EDUCATION AND PRODUCTION Effects of Conjugated Linoleic Acid. 1. Fatty Acid Modification of Yolks and Neonatal Fatty Acid Metabolism1 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 constant dose of pooled rooster semen to evaluate changes in chick liver and yolk fatty acid metabolism during neonatal growth. At hatch and through 6 d of age, there were no significant differences in breakout data (fertility and numbers of early-, mid-, or late-dead chicks) or chick BW, respectively. However, Group B chicks exhibited an increase in liver 18:3n3 and 22:1n9 and a decrease in 20:3n6 and 22:5n3 fatty acids when compared with chicks from Groups A and D. Also noted for Group B chicks, yolk 18:0 fatty acid was higher than that for Group A and D chicks. These results suggest that CLA alters lipid metabolism in growing chicks.
ABSTRACT The purpose of this study was to evaluate the effects of conjugated linoleic acids (CLA) on neonatal fatty acid metabolism. In this study, layer hens (n = 40) were divided into four equal groups and subjected to the following treatments. Group A served as the control group, Group B received 1 g CLA every other day, Group C received 1 g CLA every 4th d, and Group D was shamsupplemented with 1 g safflower oil every other day. After 4 mo of feeding, Group B hens exhibited an increase in BW and egg size; however, there were no differences noted in feed consumption among the various treatment groups. At the same time, hens were inseminated with a
(Key words: chick, conjugated linoleic acids, fatty acids, lipids) 2000 Poultry Science 79:817–821
utilized and regulated in a specific fashion during embryogenesis. Recently, a class of fatty acid has been discovered called conjugated linoleic acids (CLA). The CLA isomers of linoleic acid occur naturally in various foods but are highest in beef and dairy products (Chin et al., 1992). The CLA isomers are reported to possess potent biological effects (Ito and Hirose, 1989; Ip et al., 1994, 1996; Lee et al., 1994; Park et al., 1997; Li and Watkins, 1998) and have been shown to be incorporated in tissue lipids (Sugano et al., 1997; Li and Watkins, 1998). As CLA incorporates into membrane phospholipids, it may compete with other unsaturated fatty acids in the formation of arachidonic acid and, thereby, will likely alter eicosanoid biosynthesis (Abou-El-Ela et al., 1989; Leyton et al., 1991). Li and Watkins (1998) suggested that CLA inhibits the action of ∆ 9-desaturase, thus potentially altering the balance of specific fatty acids used during embryogenesis. Ahn et al. (1999) demonstrated that CLA can be enriched in egg yolks and likely alters egg characteristics. As stated previously, CLA isomers have been shown to have a wide range of effects in a biological system, and, more specifically, CLA isomers potentially inhibit ∆ 9desaturase activity, an enzyme known to be activated during early lipid utilization in chicks. Therefore, the fo-
INTRODUCTION Yolk-derived lipids during chick developed are important because the embryo derives more than 90% of its energy requirements from the oxidation of yolk lipids (Romanoff, 1960). It is during the incubation process that numerous changes occur in yolk fatty acid composition (Noble and Cocchi, 1990); these changes are facilitated by the chick at various points during development (Latour et al., 1998). More specifically, during incubation, there is a marked decrease in phospholipids that contain high concentrations of docosahexaenoic and stearic fatty acids, which leads to an increase in the concentrations of species containing oleic and palmitic acids. In comparison with the yolk contents, the yolk sac membrane has a marked increase in arachidonic acid (Noble and Moore, 1967). Noble and Shand (1985) showed that ∆ 9-desaturase, the enzyme capable of converting (18:0) stearic acid to (18:1) oleic acid activity increases during the early stages of yolk utilization, thereby suggesting that yolk fatty acids are
Received for publication June 30, 1999. Accepted for publication March 6, 2000. 1 Agriculture Research Project Number 16,031. 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: CLA = conjugated linoleic acids.
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cus of this study was to investigate the effects of CLA in the developing chick in terms of fatty acid utilization and growth.
MATERIALS AND METHODS Animals and Diets Forty Single Comb White Leghorns layer hens (43 wk of age) were equally and randomly assigned among four treatments. The BW of all hens on each treatment were equal at the onset of this experiment. Within each treatment, two hens were housed together to provide five replicates per treatment. Layers assigned to Group A served as the control 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 determined to be 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). All supplements were stored at 4 C prior to administration. Feed and water were provided ad libitum. Feed consumption, BW, and egg hen day production were monitored weekly.
Insemination of Hens and Egg Collection After 4 mo of feeding, hens were inseminated from a pool of collected rooster semen. Hens were inseminated twice within a 1-wk period prior to first egg collection. At each insemination, hens received 3 × 105 sperm. Eggs from the various hen groups were gathered and incubated. At 21.5 d of incubation, breakout data were collected to determine percentages of hatchability and numbers of early-, mid-, and late-dead chicks.
Organ and Fatty Acid Analysis Yolks and livers from chicks in Groups A, B, and D were subjected to fatty acid analysis at hatch and at 6 d of age. From these tissues (yolk and liver), lipids were extracted by using chloroform/methanol (2:1, vol/vol) and then saponified to determine the fatty acid methyl esters profile by the method of Li and Watkins (1998).
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Nu-Check-Prep, Elysian, MN 56028. Matreya Inc., Pleasant Gap, PA 16823.
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TABLE 1. Effects of dietary fat [conjugated linoleic acid (CLA)] on hen and egg weight Group1
Hen weight
Egg weight
A B C D SEM2
(kg) 1.60b 1.83a 1.67ab 1.56b ± 0.19
(g) 59.2ab 61.3a 55.2bc 53.5c ± 1.00
a–d Means within a column for each variable with no common letter differ (P < 0.05). 1 Group A = control of 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.
The fatty acid methyl ester samples were identified by comparison of their retention times with authentic standards.4,5
Statistical Analysis Discrete variables (breakout information following hatch) were analyzed by Chi-square analysis, and the continuous variables were analyzed by ANOVA for a completely randomized factorial design. The 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 No differences were noted in the hens for feed consumption, egg production, fertility, hatchability of fertile eggs, or breakout data (numbers of early-, mid- or latedead chicks). However, at the end of the study, Group B hens were significantly heavier than those in Groups A and D; Group C hens were not different than those in other groups (Table 1). Also, there were differences in egg weight at setting for incubation among the four groups of hens (Table 1). (Eggs were collected following the 4-mo feeding period.) Eggs collected from Group B hens had similar weights as eggs collected from Group A hens, but they were significantly larger than those collected from Group C and D hens. At hatch, however, and through 6 d of age, there were no differences in chick BW (data not shown). There were significant treatment and time (hatch vs 6 d of age) effects on fatty acids in yolk and liver samples. No significant interactions (treatment by time) were observed in yolk or liver fatty acids. Only the yolks and livers of chicks hatched from Groups A, B, and D were analyzed. Group B chicks exhibited the highest level of yolk 18:0 fatty acid when compared with Group A and D chicks (Table 2). No other differences were noted by treatment; however, the metabolism of various fatty acids as a function of time (hatch vs 6 d of age) was affected.
CONJUGATED LINOLEIC ACID AND FATTY ACID MODIFICATION AND METABOLISM
That is, there were significant increases in yolk 18:1, 20:4n6, 22:0, 22:6n3, total monounsaturates, and total n3 fatty acids when comparing hatch and Day 6 values (Table 2). At the same time, significant decreases were noted in yolk 16:0, 16:1n7, 18:2n6, 18:3n6, 18:3n3, 18:2 (9– 11, conjugated double bonds), 18:2 (10–12, conjugated double bonds), saturates, polyunsaturates, and total n6 fatty acids and the ratio of n6 to n3 fatty acids (Table 2). Chick liver fatty acid content was affected by hen treatment. Chicks from Group B hens exhibited higher concentrations of liver 18:3n3 and 22:1n9 fatty acids compared with those from Groups A and D. Conversely, livers of Group B chicks had lower concentrations of 20:3n6 and 22:5n3 fatty acids when compared with liver fatty acid values in chicks from Groups A and D. Liver fatty acids were also affected by time (hatch vs 6 d of age), but no significant interactions were observed. More specifically, there were significant increases at Day 6 compared with hatch values for liver 16:0, 16:1n7, 18:0, 20:0, 20:1n9, 20:2n6, 20:3n6, 22:4n6, and saturated fatty acids as well as an increased saturated to polyunsaturated ratio (Table 3). At the same time, decreases were observed at Day 6
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vs hatch values for liver 18:1, 18:3n3, 20:4n6, 22:1n9, and monounsaturated fatty acids (Table 3).
DISCUSSION The enhanced performance observed in Group B hens (i.e., increased BW with no change in feed consumption) may be partially explained by altered physiological function, specifically reduced production of prostaglandins. Cook et al. (1993) demonstrated that chicks challenged with an endotoxin (lipopolysaccharide), which is known to affect growth negatively, did not affect chicks supplemented with CLA in terms of growth depression. The researchers hypothesized that CLA alters prostaglandin production and favors muscle. When prostaglandin E2 is applied to muscle, it begins a wasting process (Rodemann and Goldberg, 1982), and, in a recent study, it was shown that CLA decreases prostaglandin production in a number of tissues (Cunningham et al., 1997), favoring muscle integrity. In the present study, there were notable differences in fatty acid utilization in the yolk and liver. The avian
TABLE 2. Effects of hen-induced dietary fat [conjugated linoleic acid (CLA)] on hatched chick yolk fatty acid composition (percentage of yolk weight) Group1 Fatty acid type
A
B
D
Hatch
6d Posthatch
Pooled SD2
16:0 16:1n7 17:0 18:0 18:1 18:2n6 18:3n6 18:3n3 18:2 (9,11) 18:2 (10,12) 20:0 20:1n9 20:2n6 20:3n6 20:4n6 22:0 22:4n6 22:6n3 SAT4 MONO5 PUFA6 n-67 n-38 n-6/n-3 SAT/PUFA
19.78 1.75 0.15 9.70b 44.17 17.15 0.08 0.47 ND3 ND ND 0.21 0.19 0.22 2.68 1.41 0.20 0.72 31.04 46.13 21.70 20.51 1.19 19.21 1.43
22.33 1.37 0.20 13.24a 38.29 17.23 0.07 0.41 0.32 0.29 0.01 0.18 0.16 0.16 2.04 0.95 0.12 0.47 36.72 39.83 21.27 20.39 0.88 27.18 1.74
20.99 1.73 0.05 9.68b 41.62 17.62 ND 0.48 ND ND ND 0.11 0.08 0.14 2.61 2.15 0.37 0.50 32.87 43.46 21.80 20.82 0.98 29.39 1.51
22.60x 1.42x 0.12 11.32 37.2y 19.82x 0.05x 0.52x 0.17x 0.16x 0.01 0.16 0.15 0.13 1.87y 1.58y 0.12 0.17y 35.63x 38.78y 23.15x 22.46x 0.69y 35.15x 1.55
16.22y 1.23y 0.15 10.71 49.12x 13.91y 0.01y 0.24y 0.07y 0.05y ND 0.21 0.16 0.25 3.17x 2.13x 0.34 0.87x 29.20y 50.55x 19.05y 17.94y 1.11x 17.48y 1.54
2.51 1.62 0.13 0.84 3.61 2.02 0.06 0.15 0.10 0.08 0.02 0.14 0.12 0.13 0.54 1.01 0.19 0.15 3.58 3.58 1.83 1.75 0.14 6.71 0.23
a–b Means within a row for Groups A, B, and D with no common letter differ because of supplement treatments (P < 0.05). x–y Means within a row for time (hatch vs. 6 d) with no common letter differ (P < 0.05). 1 Group A = control hens; Group B = hens fed 1 g CLA every other day; Group D = hens fed 1 g safflower oil every other day. 2 Standard deviation is based on a pooled estimate of variance. 3 ND = non-detectable. 4 SAT = saturated fatty acids. 5 MONO = monounsaturated fatty acids. 6 PUFA = polyunsaturated fatty acids. 7 n-6 = n-6 fatty acids. 8 n-3 = n-3 fatty acids.
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LATOUR ET AL. TABLE 3. Effects of hen-induced dietary fat [conjugated linoleic acid (CLA)] on chick liver fatty acid composition (weight percentage) Group1 Fatty acid type
A
B
D
Hatch
6d posthatch
Pooled SD2
16:0 16:1n7 17:0 18:0 18:1 18:2n6 18:3n6 18:3n3 18:2 (9, 11) 18:2 (10, 12) 20:0 20:1n9 20:2n6 20:3n6 20:4n6 22:1n9 22:4n6 22:5n6 22:5n3 22:6n3 SAT4 MONO5 PUFA6 n-67 n-38 n-6/n-3 SAT/PUFA
15.36 2.00 0.16 14.23 28.43 13.11 0.14 0.06b ND3 ND 0.17 0.70 0.80 7.33a 2.32 0.57b 0.41 0.47 0.21b 1.96 29.92 31.52 26.82 24.59 2.23 11.58 1.08
17.94 1.87 0.65 15.05 31.06 15.16 0.11 0.30a 0.20a 0.20a 0.13 0.59 1.04 4.41b 3.41 1.96a 0.45 0.30 0.06b 1.86 33.76 35.48 27.50 25.28 2.22 13.78 1.27
15.75 2.11 0.48 14.74 34.54 13.91 0.09 0.1b ND ND 0.20 0.74 1.42 7.79a 3.64 ND3 0.65 0.46 0.91a 1.74 31.16 37.39 30.72 27.96 2.76 10.31 1.01
10.03y 0.82y 0.52 10.71y 42.50x 14.07 0.05 0.25x 0.17a 0.17a 0.02y 0.14y 0.15y 3.54y 4.93x 1.68x 0.18y 0.52 0.36 2.04 21.28y 45.15x 26.45 23.79 2.65 10.21 0.83y
21.44x 2.89x 0.39 17.8x 22.53y 14.17 0.16 0.10y 0.00 0.00 0.27x 1.08x 1.81x 8.58x 1.75y 0.32y 0.75x 0.31 0.38 1.71 39.89x 26.82y 29.73 27.54 2.19 13.40 1.37x
4.07 1.31 0.48 2.36 5.32 2.54 0.13 0.08 0.01 0.01 0.11 0.19 0.58 1.56 1.51 0.97 0.40 0.27 0.17 0.81 4.97 6.55 4.79 4.25 0.82 4.30 0.29
a–b Means within a row for Groups A, B, and D with no common letter differ because of supplement treatments (P < 0.05). x–y Means within a row for time (hatch vs. 6 d) with no common letter differ (P < 0.05). 1 Group A = control hens; Group B = hens fed 1 g CLA every other day; Group D = hens fed 1 g safflower oil every other day. 2 Standard deviation is based on a pooled estimate of variance. 3 ND = non-detectable. 4 SAT = saturated fatty acids. 5 MONO = monounsaturated fatty acids. 6 PUFA = polyunsaturated fatty acids. 7 n-6 = n-6 fatty acids. 8 n-3 = n-3 fatty acids.
embryo derives more than 90% of its energy from the oxidation of yolk lipids (Romanoff, 1960), and it is during the incubation process that numerous changes occur in yolk fatty acid composition (Noble and Cocchi, 1990). These changes in fatty acid composition are used by the developing chick at various points during development (Latour et al., 1998). For example, fatty acids in the 20– 22 carbon range of both the n-3 and n-6 series, and particularly the 20:4n6 and 22:6n3 fatty acids, play essential roles in embryonic or neonatal neural development of most vertebrates (Neuringer et al., 1988). In the present study, there was an approximate 20% decline in yolk saturates, polyunsaturates, n-6 fatty acids, and the n-6 to n-3 ratio when comparing hatch values to those at 6 d of age. Conversely, yolk n-3 concentrations increased by 160% by Day 6 when compared with hatch values. The current study suggests that yolk unsaturated fatty acids (polyunsaturated fatty acids, n-6, and, to a lesser degree, saturated fatty acids) are used up rapidly during the first few days posthatch when compared with n-3 fatty acids. The metabolism of liver fatty acids was uniquely different from
that observed in yolks; monounsaturated and saturated or polyunsaturated fatty acid concentrations decreased by 40 and 163%, respectively. At the same time, liver saturated fatty acid content increased by 187%. Noble and Ogunyemi (1989) stated that yolk and liver lipid content can be extensively mobilized through the first few days posthatch, which is confirmed by the present study. However, chicks from Group B hens, in which the highest concentration of CLA was used, altered the metabolism of yolk sac contents. For example, chicks at hatch and 2 d of age from Group B hens exhibited little change in relative yolk sac weight when compared with all of the other groups (Latour et al., 2000). Apparently, the initial steps in the sequence of events that facilitates yolk lipid removal during the early neonatal period were impaired in chicks from Group B hens, but they quickly adapted by 6 d of age. Li and Watkins (1998) suggest that CLA inhibits the action of ∆ 9-desaturase, the enzyme capable of converting stearic acid (18:0) to oleic acid (18:1). Perhaps the increase in yolk 18:0 fatty acid of Group B chicks can be partially explained by the modification of ∆
CONJUGATED LINOLEIC ACID AND FATTY ACID MODIFICATION AND METABOLISM
9-desaturation. As stated earlier, ∆ 9-desaturase activity increases during the early stages of yolk utilization and is believed to be associated with stabilization of the lipoproteins prior to exiting the yolk proper (Noble and Shand, 1985). For more information regarding CLA and chick lipoproteins see Latour et al. (2000). The present study demonstrates that 0.5% CLA increases the BW of hens and alters the usage of specific fatty acids in the neonatal chick. The BW, feed consumption, and survival of chicks of the various groups were not affected by treatment; however, effects on other grow out parameters were not determined.
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