Changes in yolk sac membrane absorptive area and fat digestion during chick embryonic development

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1 Changes in yolk sac membrane absorptive area and fat digestion during chick embryonic development L. Yadgary, O. Kedar, O. Adepeju, and Z. Uni1 Department of Animal Science, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot 76100, Israel ABSTRACT The capacity of yolk sac (YS) utilization by the chick embryo may be affected by structural changes in the YS membrane (YSM) and by the mechanisms within its cells for digestion, absorption, and transfer of nutrients. Two experiments were conducted to examine structural and digestive changes in the YS of the broiler chick embryo; weights of embryo, YS, and YSM, as well as the total area of the YSM and the absorptive area of the YSM, were measured between embryonic day (E) 5 and E21. In addition, fat content, lipase activity, and bile acid concentration in the YSM and YS contents (YSC) were measured between E11 and E21. Results showed that YSM weight increased from 0.19 g on E5 to 6.46 g on E15, and decreased by 3.74 g between E17 and E21. The absorptive YSM area increased from 536 mm 2 on E5 (51% of total YSM area) to 6,370 mm 2 (86% of total area) on E17, and decreased to 4,439 mm 2 on E21 (85% of total area). The smaller YSM area between E17 and E21 did not decrease the rate of YS fat utilization, which could suggest that YSM mechanisms for fat absorption, digestion, and secretion increased during that period. Total YSM lipase activity relative to fat content (units per g of YSM fat) increased from approximately 1,000 units on E15 to 1,500 units on E21. The detection of lipase in the YSM lends support to the hypothesis that YS lipids are hydrolyzed in the lipolysosomes of the YSM. The current study also confirmed for the first time that bile acids are present in the YS, with levels that ranged from 0.61 to 1.06 µmol/g in the YSM, and may suggest that bile is synthesized in the YSM of the chick embryo. Results of the current study contribute to our understanding of the developmental changes that affect YS functionality and could give insight into the coordination between the embryo s demands and YSM morphological, absorptive, digestive, and secretive changes. Key words: yolk sac membrane, absorptive area, lipase, bile acid, nutrient utilization 2013 Poultry Science 92 : INTRODUCTION The yolk sac (YS) provides the chicken embryo with essential nutrients for its growth. Protein, fat, carbohydrates, and minerals are stored in the yolk of the chicken hen egg for future utilization by the embryo (Moran, 2007; Uni et al., 2012). The utilization of these nutrients is enabled by an extra-embryonic membrane that surrounds the yolk, the YS membrane (YSM). During the first week of incubation, the inner endodermal layer of the YSM increases in area and spreads over the surface of the yolk, concomitant with the vascularization of the YSM by the outer supportive mesodermal layer. By embryonic d (E) 10, the entire yolk is surrounded and vascularized, and the endodermal cells of the YSM have developed into columnar epithelial absorptive cells (Speake et al., 1998a). These endodermal epithelium 2013 Poultry Science Association Inc. Received November 7, Accepted February 20, Corresponding author: uni@agri.huji.ac.il cells (EEC) perform crucial absorptive, metabolic, and secretive processes that relate to the delivery of nutrients from the YS to the embryo; nutrients are absorbed into the EEC from the YS content (YSC), undergo different metabolic, digestive, and reassembly processes, and are then secreted to the blood circulation of the embryo (Noble and Cocchi, 1990). The utilization of nutrients from the YS by the embryo is affected by the embryonic demands for nutrients and by the capacity of the YSM to deliver these nutrients (i.e., the maximum amount of fat, protein, water, carbohydrates, and minerals that the YSM can absorb from the YSC and transfer to the embryo per day). This capacity is dependent upon morphological, digestive, metabolic, and molecular changes in the YSM that occur during incubation. Among them are the following: Structural changes such as the development of endodermal surface area and the formation of elaborate villus-like folds (Romanoff, 1960); 1634

2 Changes in digestive processes in the YSC and in mechanisms controlling nutrient transport from the YSC into the EEC, such as receptor-mediated endocytosis of lipoproteins and transporters for amino acids (Lambson, 1970; Hermann et al., 2000; Yadgary et al., 2011); and Changes in digestion and transfer of nutrients in the EEC that play a pivotal role in YS utilization. Of great importance is the digestion and transport of fat, the main energy source for the chick embryo (Speake et al., 1998b); lipoproteins of a maternal liver origin [containing apo-very low density lipoproteins (VLDL)-II] are hydrolyzed, reesterified, and resynthesized in the EEC into very low density lipoproteins (VLDL containing apob), which are then exocytosed to the blood and transferred to the embryonic tissues (Kanai et al., 1996; Murray et al., 1999; Powell et al., 2004). Although the process of reesterification and reassembly of lipoproteins has been extensively studied in the YSM, few studies have described the activity and involvement of lipase, an enzyme that cleaves fatty acids from triglycerides and phospholipids, in the hydrolysis of lipids in the YSM of the broiler chick embryo. Furthermore, although it was speculated that the change in the color of the YS from yellow to green during the last days of incubation was due to the presence of bile acids (Surai and Speake, 1998; Speake, 2006), no study has corroborated this assumption. Such a study is needed to elucidate whether bile acids may have a role in emulsifying lipids in the YS. Moreover, although YSM morphological changes may have a profound effect on nutrient utilization, few studies have been conducted on morphological changes of the YSM and of its absorptive area during broiler embryo development. The current study aims to document changes in the YSM that are associated with YS utilization. Therefore, 2 experiments were conducted to examine morphological changes as well as digestive changes during incubation of the broiler embryo. In the first experiment, the embryo, YS, and YSM were weighed on E5, E7, E10, E13, E15, E17, E19, and at hatch (E21); The total area of the YSM, and within the total area, the absorptive area of the YSM (wherein absorptive cells and structures such as EEC and villus-like folds develop in the YSM, Figure 1) were measured, and the relationship between YSM absorptive area and YSM weight was evaluated. In the second experiment, the YSM and YSC were sampled on E11, E13, E15, E17, E19, and E21, and lipase activity, bile acid concentration, and fat content were measured in the YSM and YSC. YOLK SAC MEMBRANE CHANGES obtained from a commercial breeder farm (Brown, Hod Hasharon, Israel). Eggs were incubated in a Petersime hatchery at the Faculty of Agriculture of the Hebrew University under standard conditions (37.5 C, 60% RH). Ten eggs, representing the weight distribution of the eggs at set, were selected at E5, E7, E10, E13, E15, E17, E19, and E21 (day of hatch). Embryos were killed by cervical dislocation; the YS was removed and the embryo and YS were weighed. The YS was carefully placed on a transparent paper that was on top of a moderately concave plate (so the YSC would pour out of the YS without structural damage to the YSM), and was then gently spread out to its maximum area. The edges of the YSM were then taped to the paper to avoid shrinkage of the YSM area. A transparent paper was placed on the YSM, and the top absorptive side of the YSM was scanned as a computer image. The YSM was then removed from the paper to determine its weight. The total area of the YSM (mm 2 ) and the absorptive area of the YSM (mm 2 ; Figure 1) were digitally quantified using the digital imaging software cell B 2.3 (Olympus, Hamburg, Germany). Experiment Fertile eggs (n = 120, mean weight = 60.3 g, SD = 4.3 g), from 30-wk-old broiler breeder Cobb hens, were obtained from a commercial breeder farm (Brown, Hod Hasharon, Israel). Eight eggs were selected for fresh yolk analysis: fresh yolk was weighed, homogenized, and stored at 20 C. The remaining eggs were incu- Experiment 1 MATERIALS AND METHODS Fertile eggs (n = 150, mean weight = g, SD = 3.97 g) from 35-wk-old broiler breeder Cobb hens were Figure 1. A scanned image of the yolk sac membrane (YSM) on d 17 of chick embryonic development. The YSM is spread out to its maximum area (see Materials and Methods) for total and absorptive YSM measurements. Total YSM area = absorptive area + nonabsorptive area.

3 1636 YADGARY et al. bated in a Petersime hatchery at the Faculty of Agriculture of the Hebrew University under standard conditions. Eight eggs, representing the weight distribution of the eggs at set, were selected at E11, E13, E15, E17, E19, and E21. Embryos were killed by cervical dislocation, the YSC was separated from the YSM, and both were weighed, homogenized, and stored at 20 C for fat content, lipase activity, and bile acid analyses. Lipid Analysis. Total lipids were extracted using a modified method of Folch et al. (1957), as previously described by Yadgary et al. (2010). Briefly, 10 ml of chloroform-methanol (2:1 vol/vol) was added to 0.5 ml of the fresh yolk, YC, and YSM homogenates. After 30 min, 2 ml of distilled water was added. Following overnight incubation at 4 C, 5 ml of the bottom layer was transferred to glass tubes (weighed in advance) and oven-dried at 105 C. The tubes with the remaining fat were weighed and the weight of fat per gram of fresh yolk, YC, and YSM was calculated. Lipase Activity Analysis. Lipase activity was determined by a colorimetric method using a lipase reagent set (Pointe Scientific Inc., Canton, MI). Briefly: Samples (0.1 g each) of homogenized fresh yolk, YSC, and YSM were transferred to separate microcentrifuge tubes. Beads were added, along with 0.4 ml of distilled water, and the mixture was blended using a mini beadbeater (Biospec Products, Bartlesville, OK). To 2.5-μL aliquots of the fresh yolk, YSC, and YSM homogenates, 150 μl of lipase substrate reagent was added and incubated for 5 min at 37 C. Then, 50 μl of lipase activator was added and incubated for 5 min at 37 C, after which the rate of increase in absorbance per minute was read at 550 nm. The activity of lipase was determined per g of YSM or YSC [units (U) per gram of tissue], and the total activity of lipase in the fresh yolk, YC, and YSM was calculated (U). Bile Acid Analysis. Bile acid concentration was determined using the Total Bile Acids Assay Kit (Diazyme, San Diego, CA). Briefly, samples (0.1 g each) of homogenized YSC and YSM were transferred to separate microcentrifuge tubes. Beads were added, along with 0.4 ml of distilled water, and the mixture was blended using a mini bead-beater. To 2.5-μL aliquots of YC and YSM homogenates, 150 μl of Thio-NAD was added and incubated for 3 min at 37 C. Then, 50 μl of 3-α-hydroxysteroid dehydrogenase was added, after which the absorbance was read at 405 nm for 2 min. The concentration of bile acids (μmol/g) was determined, and the total amount of bile acids in the YSC and YSM was calculated (μmol). Statistical Analysis All data were subjected to one-way ANOVA. Differences among means were tested by contrasts using Student s t-test. Values are presented as means ± 95% CI. All statistical analyses were carried out using JMP 8 software (2007 version, SAS Institute Inc., Cary, NC). RESULTS AND DISCUSSION The capacity of YS utilization may be affected by YSM structural changes and by the mechanisms within its cells for digestion and absorption. To achieve an optimal YS utilization by the embryo, a good understanding of the digestive, absorptive, and secretive processes that take place in the YS is needed. We examined 2 aspects of YS utilization the absorptive area of the YSM tissue and the digestion of lipids by lipase and bile acids. In the second half of incubation, after all extraembryonic tissues have been fully developed and the YSM has totally surrounded the yolk, the chick embryo begins its rapid growth (Figure 2A) and extensive YS utilization (Figure 2B). To adjust to the growing nutritional demands of the embryo and to the spatial changes inside the egg, the YSM is in a continuous stage of change (Romanoff, 1960; Freeman and Vince, 1974). In the current study, YSM weight increased from 0.19 g on E5 to 1.44 g on E10, rapidly increased by 5 g between E10 and E15, and decreased by 2.6 g between E17 and E21 (Figure 3A). A similar pattern was observed by Romanoff (1960), Yadgary et al. (2010), and Yadgary and Uni (2012). To elucidate the changes in YSM weight, we measured the total YSM area and the absorptive YSM area. Total and absorptive YSM area increased and decreased in a similar but not identical pattern to YSM weight. Total YSM area increased from 1,042 mm 2 on E5 to 4,183 mm 2 on E10, increased by 4,500 mm 2 between E10 and E15, and then decreased by 3,300 mm 2 until E21 (Figure 3B). The absorptive area of the YSM increased from 536 mm 2 on E5 (51% of total YSM area) to 6,370 mm 2 on E17 (86% of total area), and decreased by 1930 mm 2 from E17 to E21 (Figure 3B). The mean YSM weight per each examined embryonic day was found to be highly correlated to the means of absorptive YSM area (r = 0.96). Our results indicate that changes in YSM weight are associated with structural changes in absorptive YSM area (increase or decrease) rather than with accumulation or depletion of yolk content inside the EEC (as suggested by Romanoff, 1960). It could be suggested that in future studies YSM weight (easy and fast to measure) can be used to evaluate differences in absorptive area (difficult to measure and time consuming) between eggs from different genetic lines, hen ages, or incubation treatments. It should also be noted that a more accurate estimate of the absorptive surface area of the YSM could be calculated by a mathematical model that will combine similar methods of area measurements as those used in the current study with surface area measurements from histological cross sections, such as cell area, villus area, and number of microvilli per cell (Uni et al., 2000; Kisielinski et al., 2002). The structural changes of the YSM, which were observed in the first experiment, act in accordance with

4 Figure 2. Weights of (A) embryo, and (B) yolk sac (YS; membrane + content), during chick embryonic development (n = 10). Data are expressed as means ± 95% CI. Means with different letters (a g) differ significantly (P < 0.05) between days. the growing embryonic demand for nutrients toward hatch. A larger area to absorb and transfer YSC nutrients is needed as the embryo develops; changes in the germ layer of the YSM and proliferation of the EEC are most likely involved in the process (Romanoff, 1960). However, despite the growing nutritional needs of the YOLK SAC MEMBRANE CHANGES 1637 embryo, in the last 2 to 3 d of incubation, the total and absorptive area of the YSM decreases (Figure 3B) because the YS is due to be redrawn into the embryo s body cavity just before hatch. This breakdown of the YSM is probably facilitated by an extensive apoptotic process in the EEC (Romanoff, 1960; Yadgary et al., 2010). The smaller YSM area to absorb and secrete nutrients did not decrease the rate of YS fat utilization between E17 and E21 (Figure 4), which could suggest that YSM mechanisms for fat absorption, digestion, and secretion increased during this period. The utilization of fat from the YS by the chick embryo provides the main energy source for tissue development during incubation (Noble and Cocchi, 1990). In the last week of incubation, YS fat is extensively used and its levels decrease by more than 50%, as was observed in the current trial (Figure 4). The YS fat utilization is affected, among other things, by the digestive capacity of hydrolytic enzymes in the YSC and in the YSM. Accordingly, in the second experiment, we examined the activity of the digestive enzyme lipase and the presence of bile acids in the YSC and YSM. Lipase and bile acids were detected in the YSM as well as in the YSC. Lipase was also detected in the yolk on day of set (60 U/g); however, higher levels of activity were found in the YSM and YSC during incubation. Lipase levels of activity per 1 g of YSM decreased from 450 U/g on E11 to approximately 250 U/g on E13 with no significant change between E13 and E21 (Figure 5A). Lipase activity in the YSM lends support to the hypothesis that YS lipids are hydrolyzed in the lipolysosomes of the EEC into free fatty acids, partial glycerides, and glycerol (Kanai et al., 1996), which are then transferred to the endoplasmic reticulum, reesterified to form triglycerides and phospholipids, and assembled to VLDL particles (Murray et al., 1999; Powell et al., 2004). During incubation, bile acid concentration in the YSM ranged from 0.61 to 1.06 µmol/g, with a significant increase of 0.3 µmol/l between E11 and E13 (Figure 5B). Bile acids in the YSM could serve as an emul- Figure 3. Weight and area measurements of the yolk sac membrane (YSM) from embryonic d 5 to 21 (hatch; n = 10). (A) YSM weight (g), and (B) total YSM area (mm 2 ); and absorptive YSM area (mm 2 ). Data are expressed as means ± 95% CI. Means (within weight, total area, and absorptive area) with different letters (a g) differ significantly (P < 0.05) between days.

5 1638 YADGARY et al. Figure 4. Fat levels in the yolk sac (YS; membrane + content), YS membrane (YSM), and YS content (YSC) from embryonic d 11 to 21 (hatch; n = 8). (A) fat concentration (mg/g), and (B) fat amount (g). Data are expressed as means ± 95% CI. Means (within YS, YSM, and YSC) with different letters (a e) differ significantly (P < 0.05) between days. sifier of lipids, thus increasing the surface area of fat globules, making them more available to the action of lipase in the lipolysosomes of the YSM. To our knowledge, the present study is the first to verify the hypothesis that bile acids are found in the YSM of the chick embryo (Surai and Speake, 1998; Speake, 2006). Lipase activity and bile acids concentration were lower in the YSC compared with the YSM (Figure 5A, 5B). However, similar levels were found in the YSM and the YSC on E21, after a moderate increase in YSC lipase activity and a substantial increase in YSC bile acid concentration between E15 and E21. Surai and Speake (1998) suggested that the change of color of the YS from yellow to green during the final days of embryonic development could be due to a recycling of bile, which was secreted from the gallbladder to the intestine, into the YSC; this hypothesis is supported by a study on quail embryos that demonstrated that a part of the amniotic fluid reaches the YSC through the intestine (Yoshizaki et al., 2002). However, the higher levels of lipase and bile in the YSM compared with the YSC, as well as evidence for the ability of the EEC of the YSM to synthesize taurine, an organic acid that conjugates bile acids to form water-soluble bile salts (Chapeville and Fromageot, 1957), may indicate their synthesis in the YSM. The YSM has been previously shown to express genes that are involved in digestive and biochemical functions associated exclusively with the liver and the intestine (Yadgary et al., 2011; Speier et al., 2012; Yadgary and Uni, 2012). Nonetheless, the possibility that the complex series of enzymatic reactions involved in bile production occur in the YSM should be examined on a protein level and a transcriptional level. We also speculate that a major part of the enhanced levels of lipase activity and bile acids in the YSC during incubation, as compared with the fresh yolk, may be explained by a secretion of lipase and bile from the YSM to the YSC. The YSM s EEC, which have undergone the apoptotic process mentioned above, lose anchorage and shed into the YSC, and subsequently lipase and bile reach the YSC. It could also be suggested that toward hatch and after hatch, due to the nonspecific transfer of YSC directly to the intestine (Esteban et al., 1991; Yadgary and Uni, 2012), lipase and bile acids of YSM origin may reach the intestinal lumen of the embryo and hatchling and further promote lipid digestion and absorption. Escribano et al. (1988) reported that the activity of lipase per gram of yolk may be affected by compositional and structural changes in the YS, and suggested that a better way to express lipase activity would be per unit of fat. We calculated the total activity of lipase (Figure 5C) as well as the total amount of bile acids in the YSM or YSC (Figure 5D), and as suggested by Escribano et al. (1988), we calculated the activity of lipase per g of fat in the YSM or YSC (Figure 5E), as well as bile acid to fat ratio in the YSM or YSC (Figure 5F). Total lipase activity and bile acids corresponded with changes in YSM and YSC weight (Figure 5C, 5D), whereas lipase activity and bile acids relative to fat content increased in the YSM and YSC between E17 and E21 (Figure 5E, 5F). Further studies may help elucidate whether an increase in digestion may be coordinated with the breakdown of the YSM to maintain an adequate supply of fat to the embryo. In summary, YS utilization is affected by the morphological and digestive changes occurring in the YS. The total area of the YSM and its absorptive area increased up to E17 and decreased until hatch. These changes may relate to the increasing demand for nutrients from the YS but may also be related to the assimilation of the YS into the embryo s body cavity toward hatch. Lipase activity was detected in the YSM and is probably associated with the lysosomal digestion of lipoproteins in the EEC; this digestion is apparently aided by bile acids that for the first time have been detected in the YSM. Lipase and bile acids were also found in the YSC, their possible transfer to the intestine may have a role in the digestion of fat in the intestine of the embryo and hatchling. Further research is needed to elucidate whether lipase and bile acids are synthesized in the YSM, as well as to better understand the coordination

6 YOLK SAC MEMBRANE CHANGES 1639 Figure 5. Lipase and bile acids levels in the yolk sac membrane (YSM), and yolk sac content (YSC) from embryonic d 11 to 21 (hatch; n = 8). (A) lipase activity (U/g), (B) bile acids concentration (µmol/g), (C) total lipase activity (U), (D) total bile acids (µmol), (E) lipase activity relative to fat content (U/g of fat), and (F) bile acids relative to fat content (µmol/g of fat). Data are expressed as means ± 95% CI. Means (within YSM and YSC) with different letters (a d) differ significantly (P < 0.05) between days. between the embryo s demands and the morphological, absorptive, digestive, and secretive changes in the YSM, to achieve an efficient transfer of nutrients and an optimal development of the broiler embryo. REFERENCES Chapeville, F., and P. Fromageot Formation of sulfite, cysteic acid & taurine from the sulfate of the embryonic egg. Biochim. Biophys. Acta 26: Escribano, F., B. E. Rahn, and J. L. Sell Development of lipase activity in yolk membrane and pancreas of young turkeys. Poult. Sci. 67: Esteban, S., J. Rayo, M. Moreno, M. Sastre, R. Rial, and J. Tur A role played by the vitelline diverticulum in the yolk sac resorption in young post hatched chickens. J. Comp. Physiol. B 160: Folch, J., M. Lees, and G. H. Sloane Stanley A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: Freeman, B. M., and M. A. Vince Development of the Avian Embryo. Chapman and Hall, London, UK. Hermann, M., M. G. Mahon, K. A. Lindstedt, J. Nimpf, and W. J. Schneider Lipoprotein receptors in extraembryonic tissues of the chicken. J. Biol. Chem. 275: Kanai, M., T. Soji, E. Sugawara, N. Watari, H. Oguchi, M. Mustasabra, and D. C. Herbet Participation of endodermal epithelial cells in the synthesis of plasma LDL and HDL in the chick yolk sac. Microsc. Res. Tech. 35: Kisielinski, K., S. Willis, A. Prescher, B. Klosterhalfen, and V. Schumpelick A simple new method to calculate small intestine absorptive surface in the rat. Clin. Exp. Med. 2: Lambson, R. O An electron microscopic study of the entodermal cells of the yolk sac of the chick during incubation and after hatching. Am. J. Anat. 129:1 19. Moran, E. T., Jr Nutrition of the developing embryo and hatchling. Poult. Sci. 86: Murray, A. M., R. Denis, and B. K. Speake Acyltransferase activities in the yolk sac membrane of the chick embryo. Lipids 34: Noble, R. C., and M. Cocchi Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29: Powell, K. A., E. A. Deans, and B. K. Speake Fatty acid esterification in the yolk sac membrane of the avian embryo. J. Comp. Physiol. B 174:

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