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PRODUCTION, MODELING, AND EDUCATION |
datliolu
lu*
. Uysal
* Ege University, Faculty of Agriculture, Department of Animal Science, 35100 Izmir, Turkey; Celal Bayar University, Engineering Department, 45140 Manisa, Turkey; Laboratory for Physiology, Immunology, and Genetics of Domestic Animals, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium; Institute of Technology, Food Engineering Department, 35430 zmir, Turkey; and # Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061
1 Corresponding author: servet.yalcin{at}ege.edu.tr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: parental age • heat acclimation • embryo composition • yolk sac composition • heat stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Age of hen (Nielsen, 1998; Latour et al., 2000) influences the fatty acid composition of eggs and lipid utilization by embryos. The rate of lipid mobilization from the yolk into the yolk sac membrane is lower in younger than older breeders (Noble and Connor, 1984; Noble et al., 1986; Tullett and Noble 1989; O’Sullivan et al., 1991), with the percentage of lipid and dry matter in embryos increasing with age of parents. Similarly, in duck embryos, Braun et al. (2001) reported that both the relative number and size of liver lipid droplets in embryos increased with age of breeder.
Palmitoleic acid (16:1n-7) levels, relative to total fatty acids in yolks, were higher in unincubated eggs from 36 wk than 51- and 64-wk breeders (Latour et al., 1998). Also, relative stearic (18:0) and arachidonic (20:4n-6) acid levels were higher in eggs from breeders at 26 than 28 and 30 wk (Burnham et al., 2001), whereas C20–22 series were higher in eggs from breeders at 21 than 57 wk (Nielsen, 1998). Variation in fatty acid composition of egg yolk occurs during the incubation; Latour et al. (1998) noted that incubation effects on yolk palmitoleic (16:1n-7), oleic (18:1n-9), arachidonic (20:4n-6), and to a lesser degree linoleic (18:2n-6) acids concentrations in eggs from younger than older parents.
Recent studies have focused on acclimation of poultry to extreme posthatch temperatures by exposing embryos to lower or higher incubation temperatures (e.g., Tzschentke and Basta, 2002; Yahav et al., 2004b; Yalçin et al., 2005). Feast et al. (1998) reported reduced yolk lipid uptake in embryos exposed to lower incubation temperatures. Although physiological responses of embryos to higher incubation temperatures (e.g., increased allantoic fluid temperature, lower plasma corticosterone level at internal pipping stage, decreased heat production at hatch, similar plasma triiodothyronine and corticosterone levels to control chicks at hatch) have been reported (Janke et al., 2002; Moraes et al., 2004; Yahav et al., 2004a), information is lacking on the effect of higher incubation temperature on embryo composition and residual fatty acid profiles of chicks. The present study aimed to evaluate embryo composition as well as to evaluate the changes in egg yolk fatty acid composition during embryonic development as a function of incubation temperature and age of breeders.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There were 276 eggs/breeder age per flock. Before incubation, 5 eggs from each parental age and flock were sampled for analysis of yolk fatty acid content.
Eggs were divided equally into 2 groups and placed into 2 incubators. One incubator was maintained at 37.8°C from d 1 to 18 of incubation (CONT). In the second incubator eggs were heat-acclimated (HA) at 38.5°C for 6 h daily from d 10 to 18 of incubation. From d 18 to hatch, the temperature was 37.5°C for both groups. Relative humidity was maintained at 65% in both incubators. There were 3 replicate trays in each subgroup (46 eggs/tray).
On d 14 and 18 of incubation, after 6 h of daily heat treatment, 5 eggs/parental age per flock per incubation temperature were randomly selected (30 eggs/incubation temperature). Eggs were opened, the embryos removed, and they were cleaned of yolk sac and membrane. They were then killed by cervical dislocation. The procedure was repeated on day of hatch with 5 randomly selected chicks/parental age per flock per incubation temperature subclass, after which chicks were dried in the incubators. Yolk sacs of chicks were removed for fatty acid determinations. Embryos, chicks, and yolk sacs were weighed and stored at –20°C before analyses.
Embryos and chicks were dried at 110°C for 24 h and their dry matter calculated as the differences between wet and dry weights divided by wet weight. Embryo N content was determined by the Kjeldahl method, and CP was calculated as N x 6.25. Embryo and chick lipid contents were determined by AOAC (1990). Protein and fat content were expressed as percentage of total dry matter.
Fatty Acid Analyses of Yolk Sac of Chicks
An aliquot of egg yolk was weighed, and lipid was extracted with chloroform:methanol (2:1, vol/vol; Folch et al., 1957) and total lipids methylated by sodium methoxide in ethanol. One hundred milligrams of lipid extracted from egg yolk was placed in a 15-mL screw-cap tube, 0.2 mL of NaOH-MeOH (0.5 N) was added, vortex-mixed, and heated at 100°C for 5 min. The tube was cooled in cold water, 0.5 mL of HCl-MeOH (4%) was added to the sample, vortex-mixed, and held at room temperature for 5 min. Approximately 5 mL of isooctane and 3 mL of distilled water were added to extract the fatty acid methyl esters (FAME). The mixture was shaken for 10 min, centrifuged at 4,000 x g for 5 min, and then the upper phase was collected, dried with sodium sulfate, and concentrated with nitrogen gas for gas chromatograph (GC) analysis.
A gas chromatograph (Agilent 6890N Series) equipped with an autosampler (Model 7683B), GC ChemStation, and flame ionization detector (Agilent Technologies, Inc., Wilmington, DE) was used to analyze the FAME. The GC was operated at a temperature of 150°C for 2 min, followed by heating at 3°C/min to 210°C and holding for 20 min. A DB-23 column was used for the analysis (60 m, 0.25-mm i.d., 0.25-µm film thickness; Agilent Technologies Inc.). The injector and flame ionization detector were maintained at 220 and 250°C, respectively. Identification of sample FAME was achieved by comparing the retention times to FAME standards (Mixture ME-100; Greyhound Chromatography and Allied Chemicals, Birkenhead, Merseyside, UK).
Statistical Analyses
Data from the 2 flocks/breeder age were pooled, because differences between flocks/breeder age were not significant. One-way ANOVA was performed to analyze the effects of breeder age on fatty acid composition before incubation (SAS, 1999). Comparisons of effects of parental age and incubation temperature on fatty acid composition were made by 2-way ANOVA. Multiple mean comparisons were made by Tukey’s studentized range test. Statements of statistical significance were based on P < 0.05 unless otherwise stated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mean moisture content was 87.7, 82.9, and 81.0% on d 14, 18, and at hatch, respectively (Table 1
). Incubation temperature and parental age significantly affected embryo moisture content on d 14. A significant incubation temperature x parental age interaction showed that although HA resulted in an increase in moisture content, it was the case only for embryos from younger parents with values for 42- and 65-wk parents similar for HA and CONT embryos (Table 2). On d 18, there was no parental age effect on embryo moisture for CONT. At HA, the highest moisture content was obtained for embryos from 32-wk parents. At hatch, incubation temperature had no effect on moisture content; however, moisture was higher for embryos from 32-wk than 42- and 65-wk parents.
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Fat content of embryos changed with age of embryo, increasing from 20.8 to 25.4% between d 14 and 18 and then decreasing to 22.1% from d 18 to hatch. There were no effects of incubation temperature and parental age on fat content of embryos on d 14. On d 18, fat content of HA embryos increased. A significant interaction between incubation temperature and parental age showed that there were no differences for fat content of embryos for CONT eggs; however, fat content of embryos from 42-wk parents increased at HA temperatures (Table 2). On day of hatch, fat content was higher for embryos from 42- and 65-wk than 32-wk parents (Table 1).
Egg Yolk Fatty Acid Composition Before Incubation
Mean fatty acid compositions of egg yolk before incubation by parental age are presented in Table 3. Palmitic (16:0), oleic (18:1n-9), and linolenic (18:2n-6) acids were the major fatty acids, ranging from 17.94 to 36.88%. This was followed by stearic (18:0), palmitoleic (16:1n7), and cis-11,14,17-eicosatrienoic (20:3n-3) acids ranging from 2.21 to 9.53%, with the remaining fatty acids <1.0%.
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Although egg total monounsaturated fatty acid composition was similar across parental ages, palmitoleic (16:1n-7) acid was higher in eggs from the younger parents, and conversely, cis-11-eicosenoic acid was higher in eggs from the older parents. Total PUFA was lower in eggs from 32-wk than 42- and 65-wk parents. Linoleic acid (18:2n-6), as a major PUFA in egg, changed with parental age similar to total PUFA. Cis-11,14-eicosadienoic acid (20:2n-6) and cis-11,14,17-eicosatrienoic (20:3n-3) contents of yolk were higher for eggs from 65-wk than 32- and 42-wk parents. Parental age had no effect on DHA (22:6n-3) and eicosapentaenoic acid (20:5n-3) contents of egg. The ratio between saturated and unsaturated fatty acids was higher in eggs from younger parents than in eggs from either 42- and 65-wk parents.
Residual Yolk Sac Fatty Acid Composition of Chicks at Hatch
When yolk fatty acid profiles were compared before and after incubation, there was no change in the total saturated fatty acids (35.21 vs 35.23%; data not shown in tables). However, total monounsaturated fatty acid content of yolk was 34.9% before incubation and increased to 36.5% after incubation in the yolk sac of embryos. This increase resulted mainly from an increase in the oleic acid (18:1n-9) content in the yolk sac of chicks (34.7 vs 36.3%). Compared with before incubation, DHA (22:6n-3) and cis-11,14,17-eicosatrienoic (20:3n-3) contents reduced significantly from 0.9 to 0.3% and from 2.4 to 1.6%, respectively, whereas linoleic acid (18:2n-6) increased from 19.9 to 21.0% after incubation. However, total PUFA content was similar before (24.49%) and after incubation (24.18%).
The fatty acid composition of the residual yolk sac (Table 4) generally resembled that of yolk before incubation. Similar to yolk before incubation, palmitic (16:0), palmitoleic (16:1n-7), stearic (18:0), oleic (18:1 n-9), and linoleic (18:3 n-3) acids were the major components in the residual yolk, and the main C20–22 polyunsaturates were cis-11,14,17-eicosatrienoic (20:3n-3) and DHA (22:6n-3).
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). Parental age significantly affected pentadecanoic (15:0) and heptadecanoic (17:0) acids in the residual yolk sac, being lower in chicks from 32-wk than 42-wk parents. The HA slowly increased pentadecanoic acid (15:0) content of residual egg yolk sac. Treatments had no effect on arachidic acid content (Table 4). A significant interaction for palmitic acid content of residual yolk sac resulted from chicks hatched from 32-wk parents having higher palmitic acid content in the yolk sac when incubated at HA (Table 5).
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Total PUFA contents of yolk sac of chicks were similar among parental ages and between incubation temperatures. Interactions between these 2 main effects were significant for linolenic acid (18:3n-3); cis-11,14-eicosadienoic (20:2n-6); cis-8,11,14-eicosatrienoic acids (20:3n-6); and DHA (22:6n-3) contents. Linoleic acid (18:3n-3) was higher in the residual yolk sac of chicks from 32-wk parents incubated at HA than the CONT, whereas the pattern was reversed for chicks from 65-wk parents. Conversely, HA decreased 20:3n-6 and 22:6n-3 contents of residual yolk of chicks from younger parents but increased those fatty acid contents in yolk sac of chicks from older parents. Cis-11,14-eicosadienoic acid (20:2n-6) content increased in eggs from 42-wk parents when eggs were incubated at HA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation temperature had no effect on protein content of embryos, suggesting that the higher incubation temperature did not alter protein accumulation by the embryo. Moreover, protein-to-water ratios were similar among and between treatments during incubation. The protein in the dried samples was higher on d 14 of incubation and decreased until d 18, which occurred with an increase in fat content. The protein-to-fat ratio was lower on d 18 (2.65) than d 14 (3.56) of incubation with a small and nonsignificant change in protein from d 18 to hatch. The decrease in the fat content led to lower organic material and greater inorganic material at hatch. The decrease in relative fat content at hatch compared with d 18 may be related to the increased heat production in the embryos, linked to the hatching process.
Higher fat content in chicks from 42- and 65-wk parents would be expected due to an association of parental age with egg yolk fat. McNaughton et al. (1978), however, noted that when egg weights were the same there were no differences in yolk composition between 29- and 50-wk parents. When we analyzed for fat content excluding chick weight (using chick weight as a covariate) there was no change in the results observed for fat content. Thus, parental age influenced fat mobilization/utilization independent of chick weight, whereas incubation temperature had no apparent effect on this process. Similarly, Yafei and Noble (1990) reported a reduced yolk lipid absorption in embryos from the 23-wk parents. Yalçin et al. (2008) found heavier livers in embryos and chicks from older parents regardless of incubation temperature. Thus, the higher fat content of the embryos and chicks from older parents was associated with heavier liver weights of those embryos and chicks.
The pattern of change in the concentration of DHA (22:6n-3) and cis-11,14,17-eicosatrienoic (20:3n-3) in the residual yolk sac of chicks compared with egg yolks before incubation was markedly different from that of the other fatty acids. A consistent decrease observed in DHA and cis-11,14,17-eicosatrienoic fatty acids across the breeder ages (i.e., 60 and 30% less for DHA and cis-11,14,17-eicosatrienoic, respectively) may have been a result of prefential uptake from the yolk. Residual yolk sac 14.0, 20:0, 20:3n-3, 20:4n-6, and 20:5n-3 fatty acid contents were independent of parental age and incubation temperature. Pentadecanoic (15:0) and heptadeconoic (17:0) acids were more efficiently transported to the embryo from the yolk sac from breeders at 32 wk than those from 42 and 65 wk. The HA treatment decreased transported levels of pentadeconoic acid (15:0) and palmitoleic (16:1n-7) acids regardless of parental age. These changes in yolk fatty acid transportation may be due to activities of enzymes and lipoprotein transport (Speake et al., 1998).
Important observations in our experiment were changes in linolenic (18:3n-3) acid and DHA (22:6n-3) in yolk sacs of chicks from younger and older parents when eggs were incubated at higher temperatures. The DHA content in residual yolk sac was considerably higher in chicks from older parents incubated at HA, whereas, in contrast, levels of 18:3n-3 were lower. Also, chicks from younger parents in the HA treatment had lower transported 18:3n-3 and higher levels of transported DHA. It may be concluded that, although higher levels of 18:3n-3 were transported to the chick, they did not trigger its proper conversion to DHA in yolk sac of chicks from older parents when eggs were incubated at high temperatures. These results support previous conclusions (Maldjian et al., 1995; Speake et al., 1998; Speake and Wood, 2005) that DHA is preferentially taken up from the yolk, which is related to the specific requirements of tissue. Moreover, previous results indicated that heat-acclimated embryos and chicks, at the internal pipping stage and at hatch, exhibited adaptive physiological responses (Yalçin et al., 2008). Furthermore, actual environment during incubation influences the development of respective physiological control systems via changes in neuroorganization (Tzschentke and Plagemann, 2006). Tzschentke and Basta (2002) showed that exposure to higher incubation temperatures changed postnatal thermosensitivity of the preoptical area of anterior hypothalamus neurons in ducks. Because of the important role of DHA in the functional development of neural tissues, that this process occurred during the higher incubation temperature may be related to a protective strategy and thus contributes to heat adaptation by the chick. Shmeeda et al. (2002) also suggested that if DHA influenced membrane permeability properties, it would directly affect stress endurance during heat challenge and prevent dehydration of the cells. Because there were no flock effects within breeder age, the results may be attributed to the higher incubation temperature effects on yolk fatty acid uptake. The question is raised if the higher incubation temperature reduced the need of DHA of embryos from older parents. Our results also show that although HA had no effect of chick composition at hatch, residual yolk composition may be changed by heat acclimation of embryos during incubation and that breeder age may have additional effects.
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ACKNOWLEDGMENTS |
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tirma Kurumu (project no. 155 O 044) and Ege University Scientific Research Projects (project no. 2005 ZRF 039). Veerle Bruggeman is a postdoctoral fellow of the Fonds Wetenschappelijk Onderzoek-Vlaanderen (Belgium). Received for publication October 23, 2007. Accepted for publication March 1, 2008.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
