASSESS THE PASSAGE RATE OF LAYER RATION AND ALFALFA DIETS Introduction Molting in the vin species cn e defined s the periodic shedding nd replcement of fethers, nd the rejuvention of the reproductive system (Svihus et l., 2002). While the process occurs nturlly in chickens, it is usully for prolonged period of time during which the irds stop lying. This poses prolem for the commercil industry nd solution is to force molt the hens in n effort to prevent economic losses due to low egg production. Severl diets hve een developed which induce molting without resorting to conventionl feed deprivtion which hs een strongly opposed y niml welfre groups (Wkeling, 1978; Shippee et l., 1979; Arrington et l., 1967). Lnders et l. (2005) hve shown tht lflf cn effectively induce molt nd Woodwrd et l. (2005) confirmed tht lflf molt diets limited infesttion of Slmonell in lying hens. The effectiveness of molt rtions is determined to lrge extent y the rte of pssge of the feed through the gstrointestinl (GI) trct nd the length of time the feed stys in the vrious GI trct regions. Therefore the evlution of vrious molt diet progrms is dependent on the development of methods to study these digestion kenetics. Using indigestile flow mrkers to mrk feed ingredients nd then collecting the excret t predetermined times is common mens of otining pssge rte. Tukey et l (1958) oserved tht in chickens, mrkers first ppered in the excret within 2 to 2.5 h fter

153 141 intke nd tht most of the mrker ws usully excreted within 12 h. The use of indigestile mrker methods requires tht the mrker dose is dministered under stedy stte conditions nd tht the mrker itself is impervious to GI trct conditions, especilly cidity. Tetrvlent hfnium (Hf) ppers to ind with onds tht re very resistnt to proton displcement, ut this hs not een demonstrted in chickens. Feed pssge rte cn vry in different segments of the GI trct nd is ffected y the composition of the feed. To mesure the pssge time in ech comprtment of the trct Vn der Klis nd Vn Vorst (1993) fed irds nd killed them t different time intervls, then crefully dissected them nd removed the contents of ech comprtment to nlyze. This method of mesuring men residence time of the rtions is less sensitive to non-stedy stte conditions, ut still requires indelile flow mrkers. The purpose of the present work ws to evlute the potentil for using Hf s stle isotope indigestile mrker nd its ppliction to mesurements using the methods mentioned. To this end we dministered three differing mrked diets to groups of lying hens, collected fecl mteril t selected intervls nd collected digest from vrious portions of the GI trct (Figure 7.1) following the scrifice of the irds. We then mesured the neutron ctivtion nlysis determintion limit for hfnium in the pplicle fecl mteril nd digest, determined optimum concentrtion for dosing solutions, nd performed preliminry pssge rte studies in lying hens.

154 142 Crop Proventriculus Duodenum Gizzrd Jejunum Meckel s Diverticulum Ileum Ileo-cecl Junction Cec Cec Picture y P. Herrer nd C. Dunkley (2004) Figure 7.1. Picture of the hens gstrointestinl trct. Ingest were removed from the Crop, Gizzrd, Ileun, Jejunum, nd Cec.