Effects of In Ovo Feeding of Carbohydrates and β-hydroxy-β-methylbutyrate on the Development of Chicken Intestine 1

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1 Effects of In Ovo Feeding of Carbohydrates and β-hydroxy-β-methylbutyrate on the Development of Chicken Intestine 1 E. Tako,* P. R. Ferket, and Z. Uni*,2 *The Faculty of Agricultural, Food and Environmental Quality Sciences, the Department of Animal Sciences, Hebrew University of Jerusalem, PO Box 12, Rehovot, 76100, Isarel; and North Carolina State University, Department of Poultry Science, College of Agriculture and Life Sciences, Raleigh, North Carolina ABSTRACT Early development of the digestive tract is < 0.05) 48 h after IO feeding in all the IO fed embryos, crucial for achieving maximal growth and development of chickens. Because the late-term embryo naturally consumes the amniotic fluids, insertion of a nutrient solution into the embryonic amniotic fluid [in ovo (IO) feeding] whereas at day of hatch and at d 3 the CHO+HMB IO group had the highest maltase activity (P < 0.05), which was approximately 50% greater than control embryos. These observations indicated that small intestines of IO may enhance development. This study examined the effect of IO feeding on d 17.5 of incubation of carbohydrates velopment as a conventionally fed 2-d-old chick. Body fed hatchlings were functionally at a similar stage of de- (CHO) and β-hydroxy-β-methylbutyrate (HMB) on small weight of all IO fed hatchlings was greater than controls, and these differences (P < 0.05) were sustained until the intestinal development of chickens during the pre and end of the experiment (10 d). At d 10 chicks that were posthatch periods. Results shows that 48 h post-io feeding procedure all IO feeding treatments exhibited in- IO fed with CHO had BW that were 2.2% higher, whereas HMB and CHO+HMB IO fed chicks showed 5 to 6.2% creased villus width and surface area compared with the BW increase, respectively, compared with controls. The control group. At d 3 posthatch the surface area of an current study shows that the administration of exogenous average villi was increased by 45% for the HMB IO group nutrients into the amnion enhanced intestinal development and by 33% for the CHO and CHO+HMB IO groups compared with controls (noninjected fertile eggs). The activity of jejunal sucrase-isomaltase (SI) was higher (P by increasing the size of the villi and by increasing the intestinal capacity to digest disaccharides. This advantage probably leads to higher BW in IO fed chicks. (Key words: chicken, in ovo feeding, small intestine, sucrase-isomaltase) 2004 Poultry Science 83: INTRODUCTION The intestinal epithelium is a complex system of multiple cell types undergoing continual renewal and change with well-orchestrated patterns of gene expression during the processes of development and differentiation. This system has a major role in determining the developmental potential of the hatched chick (Bayer et al., 1975; Uni et al., 1995, 1998). Chick growth and development are dependent upon nutrient digestion and absorption, which is a direct result of the functional and morphological development of the small intestine (Baranylova and Holman, 1976; Sell et al., 1991; Akiba and Murakami, 1995; Yamauchi et al., 1996; Uni, 1999). In chickens, the first days after hatch are a critical period for development because a major change 2004 Poultry Science Association, Inc. Received for publication April 14, Accepted for publication August 31, This research was supported by Research Grant Award IS from BARD, The United States-Israel Binational Agriculture Research and Development. 2 To whom correspondence should be addressed: uni@agri.huji.ac.il. occurs in the source of nutrients as the yolk is replaced by an exogenous diet (Noble and Ogunyemi, 1989; Noy and Sklan, 1998). Because the intestine is the nutrient supply organ, the sooner it achieves its functional capacity the earlier the young chick can use dietary nutrients and grow according to its genetic potential (Uni et al., 2003b; Uni and Ferket, 2004). Chicken embryos have limited ability to digest and absorb nutrients prior to hatch, as reflected by relatively low mrna levels of sucrase-isomaltase (SI) and l-aminopeptidase and the ATPase and sodium glucose transporter (SGLT-1) in the small intestinal mucosa (Uni et al., 2003b). This absorption capability increases close to hatch and continues to increase during the first few days posthatch (Uni et al., 1999; Sklan, 2001; Uni et al., 2003b). Villus height increases by 200 to 300% from d 17 of incubation until hatch. During the posthatch period the small intestine weight Abbreviation Key: CHO = carbohydrates: maltose, sucrose, dextrin, NaCl; CHO+HMB = maltose, sucrose, dextrin, NaCl, β-hydroxy-β-methylbutyrate; HMB = β-hydroxy-β-methylbutyrate, NaCl; HMG-CoA = β- hydroxy-β-methylglutaryl coenzyme A; SI = sucrase-isomaltase. 2023

2 2024 TAKO ET AL. increases faster than the body mass (Katanbaf et al., 1988; Sell et al., 1991; Sklan, 2001). This rapid intestinal growth is due to accelerated processes of enterocyte proliferation and differentiation (Geyra et al., 2001a). In addition, the intestinal crypts, which begin to form at hatch, are clearly defined several days posthatch, increasing in cell numbers and size (Uni et al., 2000; Geyra et al., 2001a). Previous studies have shown that feeding immediately posthatch leads to an acceleration of small intestinal morphological development (Noy and Sklan, 1998), whereas late access to external feed results in delayed development of the small intestine mucosal layer (Uni et al., 1998; Geyra et al., 2001a; Uni et al., 2003b). Furthermore, birds denied access to first feed for 24 to 48 h have decreased villi length (Yamauchi et al., 1996), decreased crypt size and crypts per villi, and decreased enterocyte migration rate (Geyra et al., 2001b). In addition, delayed access to feed for 48 h posthatch resulted in changes in mucin dynamics, which probably affected the absorptive and protective functions of the small intestine (Uni et al., 2003a). Thus, because immediate access to feed after hatch is critical for the development of the intestine, nutrient supply during the prehatch period [at d 17 or 18 of incubation, using in ovo (IO) feeding] (Uni and Ferket, 2004) would be expected to enhance development of the small intestine. Many possible nutrient supplements could be included in the IO feeding solution. Carbohydrates (CHO) would be used as a source for glucose, which is crucial for hatchling development (Moran, 1985). Sodium ions are important for the brush border transporter action (Gal-Gerber et al., 2000; Currid et al., 2004). In addition other nutrients, such as minerals, vitamins, and enteric modulators are good candidates for IO feeding (Uni and Ferket 2004). From the research reported by Nissen et al., (1994) β- hydroxy-β-methylbutyrate (HMB), a leucine metabolite, decreased chicken mortality and increased carcass yield. HMB is produced through the metabolism of α-ketoisocaproate, which is oxidized by α-ketoisocaproate dioxygenase to produce free HMB within the cell cytosol (Sabourin and Bieber, 1982; Sabourin and Bieber, 1983). Studies have shown (Van Koevering and Nissen, 1992) that small amounts of HMB are synthesized endogenously in the body, as approximately 5% of leucine metabolism normally proceeds through this pathway. It was proposed by Nissen and Abumarad (1997) that HMB is converted to β-hydroxyβ-methylglutaryl coenzyme A (HMG-CoA) in some tissues and serves as a key carbon source for de novo cholesterol synthesis in tissues, which is necessary to maintain maximal cell function. Furthermore, these researchers suggested that in situations of stimulated growth or differentiation HMG-CoA may be rate limiting for cholesterol synthesis, which could limit cell growth or function. Therefore, it was proposed that feeding HMB could provide a saturating source of cytosolic HMG-CoA for cholesterol synthesis and, in turn, allow maximal cell growth and function. 3 Gold Kist Hatchery, Silver City, NC. The present study examined the hypothesis that IO feeding will accelerate development of the digestive tract during the pre- and posthatch periods. The effect of providing feeding solutions, which contained sodium chloride, CHO, and HMB on intestinal development was studied from the last quarter of embryonic incubation until d 3 posthatch by measuring morphological parameters of the intestinal mucosal layer and by evaluating activity of SI, a major brush border digestion enzyme, MATERIALS AND METHODS Incubation and IO Feeding Fertile eggs (Ross Ross) were obtained from a commercial hatchery 3 from a maternal flock 35 wk in lay. The eggs were incubated under optimal conditions at the Department of Poultry Science at North Carolina State University. Ten eggs from each group were sampled at 18, 19, and 20 d of incubation, at hatch, and at d 3 posthatch. At 17.5 d of incubation, 400 eggs containing viable embryos were weighed and divided into 4 groups with equal weight frequency distribution of 100 eggs each with average egg weight of g. Each group was then injected with suitable IO feed treatment solutions (1 ml/egg) with a 21-ga needle inserted into the amniotic fluid, which was identified by candling. The 3 treatment groups included the following: 1) CHO solution: 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, and 5 g of NaCl/L, 2) HMB solution: 1 g of HMB/L in 5 g of NaCl/L, 3) CHO+HMB solution: 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1 g of HMB/L in 5 g of NaCl/L. The control was a noninjected group that paralleled routine procedures in commercial hatcheries. In addition, preliminary experiments conducted in our laboratory indicated that injection of 1 ml of 5 g of NaCl/L did not affect (P < 0.05) embryo and chick BW, intestinal development, or brush border membrane enzymatic activity. After the eggs were injected, the injection holes were sealed with cellophane tape, and eggs were placed in hatching trays such that each treatment was equally represented in each location of the incubator. Collecting Intestinal Segments The jejunum was removed as previously described (Uni et al., 2003a) and 1 cm long segments were taken and placed in 2 separate tubes: 1) fixed in 4% neutral-buffered formalin solution for histology, and 2) stored at 20 C for determination of SI activity. Bird Housing Upon hatch (hatchability was 91% in all groups), chicks of each treatment were randomly assigned to 7 pens per treatment (10 hatchlings/m 2 pen) and housed in a total confinement building at North Carolina State University. Each pen was equipped with an automatic nipple drinker and manual self-feeder. The concrete floor was bedded with wood shavings, and supplemental incandescent heat

3 EFFECTS OF IN OVO FEEDING ON INTESTINE DEVELOPMENT 2025 TABLE 1. Effect of in ovo feeding of CHO, 1 HMB, 2 and CHO+HMB 3 on small intestinal jejunal villus length, 4 width, 4 and surface area 4 from d 19 of incubation (19E) 5 until d 3 posthatch In ovo treatment 19E 20E 6 Hatch 3 d Villus length (µm) CHO ± 7.60 a ± 6.10 c ± 41.2 b ± 59.5 b HMB ± 14.7 a ± 15.3 a ± 39.6 a ± 27.5 a CHO+HMB ± 12.9 a ± 10.4 b ± 53.6 b ± 56.5 d Con ± 9.60 a ± 12.9 c ± 29.2 c ± 49.7 c Villus width (µm) CHO ± 5.50 b ± 3.90 b ± 2.70 b ± a HMB ± 6.10 a ± 2.90 a ± 9.50 a ± a CHO+HMB ± 6.80 c ± 2.90 b ± 6.50 a ± a Con ± 0.70 c ± 4.10 c ± 4.40 c ± b Villus surface area (µm 2 ) CHO 11,473 ± 900 b 20,517 ± 1,500 c 69,313 ± 2,000 c 458,708 ± 15,000 b HMB 23,394 ± 800 a 32,762 ± 1,000 a 107,451 ± 1,500 a 499,783 ± 15,000 a CHO+HMB 9,710 ± 800 b 26,939 ± 1,000 b 90,968 ± 2,500 b 456,996 ± 20,000 b Con 10,033 ± 900 b 13,548 ± 1,000 d 51,194 ± 1,000 d 345,258 ± 10,000 c a d Treatment means with different letters within a sample time are significantly different (P < 0.05). 1 Carbohydrate; 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l in 5gofNaCl/L. 2 β-hydroxy-β-methylbutyrate; 25 g of HMB/L in 5gofNaCl/L. 3 CHO+HMB = 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1gofHMB/Lin5gofNaCl/L. 4 Values are means ± SE of 5 birds with 50 villi measured for each bird. 5 Thirty-six hours post in ovo feeding procedure. 6 Day 20 of incubation. was provided to the chicks in a confined area. Body weights of all birds were recorded at hatch and at 3, 7, and 10 d posthatch. Bird Care All experimental protocols were approved by the Institutional Animal Care and Use Committee. All birds were given ad libitum access to water and diet formulated to meet NRC recommendations (1994) in all experiments. Activity of SI Enzyme activity was assayed using jejunal lysates (250 mg of tissue/5 ml of 50 mm sodium phosphate buffer, ph 7.2). Maltase (EC ) activity was assayed colorimetrically using maltose as a substrate (Dahlquist, 1964; Palo et al., 1995a,b) and expressed as millimoles of glucose released per minute per gram of jejunal wet tissue. Morphological Examination Intestinal samples (jejunum region) at 20 d of incubation, hatch, and 3 d posthatch from each treatment were fixed in fresh 4% (vol/vol) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Serial sections were cut at 5 µm and placed on glass slides. Sections were deparaffinized in xylene, rehydrated in a graded alcohol series, stained with hematoxylin and eosin, and examined by light microscopy. Morphometric measurements of villus height and width were performed with an Olympus light microscope using EPIX XCAP software. 4 Villus surface area was calculated from villus height and width at half height (Uni et al., 1998). 4 Epix Inc., Buffalo Grove, IL. 5 SAS User s Guide, 1986, Version 6, SAS Institute Inc., Cary, NC. Statistical Analysis Results were analyzed by ANOVA using the GLM procedures of SAS software. 5 Differences between treatments were compared by the Tukey test following ANOVA, and values were considered statistically different at P < Results are reported as least squares means with standard errors. RESULTS At 19 d of incubation, 36 h after IO feeding treatments, only the HMB treatment exhibited greater villus surface area compared with controls and to other IO feeding treatments (Table 1). However, 48 h post IO (20 d of incubation) feeding procedure all IO feeding treatments exhibited (P < 0.05) increased villi width and surface area compared with the control group (Table 1; Figure 1A). On the day of hatch villi length and width of all IO feeding treatments were greater than those of the controls (Table 1; Figure 1B), and on d 3 posthatch the surface area of an average villus was elevated by 45% for the HMB IO group and by 33% for the CHO and CHO+HMB IO groups compared with those of the controls (Figure 2). The activity of jejunal SI was higher (P < 0.05) 48 h after IO feeding (20 E) in all the IO fed embryos, whereas CHO and CHO+HMB IO embryos exhibited the greatest abilities to digest maltose compared with the HMB and control groups (Figure 3). On the day of hatch and at d 3 the CHO+HMB IO group had the highest maltase activity (P < 0.05), and this activity was elevated by approximately 50% compared with control embryos at d 3. No differences were observed among CHO, HMB, and control treatments at hatch; however, significant differences were observed between CHO IO fed chicks and controls at d 3. Body weight of all IO fed hatchlings was greater than in controls (Table 2), and this difference was sustained until

4 2026 TAKO ET AL. FIGURE 1. Effect of in ovo feeding on intestine morphology. Representative light micrographs of intestinal jejunal villi from a chicken embryo at d 20 (panel A; 100), day of hatch (panel B; 100), and 3 d posthatch (panel C; 40). The treatments were carbohydrates (CHO; 25 g of maltose/ L, 25 g of sucrose/l, 200 g of dextrin/l in 5 g/l NaCl); β-hydroxy-β-methylbutyrate (HMB; 1 g of HMB/L in 5 g of NaCl/L); CHO+HMB (25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1 g of HMB/L in 5 g of NaCl/L); Con = control. Bar = 100 µm. the end of the experiment (10 d). At d 10 chicks that were IO fed with CHO had BW that were 2.2% higher, whereas HMB and CHO+HMB IO fed chicks showed 5 to 6.2% BW increases, respectively, compared with controls. DISCUSSION In this study, IO feeding (the administration of exogenous nutrients into the amnion) enhanced intestinal development by increasing the size of the villi and by increasing the intestinal capacity to digest disaccharides. Based on positive preliminary experiments in our laboratories, the IO feeding solution formulation was developed to contain disaccharides (maltose and sucrose), readily digested polysaccharides (dextrin), and a leucine metabolite, HMB. In this study, CHO and HMB were presented to the late-term embryo by IO feeding. Experiments with domestic animals have demonstrated that with calves addition of HMB to feed improves carcass quality and decreases mortality (Vukovich and Dreifort, 2001; Vukovich et al., 2001). Studies have also demonstrated that dietary HMB supplementation decreases mortality and increases carcass yield in broilers, whereas addition of 0.01% HMB to broiler feed increases BW at marketing (42 d) by 1.4% when compared with control broilers (Nissen et al., 1994). Cellular studies, done on isolated muscle strips from rats and chicks have indicated that HMB inhibits proteolysis by 80% and increases protein synthesis by 20% (Ostaszewski and Nissen, 1988). Furthermore, exposure to HMB (0.01%) induces proliferation of broiler macrophages in culture (Peterson et al., 1999) and improves immunocompetance in fish through increased cell proliferation and functionality (Siwicki et al., 2000). Based on the above studies, we hypothesize that IO feeding of HMB could enhance intestinal development by enhancing the processes of proliferation and differentiation of enterocytes or by lowering the rate of protein degradation. The findings in our current study support the hypothesis that HMB influences enterocyte proliferation because the use of HMB in the IO feeding solution, with CHO or separately, resulted in a significant increase in villus surface area (Figures 1 and 2), probably due to a higher proliferation rate. Furthermore, because no significant differences in SI activity were observed between the HMB and control

5 EFFECTS OF IN OVO FEEDING ON INTESTINE DEVELOPMENT 2027 TABLE 2. Body weight 1 (g) of embryos at d 19 of incubation (19E) and hatchlings (hatch and d 3, 7, and 10) from 3 in ovo feeding treatments: CHO, 2 HMB, 3 CHO+HMB, 4 and control Body weight (g) In ovo treatment 19E Hatch Day 3 Day 7 Day 10 CHO ± 0.44 a ± 0.76 a ± 3.74 a ± 0.91 a ± 2.27 b HMB ± 0.90 a ± 0.47 a ± 1.87 b ± 1.15 a ± 2.4 a CHO+HMB ± 0.54 a ± 0.52 a ± 2.18 a ± 2.35 a ± 4.04 a Con ± 0.88 a ± 0.48 b ± 0.83 b ± 1.49 b ± 4.11 c a d Treatment means with different letters within a sample time are significantly different (P < 0.05). 1 Weight values are means ± SE of 10 embryos at 19E, 70 birds at hatch, and 50 birds at d 3, 7, and Carbohydrate; 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l in 5gofNaCl/L. 3 β-hydroxy-β-methylbutyrate; 25 g of HMB/L in 5gofNaCl/L. 4 CHO+HMB = 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1gofHMB/Lin5gofNaCl/L. chicks at day of hatch and at d 3, it is suggested that HMB does not have a direct critical effect on intestinal functionality. Carbohydrates were chosen as an important component of the IO feed solution because they are critical during the final stage of chick embryo development, prior to emergence from the shell, and very little CHO remains in the egg before hatch (Christensen et al., 1993). In addition, several studies have demonstrated that higher levels of the substrate in the intestine elevate activity of disaccharidases. In rats, after 48 h of fasting the sucrase activity decreased and increased during refeeding (Holt and Yeh, 1992). A solution containing free amino acids and glucose that was given to premature neonatal piglets has been reported to enhance maltase and sucrase activities (Petersen et al., 2002) and the feeding of maternal feeding solutions containing various levels of sucrose, glucose, and galactose increased villi length and crypt formation in piglets (Pluske et al., 1996). Therefore, we hypothesized that providing disaccharides and easily digested CHO to the embryonic intestinal tissue, which at d 17.5 E has a low capacity to digest and absorb (Uni et al., 2003b), will elevate the activity of the relevant brush border enzymes. The current results support this hypothesis and show that CHO IO fed embryos had higher maltase activity before and after hatch. Furthermore, addition of HMB to CHO led to the highest levels of sucrase activity at hatch and at d 3 compared with other IO treatments and, therefore, may suggest an enhancing effect of this combination. This finding can be explained by the effect of HMB on enterocyte proliferation and the known effect of CHO substrate on brush border enzyme activity. The morphological measurements of the jejunal villi revealed that CHO IO fed embryos had 50% elevation in villus surface area prior to hatch, whereas HMB IO fed embryos exhibited 140% elevation, and the CHO+HMB IO fed embryos were intermediate (Figure 3; Table 1). The differences remained through hatch and d 3 (Table 1), whereas the gradual reduction in the difference between IO and control treatments suggested that the effect of IO feeding on intestine morphological development is maximal at 48 h after the IO procedure. Because the villus surface area has been shown to be correlated with growth in the chicken (Sklan, 2001) the FIGURE 2. Differences (%) in average villi surface area between control and 3 in ovo (IO) treatments. Carbohydrates (CHO; 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l in 5gofNaCl/L); β- hydroxy-β-methylbutyrate (HMB; 1 g of HMB/L in 5 g of NaCl/L); CHO+HMB (25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1gofHMB/Lin5gofNaCl/L). 20E =d20ofincubation. FIGURE 3. Activity of sucrase-isomaltase at 18, 19, and 20 d of incubation (18E, 19E, and 20E, respectively); hatch; and 3 d posthatch. a c Columns with different letters differ significantly (P < 0.05). Values are means SE, n = 10. The 3 in ovo (IO) feeding treatments were carbohydrates (CHO; 25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l in 5 g of NaCl/L); β-hydroxy-β-methylbutyrate (HMB; 1 g of HMB/L in 5 g of NaCl/L); CHO+HMB (25 g of maltose/l, 25 g of sucrose/l, 200 g of dextrin/l, 1 g of HMB/L in 5 g of NaCl/L).

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