Impaired intestinal villi growth in broiler chicks with unhealed navels

Impaired intestinal villi growth in broiler chicks with unhealed navels L. T. Kawalilak, A. M. Ulmer Franco, and G. M. Fasenko 1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada, T6G 2P5 ABSTRACT By the time of hatching, the residual yolk sac (RYS) should be completely internalized into the body cavity of the chick via the navel and the navel should be closed (healed). After hatching, the RYS contents are used for general growth and development including growth of the small intestine. This research examined the relationship between the presence of navel buttons (an unhealed navel covered with a scab) and the RYS weight and intestinal villi height of broiler chicks from 0 to 5 d posthatching. Hatching eggs containing live embryos were collected at 18, 19, and 20 d of incubation (n = 5/d). At 21 d of incubation, chicks with healed navels (H) and chicks with navel buttons smaller than 3 mm in diameter (B) were obtained (n = 30/group). The BW and RYS of embryos (n = 5/d) and chicks (n = 5/treatment per day) were weighed. Samples of the small intestine (duodenum and ileum) were processed for histological evaluation to measure villi height. The trial was replicated in time 4 wk after the first collection using eggs and chicks from the same flock. Data were analyzed using SAS PROC MIXED (P 0.05). No significant differences in BW were observed between H and B chicks. Average RYS weight and percentage RYS were greater in B than in H chicks. The interaction of age and navel condition significantly influenced both duodenal and ileal villi heights. On most of the days, duodenal and ileal villi were shorter in B than in H chicks. The presence of small navel buttons at hatching is indicative of impaired absorption of the RYS content and decreased intestinal villi growth. Further research on the relationship between navel buttons, intestinal physiology, and yolk sac infections is advised. Key words: broiler chick, chicken embryo, navel, yolk sac, intestinal villi height 2010 Poultry Science 89 :82 87 doi: 10.3382/ps.2009-00284 INTRODUCTION The avian yolk sac (YS) is a highly vascularized membrane that starts to develop and surround the yolk at around 2 d of incubation (Romanoff, 1960). The function of this extraembryonic membrane is to transfer nutrients from the yolk to the developing embryo (Romanoff, 1960). At around 19 d of incubation, the YS starts to be internalized via the navel into the body cavity of the chicken embryo. Ideally, upon hatching, the YS should be fully withdrawn with the skin surrounding the navel completely healed and closed (Romanoff, 1960). However, this is not always the case. When the YS is not fully internalized, the protruding yolk prevents the navel from closing properly. One of the most common types of unhealed navels is the navel button, in which a small scab is formed over the navel (Fasenko and O Dea, 2008). Research conducted by Fasenko and 2010 Poultry Science Association Inc. Received June 8, 2009. Accepted September 11, 2009. 1 Corresponding author: gaylene.fasenko@ualberta.ca O Dea (2008) has shown that chicks with minor navel conditions (navel buttons and navels leaking fluid) had lower final broiler BW 6 wk later and higher mortality than chicks with properly healed navels. Because automation in commercial hatcheries has greatly reduced the time that hatchery workers have to examine hatchlings, only very weak chicks or chicks with obvious defects are culled. As a consequence, chicks with minor navel conditions might inadvertently be shipped to broiler farms, thus reducing the production performance of the broiler flock. Fasenko and O Dea (2008) hypothesized that the suboptimal performance of chicks with navel buttons may have been a consequence of subclinical YS infection. As a consequence, energy that would have normally been used for growth might have been diverted toward fighting off the infection. It has been reported that bacteria associated with omphalitis (YS infection) alter protein composition of the residual YS (RYS) content (Sander et al., 1998) and cause deterioration of essential nutrients that could have been used as a source of energy during the posthatch period (Rai et al., 2005). After hatching, the RYS contents are mobilized simultaneously by the chick via 2 different routes: via the 82

YS stalk into the intestine (Esteban et al., 1991) and by absorption through the YS membrane into the circulation of the chick (Noy et al., 1996; Noy and Sklan, 1998). The absorption of nutrients from the YS is essential to promote body growth (Murakami et al., 1992; Bigot et al., 2001) and also for development of the small intestine (Noy and Sklan, 1999). Earlier reports have described the normal gastrointestinal development of the chick (Sklan, 2001; Uni et al., 2003). During the last 3 d of incubation, an increase in the number of embryonic enterocytes in the small intestine causes a large increase in villi height (Uni et al., 2003). As a consequence, the surface area available for absorption of nutrients as well as the weight of the small intestine are greatly increased (Uni et al., 2003). The period immediately after hatching is also critical for intestinal growth (Geyra et al., 2001). The small intestine grows rapidly in the first 24 h after the intake of carbohydrate-rich grain-based feeds (Uni et al., 2000; Sklan, 2001). Because the small intestine is a critical digestive organ involved in nutrient absorption and posthatch growth, the development of this organ is essential to hatchling health and performance. The main objective of this research was to study the relationship between the presence of small navel buttons at hatching with the RYS weight and intestinal villi height during the first 5 d posthatch. The hypotheses tested were that 1) the RYS of chicks with small navel buttons would be larger than those of chicks with healed navels and 2) the villi of the duodenum and ileum of chicks with small navel buttons would be shorter than the villi of chicks with healed navels. MATERIALS AND METHODS The experimental protocol was approved by the Animal Care and Use Committee for Livestock at the University of Alberta, in accordance with the guidelines set forth by the Canadian Council on Animal Care (1993). Egg and Chick Collection INTESTINAL VILLI HEIGHT OF CHICKS WITH UNHEALED NAVELS 83 On 18, 19, and 20 d of incubation, live embryos (n = 5 embryos per day) were obtained from eggs produced by a 51-wk-old Ross 308 flock. On d 21 (or 504 h) of incubation, chicks with healed navels (H) and chicks with navel buttons (between 2 and 3 mm in diameter, B) were collected (n = 30 chicks per treatment). Except for the presence of the navel button, B chicks were healthy, active, and normal in appearance. Chicks belonging to the same treatment were placed together in a battery rearing cage. Water and a crumbled starter diet (23.0% CP and 3,067 kcal of ME/kg) were provided ad libitum. The research was replicated 4 wk after the initial collection (when the breeder flock was 55 wk of age) using embryos and chicks from the same flock. Embryo and Chick Weight Measurements On each of the incubation days (18, 19, and 20 d), 5 embryos were weighed, killed via cervical dislocation, and dissected. The RYS were removed from the body and the wet yolk-free body mass (YFBM) and RYS weights were recorded. On each of the days posthatching (0 to 5 d), 5 chicks per navel treatment were randomly selected and the same procedures mentioned above were applied. Tissue Preparation for Histological Analysis From each of the dissected embryos and chicks, intestinal tissue samples (1 cm long) from the medial portions of the duodenal loop and the ileo-jejunal junction (portion of intestine adjacent to the YS stalk) were collected for histological analysis. The intestinal samples were fixed in 10% buffered formalin for 72 h and then rinsed for 30 min with running tap water. The samples were then immersed for 1 h in 30% then in 50% ethanol. All samples were stored in 70% ethanol until further histological processing. The tissues were embedded in paraffin wax using a Fisher 166 MP Histomatic Tissue Processor (Fisher Scientific, Pittsburgh, PA). Serial sections (5 μm) were obtained using an AO- 820 rotary microtome (American Optical Corporation, Buffalo, NY). The sections were placed on glass slides and stained with hematoxylin and eosin. Two slides per duodenal and ileal samples from each embryo and chick were prepared. Random intestinal villi (3 villi per field) were measured from the base of the intestinal mucosa to the tip of the villus using MetaMorph v. 7.0 software (Molecular Devices, Downingtown, PA). A total of 210 villi were measured on each the duodenum and illeum of chicken embryos. A total of 720 villi were measured on each the duodenum and illeum of broiler chicks. Statistical Analysis The experimental design was a randomized block design with embryo or chick age and navel condition as main effects and breeder flock age as the blocking factor. Each embryo or chick was the experimental unit. The statistical model for the ANOVA was as follows: Y ijkl = µ + A i + N j + A i N j + B k + Є ijkl, where Y ijkl = characteristic that was measured; µ = overall mean; A i = main effect of embryo or chick age; N j = main effect of navel condition; A i N j = effect of the interaction between age and navel condition; B k = blocking factor (parent flock); and Є ijkl = random error term. All percentage data were subjected to angular transformation to stabilize variances (arc sine square root percentage transformation) before statistical analysis. The data were analyzed using the MIXED model of

84 Kawalilak et al. Table 1. Body weight, yolk-free body mass, and residual yolk sac weight of broiler chicken embryos during the last 3 d of incubation Embryo age (incubation day) n 1 BW (g) YFBM 2 (g) RYS 3 (g) RYS 4 (%) 18 10 39.3 b 26.1 c 13.2 a 34.5 a 19 10 41.1 ab 28.9 b 12.2 a 30.0 b 20 10 43.5 a 34.4 a 9.1 b 21.1 c SEM 4.1 4.6 0.7 1.8 a c Means within the same column with different superscripts differ significantly (P 0.05). 1 n = number of embryos analyzed each day. 2 YFBM = yolk-free body mass. 3 RYS = residual yolk sac. 4 RYS = (residual yolk sac/chick wt) 100. SAS (SAS Institute, 2002 2003) with a probability level set at P 0.05. Where the model indicated significance, the least squares means were separated using the PDIFF procedure of SAS. Where the interaction effect was significant, the effects of the main factors were not discussed. RESULTS AND DISCUSSION Embryo and YS Weights As expected, embryo age had a significant effect on BW during the last 3 d of incubation (P = 0.0029; Table 1). Eighteen-day-old embryos weighed less than 20-d-old embryos with 19-d-old embryo weight not differing from 18- or 20-d-old embryos. These results agree with a previous report by Uni et al. (2005), who showed no significant difference in BW between 19- and 20-dold Ross 308 chicken embryos. However, YFBM did significantly increase (P < 0.0001) with each incubation day (Table 1). Although RYS weight did not differ between 18- and 19-d-old embryos, embryos sampled at 20 d of incubation had lower RYS weight than embryos sampled at 18 or 19 d (Table 1). An abrupt acceleration in the rate of RYS absorption on the day before hatch had also been reported by Romanoff (1960). This faster RYS absorption coincides with the time of internal pipping, in which the beak of the embryo reaches the air cell thus gaining access to more oxygen. The hatching process demands great amounts of energy from the embryo; this energy is obtained through β-oxidation of lipids from the yolk (Romanoff, 1960). After internal pipping, the diffusion of oxygen through the shell no longer limits the β-oxidation of lipids, thus accelerating this process. Noble and Cocchi (1990) reported that over 80% of the total yolk lipid is absorbed during the immediate prehatching period. As a percentage of BW, the RYS decreased significantly (P < 0.0001) as embryo age increased (Table 1). This result is in agreement with previous research (Puvadolpirod et al., 1997). Chick Weight As expected, chick BW and YFBM increased significantly (P < 0.0001) with hatchling age (Table 2). The RYS weight and percentage RYS significantly decreased (P < 0.0001) during the first 3 d posthatching (Table 2). No further significant reductions in RYS were observed from 3 d onward (Table 2). Similar results have been previously reported (Chamblee et al., 1992; Puv- Table 2. Body weight, yolk-free body mass, and residual yolk sac weight during the first 5 d posthatch for broiler chicks having healed navels or navel buttons at hatching Item n 1 BW (g) YFBM 2 (g) RYS 3 (g) RYS 4 (%) Chick age (day posthatch) 0 20 47.6 f 42.2 f 5.3 a 11.1 a 1 20 51.6 e 47.9 e 3.7 b 7.1 b 2 20 56.8 d 54.8 d 2.0 c 3.5 c 3 20 62.7 c 61.8 c 1.0 d 1.5 d 4 20 67.4 b 66.1 b 1.2 cd 1.4 d 5 20 80.1 a 79.7 a 0.4 d 0.6 d SEM 1.5 1.4 0.4 0.5 Navel Healed 60 62.1 59.4 1.9 b 3.6 b Button 60 60.0 58.1 2.7 a 5.0 a SEM 1.0 0.8 0.2 0.3 a f Means within the same column with different superscripts differ significantly (P 0.05). 1 n = number of chicks analyzed each day. 2 YFBM = yolk-free body mass. 3 RYS = residual yolk sac. 4 RYS = (residual yolk sac/chick wt) 100.

INTESTINAL VILLI HEIGHT OF CHICKS WITH UNHEALED NAVELS 85 Table 3. Duodenum and ileum villi height in broiler chicken embryos during the last 3 d of incubation 1 Embryo age (incubation day) n 2 Duodenum villi height (µm) Ileum villi height (µm) 18 10 175.8 c ± 27.0 118.8 c ± 10.4 19 10 216.7 b ± 27.2 151.5 b ± 10.8 20 10 280.1 a ± 27.3 181.8 a ± 10.8 a c Means within the same column with different superscripts differ significantly (P 0.05). 1 Values represent means ± SEM. 2 n = number of embryos sampled. adolpirod et al., 1997) in different chicken strains (Arbor Acres and Peterson Arbor Acres). Chick BW and YFBM were not different between the navel treatments (Table 2). The interaction between age and navel treatment did not influence the BW or RYS parameters measured (data not shown). These results show that the presence of a small navel button did not have a detrimental effect on BW or YFBM during the first 5 d posthatch. Fasenko and O Dea (2008) also reported no significant differences in newly hatched chick BW among chicks with different navel conditions. However, these authors did find that chicks with navel buttons had lower BW at 7 and 41 d posthatching compared with chicks with normal navels. Even though a full broiler grow-out trial was not included in this experiment, we hypothesize that small daily reductions in weight gain could cumulatively contribute to lower BW after 7 d. RYS Weight It was interesting to observe that RYS weight and RYS as a percentage of chick BW were significantly greater in B than in H chicks (P = 0.0025 and P < 0.0001, respectively; Table 2). Thus, the first hypothesis that the RYS of chicks with small navel buttons would be larger than that of chicks with healed navels was accepted. Sander et al. (1998) and Rai et al. (2005) reported slower absorption of RYS in chicks suffering from YS infections. The lower absorption rate was a consequence of nutrient breakdown by bacteria present in the RYS, which rendered the RYS unabsorbable by the embryo (Sander et al., 1998). Our proposed hypothesis was based on the report and hypothesis of Fasenko and O Dea (2008), which stated that subclinical omphalitis might be affecting newly hatched broiler chicks with navel buttons. An alternative explanation to the greater RYS observed in B chicks could be related to egg yolk weight. Because initial (set) egg weight was not known, it is possible that B chicks had originated from larger eggs. These larger eggs would likely have larger yolks, which would be more difficult to be fully withdrawn into the chick body cavity than smaller yolks. Lawrence et al. (2004) reported a greater percentage of culled chicks from large eggs than from average or small eggs. Even though the authors did not specify the reasons for culling, it is likely that unhealed navels were present in these chicks. Although the possibility that B chicks hatched from large eggs cannot be definitely excluded, the lack of a significant difference between B and H hatched chick BW provides some evidence that initial egg weights were similar because there is a strong positive correlation between egg weight and chick weight (McNaughton et al., 1978). It has been reported that RYS contents are preferentially used for growth and development of the small intestine in the immediate posthatch period (Noy and Sklan, 1999). The results of the current research indicate that B chicks were not absorbing the RYS content at the same level as H chicks. Thus, the B chicks are not able to devote the same amount of nutrients for growth and development of the small intestine as the H chicks. Whether B chicks were suffering from subclinical omphalitis was not established in this research. The possibility exists that RYS absorption was impaired because of changes to the YS nutrients caused by bacteria (Sander et al., 1998). Further examination of nutrient transport mechanisms in B and H chicks is warranted to more completely understand the apparent impaired nutrient absorption in B chicks. Embryo Duodenum and Ileum Villi Heights Embryonic duodenal and ileal villi height increased significantly with each day during the last 3 d of incubation (Table 3; P < 0.0001). These changes agree with previously published research by Sklan (2001) and Uni et al. (2003). Differences in villi height observed between the above-mentioned reports and the current research are likely due to the fact that in the current research, the intestinal villi were not classified according to stages of development before measuring the villi (Uni et al., 2003). In the current research, the intestinal villi were classified based on day of incubation before killing the embryos. Chick Duodenum and Ileum Villi Heights Because of the significant effect of the interaction on villi height, the main effects of chick age and navel condition will not be discussed (data not shown). Both duodenal (P < 0.0001) and ileal (P = 0.0293) intestinal villi heights of chicks were influenced by the interaction (Table 4). The only time that B chicks had signifi-

86 Kawalilak et al. Table 4. Duodenum and ileum villi heights for the interaction of chick age (day posthatch) and navel condition for broiler chicks having healed navels or navel buttons at hatching 1 Age navel n 2 Duodenum villi height (µm) Ileum villi height (µm) 0 H 3 10 393.5 f ± 28.6 278.9 e ± 14.9 0 B 4 10 462.6 e ± 25.8 217.1 f ± 15.9 1 H 10 549.9 cd ± 30.0 260.7 e ± 15.8 1 B 10 500.1 de ± 33.3 217.1 f ± 15.0 2 H 10 561.7 cd ± 27.4 316.4 cd ± 15.8 2 B 10 464.3 e ± 28.3 306.2 d ± 14.3 3 H 10 658.7 a ± 27.3 335.9 c ± 14.5 3 B 10 612.3 b ± 26.2 317.9 cd ± 15.3 4 H 10 650.1 a ± 28.7 368.0 b ± 16.3 4 B 10 593.0 bc ± 29.2 326.5 cd ± 15.2 5 H 10 621.4 ab ± 26.8 399.2 a ± 14.5 5 B 10 655.7 a ± 26.9 358.0 b ± 18.3 a f Means within the same column with different superscripts differ significantly (P 0.05). 1 Values represent means ± SEM. 2 n = number of chicks sampled. 3 H = healed navel. 4 B = navel button. cantly taller villi height than the H chicks was for the duodenum on the day of hatch. The reason for this is unknown. On d 2, 3, and 4 for the duodenum and d 0, 1, 4, and 5 for the ileum, it was the H chicks that had taller villi than the B chicks (Table 4). This is the first time that a negative relationship between navel buttons and intestinal villi height in commercial broiler chicks has been reported. Because the surface area available for nutrient absorption is lower in the B chicks, this poses a physiological disadvantage compared with the H chicks. These results confirm the second hypothesis by showing that the presence of a small navel button is related to a reduction in duodenal and ileal villi height in broiler chicks. These results in conjunction with results showing that the B chicks had more RYS provide evidence that intestinal absorption is impaired in B versus H chicks. This reduced intestinal development of B chicks could further impair the development of the intestine; Noy and Sklan (1999) showed that efficient RYS absorption was essential for intestinal villi growth. Conclusions Collectively, the results of this study showed that chicks with small navel buttons at hatching had larger RYS and shorter intestinal villi than chicks with healed navels. If the shorter villi height in B versus H chicks during the 5 d posthatching is indicative of future intestinal development, this could explain the lower market BW in B versus H birds observed by Fasenko and O Dea (2008). To prove the hypothesis put forth by Fasenko and O Dea (2008) that subclinical omphalitis in B chicks was the cause of lower final BW, more research must be completed. Further research on navel conditions, YS infections, and intestinal physiology is advised to gain insight into the relationship of navel buttons with hatchling health. ACKNOWLEDGMENTS We acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (Ottawa, Ontario, Canada), the Alberta Poultry Industry Council (Edmonton, Alberta, Canada), and the Poultry Research Centre at the University of Alberta (Edmonton, Alberta, Canada). The in-kind donation of hatching eggs and chicks from Lilydale Hatchery (Edmonton, Alberta, Canada) is appreciated. We also thank R. Mandryk (Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada) and J. Turchinsky (Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada) for their technical support and J. Gallegos (Lilydale Hatchery, Edmonton, Alberta, Canada), A. Leszczynski (WISEST Women in Scholarship, Engineering, Science and Technology, Edmonton, Alberta, Canada), and the staff and students of the Poultry Research Centre for their assistance with this project. REFERENCES Bigot, K., S. Tesseraud, M. Taquis, and M. Picard. 2001. Alimentation néonatale et développement précoce du poulet de chair. INRA Prod. Anim. 14:219 230. Canadian Council on Animal Care. 1993. A Guide to the Care and Use of Experimental Animals. Vol. 1. 2nd ed. E. D. Olfert, B. M. Cross, and A. A. McWilliams, ed. Can. Counc. Anim. Care (CCAC), Ottawa, Ontario, Canada. Chamblee, T. N., J. D. Brake, C. D. Schultz, and J. P. Thaxton. 1992. Yolk sac absorption and initiation of growth in broilers. Poult. Sci. 71:1811 1816. Esteban, S., J. M. Rayó, M. Moreno, M. Sastre, R. V. Rial, and J. A. Tur. 1991. A role played by the vitelline diverticulum in the yolk sac resorption in young post-hatched chickens. J. Comp. Physiol. [B] 160:645 648. Fasenko, G. M., and E. E. O Dea. 2008. Evaluating broiler growth and mortality in chicks with minor navel conditions at hatching. Poult. Sci. 87:594 597.

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