- Email: [email protected]
Effects of Conjugated Linoleic Acid. 2. Embryonic and Neonatal Growth and Circulating Lipids1
Effects of Conjugated Linoleic Acid. 2. Embryonic and Neonatal Growth and Circulating Lipids1 M. A. Latour,*,2 A. A. Devitt,† R. A. Meunier,* J. J. Stewart,* and B. A. Watkins†,3 *Department of Animal Sciences and †Department of Food Sciences, Lipid Chemistry and Molecular Biology Laboratory, Purdue University, West Lafayette, Indiana 47907
(Key words: chick, conjugated linoleic acids, lipids, lipoproteins) 2000 Poultry Science 79:822–826
Hargis et al., 1991; Vilchez et al., 1991; Latour et al., 1998), which is an excellent way of determining the effects of yolk fatty acid modification on neonatal avian lipid metabolism. One of the most interesting polyunsaturated fatty acids to gain attention in recent years is conjugated linoleic acid (CLA). Conjugated linoleic acid was first recognized as an anticarcinogen after being isolated from extracts of grilled ground beef that exhibited anticarcinogenic activity against chemically induced mouse skin cancer (Ha et al., 1987). Conjugated linoleic acid occurs naturally and is reported to have significant biological effects. For example, CLA has been shown to reduce plasma low density lipoprotein (LDL) concentrations and inhibit the development of atherosclerosis in rabbits (Lee et al., 1994) and hamsters (Nicolosi et al., 1997) fed atherogenic diets. Conjugated linoleic acids are also believed to inhibit the action of ∆ 9-desaturase, an enzyme capable of converting stearic acid (18:0) to oleic acid (18:1) (Li and Watkins, 1998). Noble and Shand (1985) have demonstrated that
INTRODUCTION The avian embryo derives more than 90% of its energy requirements from the oxidation of yolk lipids (Romanoff, 1960). Therefore, the yolk and, more importantly, its constituents are very important for embryo development and growth. The relative amount of lipid material used by the developing embryo is high during the final week of incubation (Noble and Cocchi, 1990). A large proportion of the yolk sac remains unused at hatch and is sequestered in the body cavity; it is completely utilized under appropriate conditions by 5 d posthatch (Latour et al., 1996). During the first 5 d after hatching, chicks acquire yolkderived lipids via lipoproteins. The lipoprotein pattern exhibits numerous changes during the first 5 d of age before stabilizing on Day 7 (Latour et al., 1995). Manipulating egg yolk fatty acids can be accomplished by adding fat to the hen’s diet (Cherian and Sim, 1991;
Received for publication June 30, 1999. Accepted for publication March 6, 2000. 1 Agriculture Research Project Number 16,024. 2 To whom correspondence should be addressed: M. A. Latour, Purdue University, 1151 Smith Hall, West Lafayette, IN 47907-1151; email: [email protected]. 3 Corresponding author for fatty acid analysis.
Abbreviation Key: CE = cholesterol esters; CLA = conjugated linoleic acid; FC = free cholesterol; LDL = low density lipoproteins; PL = phospholipids; PRO = protein; RYW = relative yolk sac weight; TG = triglycerides; VLDL = very low density lipoprotein.
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had smaller RYW compared with Group A embryos; this difference remained through 2 d posthatch. During that period (15 d of incubation through 2 d posthatch), however, embryos and chicks from Group B hens exhibited a unique absorption pattern such that little to no yolk was utilized between hatch and 2 d posthatch, a period normally characterized by high yolk lipid utilization. Similar to the RYW effects, VLDL particles were also altered by hen-induced treatment. Specifically, at hatch, chicks from Group A hens had the highest percentage of triglycerides (TG) within their VLDL particles compared with chicks from hens under all other treatments. This trend in VLDL particles was continued at 4 d posthatch. The present study demonstrates that CLA enrichment of eggs alters relative yolk sac absorption and the composition of circulating VLDL particles.
ABSTRACT The present study was designed to investigate the effects of conjugated linoleic acid (CLA) on yolk usage and circulating very low density lipoproteins (VLDL) during incubation (Day 15) and through 6 d posthatch. Eggs enriched with CLA were obtained from hens subjected to the following treatments. Group A hens served as the control group, Group B hens received 1 g CLA every other day, Group C hens received 1 g CLA every 4th d, and Group D hens were sham-supplemented with 1 g safflower oil every other day. Enrichment with CLA did not effect fertility, hatch of fertile, BW, or yolkfree BW of embryos or chicks. However, there were significant changes in relative yolk sac weight (RYW) and composition of circulating VLDL particles. Across all dietary treatments (Groups B, C, and D), 15-d embryos
CONJUGATED LINOLEIC ACID, EMBRYONIC AND NEONATAL GROWTH, AND CIRCULATING LIPIDS
MATERIALS AND METHODS Animals and Diets Forty Single Comb White Leghorn hens (43 wk of age) were randomly assigned among four treatments. Within each treatment, two hens were housed together to provide five replicates per treatment. Hens assigned to Group A served as the control (CON) group and received no dietary supplement. Group B hens received 1 g CLA every other day. Group C hens received 1 g CLA every 4th d, and Group D hens were sham-supplemented with 1 g safflower oil (the gel coating surrounding the CLA capsule) every other day. Dietary supplements (CLA and safflower capsules) were stored at 4 C and administered orally on the day specified. Before the experiment began, the average feed consumption was 106 g/d per hen. Therefore, the percentages of CLA in diets for Groups B and C were approximately 0.5 and 0.25%, respectively. The upper level of CLA (0.5% of diet) was chosen because, in rats, this amount was effectively incorporated into the liver and did alter fatty acid metabolism (Belury and Kempa-Steczko, 1997). The basal diet was consistent for all treatment groups and met or exceeded all of the nutritional requirements for layers (NRC, 1994). Feed and water were provided ad libitum.
Insemination of Hens and Egg Collection After 4 mo of feeding, hens were inseminated with 3 × 105 sperm at each insemination period. Hens were inseminated twice within a 1-wk period prior to first egg collection. Eggs from the various hen groups were gathered and incubated. Fertility and hatchability of fertile eggs were determined. Also, data were obtained on numbers of early-, mid-, and late-dead chicks.
Organ Analysis At 15 d of incubation, at hatch, and at 2, 4, and 6 d posthatch, chicks were weighed, and relative yolk sac
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Beckman Coulter, Inc., Fullerton, CA 92834.
weight (RYW) and relative liver weight were determined. For both relative measurements, the following formula was used: (tissue wt/BW) × 100.
Separation of Very Low Density Lipoproteins At hatch and at 2, 4, and 6 d posthatch, chicks were bled from the jugular vein into non-heparinized tubes. Serum samples were recovered after 5 min of centrifugation. Serum was then fractionated into VLDL by using a Beckman Ti 42.2 rotor4 at 40,000 rpm for 18 h (Latour et al., 1997). The VLDL were considered to be at a density <1.016 g/mi. Within the VLDL fraction, the following parameters were evaluated: triglycerides (TG), total cholesterol (TC), free cholesterol (FC), calculated cholesterol esters [(CE) = TC − FC × 1.67], phospholipids (PL), and protein (PRO). The lipids and PRO were determined as described by Latour et al. (1997) and Markwell et al. (1978), respectively.
Statistical Analysis Chi-square analysis was used to evaluate fertility and number of early-, mid-, and late-dead chicks. The continuous variables were analyzed by ANOVA for a completely randomized factorial design. This design allowed for testing the main effects (treatment and day) and their interaction. All data were analyzed using the General Linear Models procedure of SAS (SAS Institute, 1997). Statements of significance were based on P < 0.05 unless otherwise noted.
RESULTS Across all treatment groups, there were no significant differences in fertility, hatchability, or early-, mid-, or late-dead embryos, and there were no treatment effects on chick BW (P = 0.3495) or yolk-free BW (P = 0.2291). Mean BW for chicks from hens in Groups A, B, C, and D were 47.2, 47.5, 48.6, and 46.3 g, respectively. Yolk-free BW data are shown in Table 1. Fifteen-day embryos and newly hatched chicks from hens in Group A had larger remnant yolks than did embryos from hens in Groups B, C, and D (Figure 1). Embryos from Group C hens had the next highest percentage of remnant yolk at 15 d of incubation followed by embryos from Group B and D hens. At hatch, chicks from Group A hens had the greatest percentage of RYW when compared with all other groups; chicks from Group B and C hens had statistically the smallest RYW (Figure 1). Chicks hatched from Group B hens exhibited no change in RYW at Day 2 when compared with hatch values (Figure 1). At 2 and 4 d posthatch, chicks hatched from hens in Group B had the largest percentage of RYW when compared with chicks hatched from hens in all other groups; however, by Day 6, there were no differences in RYW among the groups. Neonatal chicks exhibited numerous changes in their VLDL particles across all treatment groups. Chicks from
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the activity of ∆ 9-desaturase increases during the early stages of yolk utilization and is believed to be indirectly associated with the stabilization of lipoprotein prior to exiting the yolk proper. Therefore, alterations in ∆ 9desaturase via CLA may change the composition and concentration of circulating lipoproteins in embryos and neonatal chicks. Conjugated linoleic acids have been reported to have biological effects on circulating cholesterol, LDL, and ∆ 9-desaturase, all of which are vital to the developing embryo in terms of yolk absorption via lipoprotein metabolism, especially VLDL. Therefore, the purpose of this study was to investigate the role of CLA on yolk usage and VLDL utilization during incubation and through 6 d posthatch.
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LATOUR ET AL. TABLE 1. Effects of dietary fat [conjugated linoleic acid (CLA)] on day by treatment interaction for yolkfree BW. Yolk-free BW was determined as BW minus yolk weight. Group1 Day of age
A
B
−15 (day of incubation) 0 (day of hatch) 2 4 6 SEM2
18.99 36.16 42.78 52.81 69.42 ± 2.07
22.94 39.47 43.56 55.44 63.34 ± 2.07
C
D
18.44 36.75 43.20 59.07 73.78 ± 2.07
19.88 35.84 41.95 53.26 69.66 ± 2.07
(g)
Group C hens had the highest percentage of CE when compared with chicks from hens in Groups A and D. Chicks from Group B hens had intermediate percentages of CE compared with those from chicks from Group A and C hens (Table 2). The percentage of PRO in VLDL particles was highest for chicks from Group D hens when compared with chicks from Group A and B hens. Chicks from Group C were not different from the other groups (Table 2). Across all hen-diet treatments, the composition of VLDL particles changed in chicks at different sampling periods. The percentages of CE and PL in VLDL particles were reduced by Day 6 compared with the same percentages at hatch and 4 d posthatch (Figure 2), whereas the
percentage of FC was reduced at 4 and 6 d compared with the values observed at hatch and at Day 2 (Figure 2). Conversely, the PRO percentage increased by Day 6 compared with previous sampling points (Figure 2). Neonatal chicks also exhibited a sampling day by treatment interaction for TG. The percentage of TG in VLDL particles was highest in chicks from Group A hens when compared with newly hatched chicks from Group C and D hens, whereas chicks from Group B hens had similar percentages of TG in VLDL when compared with all others (Figure 3). There were no differences noted in chick TG at 2 or 6 d of age; however, by Day 4, the percentage of TG was highest in chicks from Group A hens compared with chicks from hens in any other group. Chicks from Group D hens had the next highest concentration of TG at Day 4 compared with the concentration observed in chicks from Group C hens. Chicks from hens in Group B had intermediate concentrations of TG compared with chicks from hens in Groups C and D (Figure 3).
DISCUSSION
FIGURE 1. Sampling day by treatment interaction for relative yolk sac weight in 15-d embryos (−7 of incubation) and for chicks at hatch and at 2, 4, and 6 d posthatch. Chicks were hatched from eggs obtained from hens fed the following diets: Group A = control hens, Group B = hens fed 1 g conjugated linoleic acid every other day, and Group D = hens fed 1 g safflower oil every other day. Symbols within a day with no common letter differ significantly (P < 0.05).
Lipoproteins transport both water-insoluble nutrients and other compounds, such as cholesterol and nonessential fatty acids, through the bloodstream from their sites of absorption or synthesis to peripheral tissues (Elkin, 1997). In newly hatched chicks, the yolk and liver are both releasing lipoprotein particles, whereas the peripheral tissues actively recruit these circulating lipoproteins. Latour et al. (1995) showed that the concentration of circulating LDL cholesterol decreased sharply following hatch, whereas the level of high density lipoprotein cholesterol increased through the completion of yolk absorption. The importance of high density lipoprotein cholesterol becoming higher than LDL cholesterol resides in the unique changes occurring during late embryonic development and neonatal growth. Tarugi et al. (1994) found that, in chicks, CE begins to accumulate in the liver during the final stages of incubation and is rapidly depleted after 2 to 7 d posthatch. The transfer of lipids between the yolk and liver is accomplished by specific lipoproteins. Lazier et al. (1994) observed that the level of mRNA for apolipoprotein B, the primary protein of VLDL and LDL, in-
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Means within a row do not differ significantly (P < 0.2291). 1 Group A = control hens; Group B = hens fed 1 g CLA every other day; Group C = hens fed 1 g CLA every 4th d; Group D = hens fed 1 g safflower oil every other day. 2 Standard error of the mean is based on a pooled estimate of variance.
CONJUGATED LINOLEIC ACID, EMBRYONIC AND NEONATAL GROWTH, AND CIRCULATING LIPIDS
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TABLE 2. Effects of dietary fat [conjugated linoleic acid (CLA)] on hatched chick very low density lipoprotein (VLDL) particles. Group1
CE2
PL3
A B C D SEM7
9.48bc 11.98ab 12.71a 8.40c ± 1.05
19.19a 19.56a 19.29a 20.19a ± 0.41
TG4
FC5
PRO6
5.55a 5.01a 5.20a 5.12a ± 0.29
14.19b 14.22b 15.83ab 18.71a ± 1.31
(% of total particle) 51.57a 49.22a 46.95a 47.56a ± 1.44
Means within a column for each variable with no common letter differ significantly (P < 0.05). Group A = control hens; Group B = hens fed 1 g CLA every other day; Group C = hens fed 1 g CLA every 4th d; Group D = hens fed 1 g safflower oil every other day. 2 CE = cholesterol ester. 3 PL = phospholipids. 4 TG = triglycerides. 5 FC = free cholesterol. 6 PRO = protein. 7 Standard error of the mean is based on a pooled estimate of variance. a–c 1
FIGURE 2. Sampling day effects on chicks’ circulating lipids within very low density lipoproteins (VLDL) at hatch and at 2, 4, and 6 d posthatch. Symbols across different days for each lipid measurement with no common letter differ significantly (P < 0.05).
ment groups. It is well known that alterations in certain fatty acids can directly change the production rate and clearance rate of lipoproteins (Lichtenstein et al., 1994) so that the changes observed (i.e., the decrease in yolk absorption between hatch and 2 d of age) in chicks from Group B hens may be attributed to changes in concentrations of fatty acids. Chicks from Group B hens exhibited little or no change in RYW when compared with chicks from the other groups. Moreover, the lack of change in chicks from
FIGURE 3. Sampling day by treatment interaction for triglycerides within very low density lipoproteins (VLDL) for chicks at hatch and at 2, 4, and 6 d of age pothatch. Chicks were hatched from eggs obtained from hens fed the following diets: Group A = control hens, Group B = hens fed 1 g conjugated linoleic acid every other day, and Group D = hens fed 1 g safflower oil every other day. Symbols within a day with no common letter differ significantly (P < 0.05).
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creases in the vascular yolk sac membrane during incubation. Kelly et al. (1979) also noted that the concentration of circulating apolipoprotein B is relatively high during incubation but decreases sharply during the final 3 d of incubation. At the same time, apolipoprotein A, the primary protein of high density lipoproteins begins to increase at a steady rate to intersect the downward production of apolipoprotein B at hatch. At hatch, cholesterol is carried equally between LDL and high density lipoprotein particles (Latour et al., 1997), but, at 3 d of age, high density lipoprotein becomes the predominate carrier of circulating cholesterol (Latour et al., 1995). In this experiment, VLDL particles differed among the various treat-
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REFERENCES Belury, M. A., and A. Kempa-Steczko, 1997. Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 32:199–204. Cherian, G., and J. S. Sim, 1991. Effect of feeding full fat flax and canola seeds to laying hens on the fatty acid composition of eggs, embryos, and newly hatched chicks. Poultry Sci. 70:917–922. Elkin, R. G., 1997. An overview of recent developments in avian lipoprotein metabolism. J Nutr. 127:793S–794S. Ha, Y. L., N. K. Grimm, and M. W. Pariza, 1987. Anticarcinogens from fried ground beef: heat altered derivatives of linoleic acid. Carcinogenesis 8:1881–1887. Hargis, P. S., M. E. VanElswyk, and B. M. Hargis, 1991. Dietary modification of yolk lipid with Menhaden oil. Poultry Sci. 70:874–883.
Kelly, J. L., O. A. Schjeide, S. Schjeide, R. Milus, and P. Alaupovic, 1979. Quantification of the major apolipoproteins in chicken and turkey serum during embryonic development. Comp. Biochem. Physiol. 65B:239–242. Latour, M. A., A. A. Devitt, R. A. Meunier, J. J. Stewart, and B. A. Watkins, 2000. Effects of conjugated linoleic acid. 1. Fatty acid modification of yolk lipids. Poultry Sci. 79:817–821. Latour, M. A., E. D. Peebles, C. R. Boyle, J. D. Brake, and T. F. Kellogg, 1995. Changes in serum lipid lipoprotein and corticosterone concentrations during neonatal chick development. Biol. Neonate 67:381–386. Latour, M. A., E. D. Peebles, C. R. Boyle, S. M. Doyle, T. Pansky, and J. D. Brake, 1996. Effects of breeder hen age and dietary fat on embryonic and neonatal broiler serum lipids and glucose. Poultry Sci. 75:695–701. Latour, M. A., E. D. Peebles, S. M. Doyle, T. Pansky, T. W. Smith, and C. R. Boyle, 1998. Broiler breeder age and dietary fat influence the yolk fatty acid profiles of fresh eggs and newly hatched chicks. Poultry Sci. 77:47–53. Latour, M. A., E. D. Peebles, C. W. Gardner, and G. Schonfeld, 1997. An evaluation of the lipoprotein composition and corresponding apolipoproteins in the newly hatched male and female broiler chick from two different aged flocks. Biol. Neonate 72:380–388. Lazier, C. B., M. Wiktorowicz, G. E. DiMattia, D. A. Gordon, R. Binder, and D. L. Williams, 1994. Apolipoprotein (apo) B and apoII gene expression are both estrogen-responsive in chick embryo liver but only apoII is estrogen-responsive in kidney. Mol. Cell. Endocrinol. 106:187–194. Lee, K., D. Kritchevsky, and M. W. Pariza, 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. Li, Y., and B. A. Watkins, 1998. Conjugated linoleic acids altered bone fatty acid composition and reduced ex vivo PGE2 biosynthesis in rats fed n-3 or n-6 fatty acids. Lipids 33:417–425. Lichtenstein, A. H., L. M. Ausman, W. Carrasco, J. L. Jenner, J. M. Ordovas, and E. J. Schaefer, 1994. Hypercholesterolemic effect of dietary cholesterol in diets enriched in polyunsaturated and saturated fat. Arterio. Thromb. Vasc. Biol. 14:168–175. Markwell, M. K., S. M. Haas, L. L. Bieber, and N. E. Tolbert, 1978. A modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206–210. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academic Press, Washington, DC. Nicholosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth, 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277. Noble, R. C., and M. Cocchi, 1990. Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29:107–140. Noble, R. C., and J. H. Shand, 1985. Unsaturated fatty acid compositional changes and desaturation during the embryonic development of the chicken (Gallus domesticus). Lipids 20:278–282. Romanoff, A. L., 1960. The Avian Embryo. MacMillan Publishing Co., New York, NY. SAS Institute, 1997. SAS威 User’s Guide: Basics. Release 6.12 Edition. SAS Institute Inc., Cary, NC. Tarugi, P., S. Nicolini, L. Marchi, G. Ballarini, and S. Calandra, 1994. Apolipoprotein B-100 production and cholesteryl ester content in the liver of developing chick. J. Lipid Res. 35:2019–2031. Vilchez, C., S. P. Touchburn, E. R. Chavez, and C. W. Chan, 1991. Effect of feeding palmitic, oleic, and linoleic acids to Japanese quail hens (Coturnix coturnix japonica). I. Reproductive performance and tissue fatty acids. Poultry Sci. 70:2484–2493.
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Group B hens when comparing Day 2 vs. hatch values is unique; that is, this period (Day 2 vs. hatch) normally represents an intense period of yolk lipid utilization and mobilization (Latour et al., 1995, 1997). Apparently, the initial steps in the sequence of events that facilitates yolk lipid removal during the early neonatal period was impaired in chicks from Group B hens. Perhaps the absorption was impaired by CLA alteration of lipid transformation; Li and Watkins (1998) suggested that CLA inhibits the action of ∆ 9-desaturase, the enzyme capable of converting stearic acid (18:0) to oleic acid (18:1). In the present experiment, yolk 18:0 fatty acid was elevated in chicks from Group B hens (Latour et al., 2000). Therefore, the possibly exists that ∆ 9-desaturase activity was compromised and perhaps lipoprotein metabolism was altered. Noble and Shand (1985) showed that ∆ 9-desaturase activity increases during the early stages of yolk utilization and is believed to be associated with stabilizing the lipoproteins prior to exiting the yolk proper. Romanoff (1960) states that the avian embryo derives more than 90% of its energy requirements from the oxidation of yolk lipids. At hatch and through the first few days after hatch, the chick continues to absorb yolk material while making a transition between primarily a lipid diet (yolk) to primarily a carbohydrate (standard ration) diet. Hence, it is during this period that many changes occur in circulating lipids (Latour et al., 1995), especially circulating VLDL. In the present study, there was a sharp decrease in the composition of VLDL particles measured (i.e., decreases in PL, CE, FC, and TG) as the chicks became older; at the same time, however, the level of protein was increasing. The decrease in lipid components within VLDL particles most likely represents the fractional rate of removal of these molecules; that is, in circulation, VLDL particles interact with peripheral tissues via cell surface receptors and, thus, give up lipid. Therefore, as the lipids are being removed, the VLDL particle becomes proportionally denser, and, thus, the percentage of protein begins to rise in VLDL particles (Figure 2). The present study demonstrates that CLA alters lipid metabolism in chick embryos. The precise mechanism underlying decreased yolk absorption at hatch and at 2 d posthatch in chicks from Group B hens remains to be determined and invites new cellular studies.
(Key words: chick, conjugated linoleic acids, lipids, lipoproteins) 2000 Poultry Science 79:822–826
Hargis et al., 1991; Vilchez et al., 1991; Latour et al., 1998), which is an excellent way of determining the effects of yolk fatty acid modification on neonatal avian lipid metabolism. One of the most interesting polyunsaturated fatty acids to gain attention in recent years is conjugated linoleic acid (CLA). Conjugated linoleic acid was first recognized as an anticarcinogen after being isolated from extracts of grilled ground beef that exhibited anticarcinogenic activity against chemically induced mouse skin cancer (Ha et al., 1987). Conjugated linoleic acid occurs naturally and is reported to have significant biological effects. For example, CLA has been shown to reduce plasma low density lipoprotein (LDL) concentrations and inhibit the development of atherosclerosis in rabbits (Lee et al., 1994) and hamsters (Nicolosi et al., 1997) fed atherogenic diets. Conjugated linoleic acids are also believed to inhibit the action of ∆ 9-desaturase, an enzyme capable of converting stearic acid (18:0) to oleic acid (18:1) (Li and Watkins, 1998). Noble and Shand (1985) have demonstrated that
INTRODUCTION The avian embryo derives more than 90% of its energy requirements from the oxidation of yolk lipids (Romanoff, 1960). Therefore, the yolk and, more importantly, its constituents are very important for embryo development and growth. The relative amount of lipid material used by the developing embryo is high during the final week of incubation (Noble and Cocchi, 1990). A large proportion of the yolk sac remains unused at hatch and is sequestered in the body cavity; it is completely utilized under appropriate conditions by 5 d posthatch (Latour et al., 1996). During the first 5 d after hatching, chicks acquire yolkderived lipids via lipoproteins. The lipoprotein pattern exhibits numerous changes during the first 5 d of age before stabilizing on Day 7 (Latour et al., 1995). Manipulating egg yolk fatty acids can be accomplished by adding fat to the hen’s diet (Cherian and Sim, 1991;
Received for publication June 30, 1999. Accepted for publication March 6, 2000. 1 Agriculture Research Project Number 16,024. 2 To whom correspondence should be addressed: M. A. Latour, Purdue University, 1151 Smith Hall, West Lafayette, IN 47907-1151; email: [email protected]. 3 Corresponding author for fatty acid analysis.
Abbreviation Key: CE = cholesterol esters; CLA = conjugated linoleic acid; FC = free cholesterol; LDL = low density lipoproteins; PL = phospholipids; PRO = protein; RYW = relative yolk sac weight; TG = triglycerides; VLDL = very low density lipoprotein.
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had smaller RYW compared with Group A embryos; this difference remained through 2 d posthatch. During that period (15 d of incubation through 2 d posthatch), however, embryos and chicks from Group B hens exhibited a unique absorption pattern such that little to no yolk was utilized between hatch and 2 d posthatch, a period normally characterized by high yolk lipid utilization. Similar to the RYW effects, VLDL particles were also altered by hen-induced treatment. Specifically, at hatch, chicks from Group A hens had the highest percentage of triglycerides (TG) within their VLDL particles compared with chicks from hens under all other treatments. This trend in VLDL particles was continued at 4 d posthatch. The present study demonstrates that CLA enrichment of eggs alters relative yolk sac absorption and the composition of circulating VLDL particles.
ABSTRACT The present study was designed to investigate the effects of conjugated linoleic acid (CLA) on yolk usage and circulating very low density lipoproteins (VLDL) during incubation (Day 15) and through 6 d posthatch. Eggs enriched with CLA were obtained from hens subjected to the following treatments. Group A hens served as the control group, Group B hens received 1 g CLA every other day, Group C hens received 1 g CLA every 4th d, and Group D hens were sham-supplemented with 1 g safflower oil every other day. Enrichment with CLA did not effect fertility, hatch of fertile, BW, or yolkfree BW of embryos or chicks. However, there were significant changes in relative yolk sac weight (RYW) and composition of circulating VLDL particles. Across all dietary treatments (Groups B, C, and D), 15-d embryos
CONJUGATED LINOLEIC ACID, EMBRYONIC AND NEONATAL GROWTH, AND CIRCULATING LIPIDS
MATERIALS AND METHODS Animals and Diets Forty Single Comb White Leghorn hens (43 wk of age) were randomly assigned among four treatments. Within each treatment, two hens were housed together to provide five replicates per treatment. Hens assigned to Group A served as the control (CON) group and received no dietary supplement. Group B hens received 1 g CLA every other day. Group C hens received 1 g CLA every 4th d, and Group D hens were sham-supplemented with 1 g safflower oil (the gel coating surrounding the CLA capsule) every other day. Dietary supplements (CLA and safflower capsules) were stored at 4 C and administered orally on the day specified. Before the experiment began, the average feed consumption was 106 g/d per hen. Therefore, the percentages of CLA in diets for Groups B and C were approximately 0.5 and 0.25%, respectively. The upper level of CLA (0.5% of diet) was chosen because, in rats, this amount was effectively incorporated into the liver and did alter fatty acid metabolism (Belury and Kempa-Steczko, 1997). The basal diet was consistent for all treatment groups and met or exceeded all of the nutritional requirements for layers (NRC, 1994). Feed and water were provided ad libitum.
Insemination of Hens and Egg Collection After 4 mo of feeding, hens were inseminated with 3 × 105 sperm at each insemination period. Hens were inseminated twice within a 1-wk period prior to first egg collection. Eggs from the various hen groups were gathered and incubated. Fertility and hatchability of fertile eggs were determined. Also, data were obtained on numbers of early-, mid-, and late-dead chicks.
Organ Analysis At 15 d of incubation, at hatch, and at 2, 4, and 6 d posthatch, chicks were weighed, and relative yolk sac
4
Beckman Coulter, Inc., Fullerton, CA 92834.
weight (RYW) and relative liver weight were determined. For both relative measurements, the following formula was used: (tissue wt/BW) × 100.
Separation of Very Low Density Lipoproteins At hatch and at 2, 4, and 6 d posthatch, chicks were bled from the jugular vein into non-heparinized tubes. Serum samples were recovered after 5 min of centrifugation. Serum was then fractionated into VLDL by using a Beckman Ti 42.2 rotor4 at 40,000 rpm for 18 h (Latour et al., 1997). The VLDL were considered to be at a density <1.016 g/mi. Within the VLDL fraction, the following parameters were evaluated: triglycerides (TG), total cholesterol (TC), free cholesterol (FC), calculated cholesterol esters [(CE) = TC − FC × 1.67], phospholipids (PL), and protein (PRO). The lipids and PRO were determined as described by Latour et al. (1997) and Markwell et al. (1978), respectively.
Statistical Analysis Chi-square analysis was used to evaluate fertility and number of early-, mid-, and late-dead chicks. The continuous variables were analyzed by ANOVA for a completely randomized factorial design. This design allowed for testing the main effects (treatment and day) and their interaction. All data were analyzed using the General Linear Models procedure of SAS (SAS Institute, 1997). Statements of significance were based on P < 0.05 unless otherwise noted.
RESULTS Across all treatment groups, there were no significant differences in fertility, hatchability, or early-, mid-, or late-dead embryos, and there were no treatment effects on chick BW (P = 0.3495) or yolk-free BW (P = 0.2291). Mean BW for chicks from hens in Groups A, B, C, and D were 47.2, 47.5, 48.6, and 46.3 g, respectively. Yolk-free BW data are shown in Table 1. Fifteen-day embryos and newly hatched chicks from hens in Group A had larger remnant yolks than did embryos from hens in Groups B, C, and D (Figure 1). Embryos from Group C hens had the next highest percentage of remnant yolk at 15 d of incubation followed by embryos from Group B and D hens. At hatch, chicks from Group A hens had the greatest percentage of RYW when compared with all other groups; chicks from Group B and C hens had statistically the smallest RYW (Figure 1). Chicks hatched from Group B hens exhibited no change in RYW at Day 2 when compared with hatch values (Figure 1). At 2 and 4 d posthatch, chicks hatched from hens in Group B had the largest percentage of RYW when compared with chicks hatched from hens in all other groups; however, by Day 6, there were no differences in RYW among the groups. Neonatal chicks exhibited numerous changes in their VLDL particles across all treatment groups. Chicks from
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the activity of ∆ 9-desaturase increases during the early stages of yolk utilization and is believed to be indirectly associated with the stabilization of lipoprotein prior to exiting the yolk proper. Therefore, alterations in ∆ 9desaturase via CLA may change the composition and concentration of circulating lipoproteins in embryos and neonatal chicks. Conjugated linoleic acids have been reported to have biological effects on circulating cholesterol, LDL, and ∆ 9-desaturase, all of which are vital to the developing embryo in terms of yolk absorption via lipoprotein metabolism, especially VLDL. Therefore, the purpose of this study was to investigate the role of CLA on yolk usage and VLDL utilization during incubation and through 6 d posthatch.
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LATOUR ET AL. TABLE 1. Effects of dietary fat [conjugated linoleic acid (CLA)] on day by treatment interaction for yolkfree BW. Yolk-free BW was determined as BW minus yolk weight. Group1 Day of age
A
B
−15 (day of incubation) 0 (day of hatch) 2 4 6 SEM2
18.99 36.16 42.78 52.81 69.42 ± 2.07
22.94 39.47 43.56 55.44 63.34 ± 2.07
C
D
18.44 36.75 43.20 59.07 73.78 ± 2.07
19.88 35.84 41.95 53.26 69.66 ± 2.07
(g)
Group C hens had the highest percentage of CE when compared with chicks from hens in Groups A and D. Chicks from Group B hens had intermediate percentages of CE compared with those from chicks from Group A and C hens (Table 2). The percentage of PRO in VLDL particles was highest for chicks from Group D hens when compared with chicks from Group A and B hens. Chicks from Group C were not different from the other groups (Table 2). Across all hen-diet treatments, the composition of VLDL particles changed in chicks at different sampling periods. The percentages of CE and PL in VLDL particles were reduced by Day 6 compared with the same percentages at hatch and 4 d posthatch (Figure 2), whereas the
percentage of FC was reduced at 4 and 6 d compared with the values observed at hatch and at Day 2 (Figure 2). Conversely, the PRO percentage increased by Day 6 compared with previous sampling points (Figure 2). Neonatal chicks also exhibited a sampling day by treatment interaction for TG. The percentage of TG in VLDL particles was highest in chicks from Group A hens when compared with newly hatched chicks from Group C and D hens, whereas chicks from Group B hens had similar percentages of TG in VLDL when compared with all others (Figure 3). There were no differences noted in chick TG at 2 or 6 d of age; however, by Day 4, the percentage of TG was highest in chicks from Group A hens compared with chicks from hens in any other group. Chicks from Group D hens had the next highest concentration of TG at Day 4 compared with the concentration observed in chicks from Group C hens. Chicks from hens in Group B had intermediate concentrations of TG compared with chicks from hens in Groups C and D (Figure 3).
DISCUSSION
FIGURE 1. Sampling day by treatment interaction for relative yolk sac weight in 15-d embryos (−7 of incubation) and for chicks at hatch and at 2, 4, and 6 d posthatch. Chicks were hatched from eggs obtained from hens fed the following diets: Group A = control hens, Group B = hens fed 1 g conjugated linoleic acid every other day, and Group D = hens fed 1 g safflower oil every other day. Symbols within a day with no common letter differ significantly (P < 0.05).
Lipoproteins transport both water-insoluble nutrients and other compounds, such as cholesterol and nonessential fatty acids, through the bloodstream from their sites of absorption or synthesis to peripheral tissues (Elkin, 1997). In newly hatched chicks, the yolk and liver are both releasing lipoprotein particles, whereas the peripheral tissues actively recruit these circulating lipoproteins. Latour et al. (1995) showed that the concentration of circulating LDL cholesterol decreased sharply following hatch, whereas the level of high density lipoprotein cholesterol increased through the completion of yolk absorption. The importance of high density lipoprotein cholesterol becoming higher than LDL cholesterol resides in the unique changes occurring during late embryonic development and neonatal growth. Tarugi et al. (1994) found that, in chicks, CE begins to accumulate in the liver during the final stages of incubation and is rapidly depleted after 2 to 7 d posthatch. The transfer of lipids between the yolk and liver is accomplished by specific lipoproteins. Lazier et al. (1994) observed that the level of mRNA for apolipoprotein B, the primary protein of VLDL and LDL, in-
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Means within a row do not differ significantly (P < 0.2291). 1 Group A = control hens; Group B = hens fed 1 g CLA every other day; Group C = hens fed 1 g CLA every 4th d; Group D = hens fed 1 g safflower oil every other day. 2 Standard error of the mean is based on a pooled estimate of variance.
CONJUGATED LINOLEIC ACID, EMBRYONIC AND NEONATAL GROWTH, AND CIRCULATING LIPIDS
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TABLE 2. Effects of dietary fat [conjugated linoleic acid (CLA)] on hatched chick very low density lipoprotein (VLDL) particles. Group1
CE2
PL3
A B C D SEM7
9.48bc 11.98ab 12.71a 8.40c ± 1.05
19.19a 19.56a 19.29a 20.19a ± 0.41
TG4
FC5
PRO6
5.55a 5.01a 5.20a 5.12a ± 0.29
14.19b 14.22b 15.83ab 18.71a ± 1.31
(% of total particle) 51.57a 49.22a 46.95a 47.56a ± 1.44
Means within a column for each variable with no common letter differ significantly (P < 0.05). Group A = control hens; Group B = hens fed 1 g CLA every other day; Group C = hens fed 1 g CLA every 4th d; Group D = hens fed 1 g safflower oil every other day. 2 CE = cholesterol ester. 3 PL = phospholipids. 4 TG = triglycerides. 5 FC = free cholesterol. 6 PRO = protein. 7 Standard error of the mean is based on a pooled estimate of variance. a–c 1
FIGURE 2. Sampling day effects on chicks’ circulating lipids within very low density lipoproteins (VLDL) at hatch and at 2, 4, and 6 d posthatch. Symbols across different days for each lipid measurement with no common letter differ significantly (P < 0.05).
ment groups. It is well known that alterations in certain fatty acids can directly change the production rate and clearance rate of lipoproteins (Lichtenstein et al., 1994) so that the changes observed (i.e., the decrease in yolk absorption between hatch and 2 d of age) in chicks from Group B hens may be attributed to changes in concentrations of fatty acids. Chicks from Group B hens exhibited little or no change in RYW when compared with chicks from the other groups. Moreover, the lack of change in chicks from
FIGURE 3. Sampling day by treatment interaction for triglycerides within very low density lipoproteins (VLDL) for chicks at hatch and at 2, 4, and 6 d of age pothatch. Chicks were hatched from eggs obtained from hens fed the following diets: Group A = control hens, Group B = hens fed 1 g conjugated linoleic acid every other day, and Group D = hens fed 1 g safflower oil every other day. Symbols within a day with no common letter differ significantly (P < 0.05).
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creases in the vascular yolk sac membrane during incubation. Kelly et al. (1979) also noted that the concentration of circulating apolipoprotein B is relatively high during incubation but decreases sharply during the final 3 d of incubation. At the same time, apolipoprotein A, the primary protein of high density lipoproteins begins to increase at a steady rate to intersect the downward production of apolipoprotein B at hatch. At hatch, cholesterol is carried equally between LDL and high density lipoprotein particles (Latour et al., 1997), but, at 3 d of age, high density lipoprotein becomes the predominate carrier of circulating cholesterol (Latour et al., 1995). In this experiment, VLDL particles differed among the various treat-
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Kelly, J. L., O. A. Schjeide, S. Schjeide, R. Milus, and P. Alaupovic, 1979. Quantification of the major apolipoproteins in chicken and turkey serum during embryonic development. Comp. Biochem. Physiol. 65B:239–242. Latour, M. A., A. A. Devitt, R. A. Meunier, J. J. Stewart, and B. A. Watkins, 2000. Effects of conjugated linoleic acid. 1. Fatty acid modification of yolk lipids. Poultry Sci. 79:817–821. Latour, M. A., E. D. Peebles, C. R. Boyle, J. D. Brake, and T. F. Kellogg, 1995. Changes in serum lipid lipoprotein and corticosterone concentrations during neonatal chick development. Biol. Neonate 67:381–386. Latour, M. A., E. D. Peebles, C. R. Boyle, S. M. Doyle, T. Pansky, and J. D. Brake, 1996. Effects of breeder hen age and dietary fat on embryonic and neonatal broiler serum lipids and glucose. Poultry Sci. 75:695–701. Latour, M. A., E. D. Peebles, S. M. Doyle, T. Pansky, T. W. Smith, and C. R. Boyle, 1998. Broiler breeder age and dietary fat influence the yolk fatty acid profiles of fresh eggs and newly hatched chicks. Poultry Sci. 77:47–53. Latour, M. A., E. D. Peebles, C. W. Gardner, and G. Schonfeld, 1997. An evaluation of the lipoprotein composition and corresponding apolipoproteins in the newly hatched male and female broiler chick from two different aged flocks. Biol. Neonate 72:380–388. Lazier, C. B., M. Wiktorowicz, G. E. DiMattia, D. A. Gordon, R. Binder, and D. L. Williams, 1994. Apolipoprotein (apo) B and apoII gene expression are both estrogen-responsive in chick embryo liver but only apoII is estrogen-responsive in kidney. Mol. Cell. Endocrinol. 106:187–194. Lee, K., D. Kritchevsky, and M. W. Pariza, 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19–25. Li, Y., and B. A. Watkins, 1998. Conjugated linoleic acids altered bone fatty acid composition and reduced ex vivo PGE2 biosynthesis in rats fed n-3 or n-6 fatty acids. Lipids 33:417–425. Lichtenstein, A. H., L. M. Ausman, W. Carrasco, J. L. Jenner, J. M. Ordovas, and E. J. Schaefer, 1994. Hypercholesterolemic effect of dietary cholesterol in diets enriched in polyunsaturated and saturated fat. Arterio. Thromb. Vasc. Biol. 14:168–175. Markwell, M. K., S. M. Haas, L. L. Bieber, and N. E. Tolbert, 1978. A modification of the lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206–210. National Research Council, 1994. Nutrient Requirements of Poultry. 9th rev. ed. National Academic Press, Washington, DC. Nicholosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and P. J. Huth, 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277. Noble, R. C., and M. Cocchi, 1990. Lipid metabolism and the neonatal chicken. Prog. Lipid Res. 29:107–140. Noble, R. C., and J. H. Shand, 1985. Unsaturated fatty acid compositional changes and desaturation during the embryonic development of the chicken (Gallus domesticus). Lipids 20:278–282. Romanoff, A. L., 1960. The Avian Embryo. MacMillan Publishing Co., New York, NY. SAS Institute, 1997. SAS威 User’s Guide: Basics. Release 6.12 Edition. SAS Institute Inc., Cary, NC. Tarugi, P., S. Nicolini, L. Marchi, G. Ballarini, and S. Calandra, 1994. Apolipoprotein B-100 production and cholesteryl ester content in the liver of developing chick. J. Lipid Res. 35:2019–2031. Vilchez, C., S. P. Touchburn, E. R. Chavez, and C. W. Chan, 1991. Effect of feeding palmitic, oleic, and linoleic acids to Japanese quail hens (Coturnix coturnix japonica). I. Reproductive performance and tissue fatty acids. Poultry Sci. 70:2484–2493.
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Group B hens when comparing Day 2 vs. hatch values is unique; that is, this period (Day 2 vs. hatch) normally represents an intense period of yolk lipid utilization and mobilization (Latour et al., 1995, 1997). Apparently, the initial steps in the sequence of events that facilitates yolk lipid removal during the early neonatal period was impaired in chicks from Group B hens. Perhaps the absorption was impaired by CLA alteration of lipid transformation; Li and Watkins (1998) suggested that CLA inhibits the action of ∆ 9-desaturase, the enzyme capable of converting stearic acid (18:0) to oleic acid (18:1). In the present experiment, yolk 18:0 fatty acid was elevated in chicks from Group B hens (Latour et al., 2000). Therefore, the possibly exists that ∆ 9-desaturase activity was compromised and perhaps lipoprotein metabolism was altered. Noble and Shand (1985) showed that ∆ 9-desaturase activity increases during the early stages of yolk utilization and is believed to be associated with stabilizing the lipoproteins prior to exiting the yolk proper. Romanoff (1960) states that the avian embryo derives more than 90% of its energy requirements from the oxidation of yolk lipids. At hatch and through the first few days after hatch, the chick continues to absorb yolk material while making a transition between primarily a lipid diet (yolk) to primarily a carbohydrate (standard ration) diet. Hence, it is during this period that many changes occur in circulating lipids (Latour et al., 1995), especially circulating VLDL. In the present study, there was a sharp decrease in the composition of VLDL particles measured (i.e., decreases in PL, CE, FC, and TG) as the chicks became older; at the same time, however, the level of protein was increasing. The decrease in lipid components within VLDL particles most likely represents the fractional rate of removal of these molecules; that is, in circulation, VLDL particles interact with peripheral tissues via cell surface receptors and, thus, give up lipid. Therefore, as the lipids are being removed, the VLDL particle becomes proportionally denser, and, thus, the percentage of protein begins to rise in VLDL particles (Figure 2). The present study demonstrates that CLA alters lipid metabolism in chick embryos. The precise mechanism underlying decreased yolk absorption at hatch and at 2 d posthatch in chicks from Group B hens remains to be determined and invites new cellular studies.