Introduction
The omega-3 long-chain polyunsaturated fatty acids (n−3 LCPUFA) have a number of important health benefits in humans, in particular in relation to inflammatory conditions such as rheumatoid arthritis and protection against cardiovascular diseases.Reference Colquhoun, Ferreira-Jardim, Udell and Eden 1 , Reference Galli and Calder 2 These effects rely on the incorporation of n−3 LCPUFA into the phospholipid fraction of the cell membrane, and subsequent release in the free fatty acid pool to give rise to bio-active mediators.Reference Jump 3 This has led to recommendations from a number of health agencies for humans to increase their consumption of fish and seafood, the richest dietary sources of these fatty acids. Despite this, consumption of these sources remains low in western countries,Reference Colquhoun, Ferreira-Jardim, Udell and Eden 1 , 4 and fish and seafood are also not environmentally sustainable sources of these fats.Reference Hannesson 5 In contrast to seafood, the global consumption of poultry, especially chicken, is steadily increasing and this is now the most popular type of meat in many societies. 6 This has led to suggestion that one strategy to increase dietary n−3 LCPUFA intake in western countries, and one which would avoid placing additional pressure on global marine resources, is to increase the n−3 LCPUFA content of chicken meat.Reference Lopez-Ferrer, Baucells, Barroeta, Galobart and Grashorn 7 , Reference Konieczka, Czauderna and Smulikowska 8
Chicken meat is naturally low in fat (~2.0%), and a poor source of n−3 polyunsaturated fatty acids (PUFA) at only ~2.5% of total fatty acids including n−3 LCPUFA at ~1.3%.Reference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9 However, we and other researchers have demonstrated that increasing the amount of n−3 PUFA in the diet, by feeding chickens diets supplemented with flaxseed (Linum usitatissimum) oil [high in the short-chain n−3 PUFA, α-linolenic acid (ALA)], results in substantial increases in the ALA content of the meat, without increasing the overall fat content.Reference Kartikasari, Hughes, Geier, Makrides and Gibson 10 Importantly, chickens also possess the hepatic enzymes required to synthesize the n−3 LCPUFA; eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from ALA.Reference Lopez-Ferrer, Baucells, Barroeta, Galobart and Grashorn 7 , Reference Barceló-Coblijn and Murphy 11 Consequently, replacing standard fat sources in formulated chicken diets such as corn oil, canola oil, soybean oil (all rich in n−6 PUFA), animal fat or their blends, with flaxseed oil reduces the dietary n−6:n−3 PUFA ratio and increases the n−3 LCPUFA content of chicken tissuesReference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9 , Reference Kartikasari, Hughes, Geier, Makrides and Gibson 10 , Reference Kartikasari, Geier and Hughes 12 – Reference Carragher, Mühlhäusler and Geier 14 , as n−6 and n−3 PUFA precursors compete for the same hepatic enzymes in their elongation and desaturation pathways.Reference Kartikasari, Hughes, Geier, Makrides and Gibson 10 , Reference Jing, Gakhar, Gibson and House 15 Despite the success of High-ALA diets for increasing the level of n−3 LCPUFA in chicken meat, the actual levels of n−3 LCPUFA which are achieved (76.5–108 mg/100 g meat)Reference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9 are still substantially lower than those in oily fish (e.g. herring, salmon and mackerel), which contains >1500 mg in the similar amount of meat. 16
Improving the efficiency through which High-ALA diets improve chicken meat n−3 LCPUFA content relies on altering the processes which regulate ALA conversion to n−3 LCPUFA and/or deposition of fatty acids into different lipid fractions.Reference Boschetti, Bordoni and Meluzzi 17 , Reference Gonzalez-Esquerra and Leeson 18 The egg yolk is the main reserve of energy and the sole source of essential fatty acids during embryogenesis,Reference Cherian 19 and previous studies have demonstrated that feeding layer hens a diet enriched in ALA increases the n−3 LCPUFA content of the eggsReference Goldberg, Ryland, Aliani and House 20 – Reference Koppenol, Delezie and Wang 23 and of the chicks at hatch.Reference Cherian and Sim 22 In addition, feeding hens a diet enriched with fish oil results in higher n−3 PUFA levels in cardiac tissues in their chicks.Reference Ajuyah, Cherian, Wang, Sunwoo and Sim 24 However, no previous studies have determined the effect of in ovo exposure to an increased supply of n−3 PUFA on the chicken’s subsequent capacity for ALA-derived n−3 LCPUFA accumulation in meat tissues. Therefore, the aim of the present study was to determine the effect of maternal dietary treatments that would expose developing chicks in ovo to an elevated level of n−3 LCPUFA, or its precursor (ALA) on the capacity of chickens to accumulate n−3 LCPUFA when fed a High-ALA diet post-hatch.
Methods
Maternal dietary treatments
A total of 324 of broiler breeder hens of the Cobb 500 strain were housed in the HiChick Breeding Company facility (Bethyl, SA, Australia). Before the study all the hens received the same commercial breeder diet. Hens were allocated to one of three dietary groups (n=108/group): Control (basal diet mixed with 4% w/w beef tallow), High-ALA (basal diet mixed with 4% w/w flaxseed oil) or Fish-Oil (basal diet mixed with 4% w/w fish-oil) (Fig. 1.). The breeder basal diet used for all feeds was purchased from Lauke Mills, Australia. The three diets contained the same proportions of fat, carbohydrate and protein, and differed only in their fatty acid composition (Table 1). All three experimental diets contained the same levels of vitamins and minerals and these either met or exceeded the recommended levels. 25 Each group of hens was housed separately with eight roosters from the same strain, and fed the diets for the duration of the experiment (Fig. 1).
Fig. 1 The methodology and numbers of birds used during different experimental phases.
Table 1 Fatty acid composition of the experimental dietsFootnote a
a Values are fatty acid group percentage of the total fatty acid.
b By mixing basal breeder hens diet with (4% w/w) beef tallow (Control), flaxseed oil-based diet or fish oil (n=3).
c By mixing finisher basal diet with (4% w/w) beef tallow (Control), flaxseed oil (n=6).
d Percentages are based on the wet weight.
e SFA, Saturated fatty acid.
f n-9 MUFA, Omega 9 monounsaturated fatty acid.
g n-7 MUFA, Omega 7 monounsaturated fatty acid.
h n-6 PUFA, Omega 6 polyunsaturated fatty acid.
i n-3 PUFA, Omega 3 polyunsaturated fatty acid.
j ALA, α-linolenic acid ALA.
k n-3 LCPUFA, Omega 3 long chain polyunsaturated fatty acid.
Egg sampling
All eggs that were laid in the 5th week of the maternal diet regimen (Control, n=132 eggs; High-ALA, n=148 eggs; Fish-Oil, n=80 eggs) were collected and stored at holding temperature to temporarily prevent initiation of embryonic development. The yolk from five eggs selected at random from each maternal diet group were collected and stored at −18°C for subsequent fatty acid analysis. The remaining eggs were transferred to the South Australian Research and Development Institute (Roseworthy, SA, Australia) and immediately placed in incubators under standardized conditions (38°C and 55% humidity, increasing to 60% in the last 4 days of incubation). After 1 week of incubation, the fertility of each egg was assessed, and non-fertile eggs and eggs with dead embryos were discarded.
Chick hatch and sampling
Chicks were hatched at ~21 days after the start of the incubation, and at 1-day old were feather-sexed into pullets and cockerelsReference Jacob 26 (Control: n=26 females, n=25 males; High-ALA: n=25 females, n=25 males; Fish-Oil: n=24 females, n=11 males). In total, 20 male and 20 female chicks in each maternal dietary group were then allocated to two separate raised floor pens (1.2×0.9 m each; n=10 chicks/pen), except for the Fish-Oil group males where the pens held six and five chicks due to the smaller number of hatchlings (Fig. 1).
The unallocated 1-day old chicks (Control: six females and five males; High-ALA: five of each sex; and Fish-Oil: four females only) were euthanized by cervical dislocation and ~1–2 g of breast and leg meat were collected in plastic vials and immediately placed on dry ice. Samples then were transferred to the laboratory where they stored at −18°C until subsequent determination of crude fat content and fatty acid composition.
Housing of broilers
A complete factorial randomized block design (3×2×2) was implemented such that one pen of male and one pen of female chicks in each of the three maternal dietary treatment were fed either the control (4% w/w beef tallow) or the High-ALA (4% w/w flaxseed oil) progeny diets for the entire 6 weeks of grow-out. The two experimental progeny diets were nutritionally identical, met requirements for healthy growth and all vitamins and minerals met or exceeded the recommended levels. 25 Broilers were reared under controlled environmental conditions with free access to feed from hoppers and water from a nipple drinker line. The room temperature was 27°C for the first 4 days then gradually decreased to 20°C and maintained until harvest, with pens heated by infrared lamps (175 W) during the first 3 weeks. Feed intake and final body weights (BW) of broilers were recorded and feed conversion rate (FCR) during the final week before slaughter was calculated. The numbers of birds that were culled or died was recorded on daily basis.
Tissue sampling
On day 42 of grow-out, three birds from each pen (n=36) were randomly selected and euthanized by cervical dislocation. Breast and leg meat tissues were sampled, frozen on dry ice and stored at −18°C for subsequent fatty acid profiling.
Fatty acid analysis
Crude lipid was extracted from a representative sample of homogenized feed, egg yolk and lean meat samples.Reference Folch, Lees and Sloane Stanley 27 The gravimetric approach was utilized to estimate total crude lipid (% of wet weight). Fatty acid profiling was performed after transmethylation of the extracted crude lipids with 1% H2SO4 in methanol at 70°C for 3 hours. Briefly, after cooling to room temperature, the resulting fatty acid methyl esters (FAME) were extracted with n-heptane (2 ml) and transferred into gas chromatography (GC) vials containing about 30 mg of anhydrous sodium sulfate and stored at −18°C until GC analysis. The FAMEs were separated using a Hewlett-Packard 6890 GC (Hewlett-Packard, CA, USA) equipped with a flame ionization detector, a split injector and a BPX-70 capillary column (50 m×0.32 mm internal diameter) with a 0.25 µm film thickness (SGE, Victoria, Australia). The operating conditions of the GC, fatty acid identification using the GLC 463 external standard (Nu-Chek Prep Inc., MN, USA) and qualitative analysis were as described previously.Reference Tu, Cook-Johnson, James, Mühlhäusler and Gibson 28
Statistical analyses
The effects of dietary treatment on the fatty acid profile were tested by one-way, two-way and three-way analysis of variance (ANOVA) for egg, chick and broiler tissues, respectively, using SPSS version 21 for Windows (IBM Corp., NY, USA). Duncan’s multiple comparison test was implemented where the ANOVA showed significant differences between groups (P<0.05). Due to the uneven number of broilers in each pen, it was not possible to reliably assess the impact of the dietary treatments on growth performance.
Results
Fatty acid composition of the experimental diets
There was no difference in the crude fat percentages between the three breeder diets or the two progeny diets. The crude fat percentage of the progeny finisher diet (fed in the last 3 weeks of broilers grow-out) was lower than the breeder hens’ diets by ~1.2%, due to the different nutritional requirements of birds, however, the fatty acid profiles of both the breeder and progeny diets similarly reflected the type of lipid added to the basal feed. Thus, the Control (beef tallow) diet comprised predominately of saturated fatty acid (SFA) and n−9 monounsaturated fatty acid (MUFA), the High-ALA diet comprised predominately of n−3 PUFA as ALA, whereas the Fish-Oil diet was the only one which contained n−3 LCPUFA (Table 1). The ratio of n−6:n−3 PUFA in the diets decreased from 5.8 in the maternal control diet to 0.8–1.0 in the flaxseed and fish oil diets (Table 1).
Productivity of breeder hens
The laying rate of the breeder hens (number of eggs/breeder) appeared to be lower in hens fed the Fish-Oil diet compared with those fed either the Control or High-ALA diets (Control, 1.22; High-ALA, 1.37; Fish-Oil, 0.74). The ratio of female:male chicks hatched, on the other hand, appeared to be higher in the Fish-Oil group compared with the other dietary treatments (Control, 0.96; High-ALA, 1.00; and Fish-Oil, 2.18). The hatchability of the eggs (number of chicks hatched/egg laid) was similar between groups (Control, 0.39; High-ALA, 0.33; Fish-Oil, 0.44).
Fatty acid composition of egg yolks
The crude fat content of egg yolks ranged from 32–38% of the yolk weight (data not shown), and did not differ between dietary treatments. The fatty acid composition of the yolk reflected that of the maternal diet (Fig. 2a). Thus, the ALA content of the yolk was higher in the High-ALA dietary group compared with the Control and Fish-Oil groups (Fig. 2b; P<0.0001), whereas the n−3 LCPUFA level in the yolk was the highest in the Fish-Oil group compared with the other treatments (Fig. 2b, P<0.0001). The yolk from hens in the Fish-Oil treatment group also contained less n−6 PUFA than both other groups (P<0.01). The ratio of n−6:n−3 PUFA of the egg yolk was 4–5-fold lower in both the High-ALA (1.3) and Fish-Oil (1.2) groups compared with the Control (5.6) group (P<0.01).
Fig. 2 Fatty acid profile (a) and omega-3 distribution (b) in egg yolk of breeder hens fed three different diets (n=5±s.e.m.). Different stars within the same fatty acid indicate significant difference (P<0.01 for n−9 and n−6) or (P<0.001 for other fatty acids) between maternal treatments.
Fatty acid composition of day-old chicks
There were significant differences (P<0.0001) in the levels of all major fatty acids in both the breast and leg tissues of the day-old chicks between dietary treatment groups, with the exception of the SFA content, which was similar between treatments. In the breast meat, the MUFA content was lower in the chicks in the High-ALA treatment group compared with both the Fish-Oil and Control treatments. The total n−3 PUFA content (ALA+n−3 LCPUFA) was highest (13.7%) in chicks in the Fish-Oil group, slightly lower (13.4%) in High-ALA and lowest (5.4%) in the Control group (Fig. 3a; P<0.0001). A similar effect was observed for the n−3 LCPUFA content, which was 11.6, 9.0 and 4.9% in the Fish-Oil, High-ALA and Control groups, respectively. The n−3 LCPUFA made up a higher proportion of total n−3 PUFA in the Fish-Oil group compared with the High-ALA group (Fig. 3b; P<0.0001). The ALA content of the chicks was also different between treatments, and was higher in the High-ALA (4.4%) and Fish-Oil (2.1%) groups compared with the Control group (0.5%) (Fig. 3b; P<0.0001). The n−6 PUFA content was ~5% higher in the Control and High-ALA chicks compared with chicks in the Fish-Oil group (Fig. 3a; P<0.0001). However, the n−6:n−3 PUFA ratio was reduced by 2.5-fold and 3.2-fold (P<0.0001) in the High-ALA and Fish-Oil groups compared with the Control group. Similar effects were observed in the leg meat, and there were no differences between sexes (Control and High-ALA groups) or interactions between chick sex and maternal dietary treatment (males of Fish-Oil group excluded) in either tissue.
Fig. 3 Fatty acid profile (a) and omega-3 distribution (b) in breast meat of female day-old chicks from three different maternal dietary treatments (n=6.5 and 4± s.e.m. for control, flaxseed and fish oil treatments, respectively). Different stars within the same fatty acid indicate significant difference (P<0.0001) between maternal treatments.
Growth of broilers
The final BW of the broilers at 42 days’ post-hatch was 2.98 kg in females and 3.64 kg in males, and the FCR in the final week before tissue collection were 1.92 and 1.81 for females and males, respectively. Only two broilers were culled or died before tissue collection throughout the entire 6-week grow-out period.
Fatty acid composition of broilers
Overall, the fatty acid composition of the leg and breast meat tissues was broadly reflective of the diets that the offspring were fed post-hatch, independent of the diet to which they had been exposed in ovo (Table 2). Therefore, tissues from broilers fed on the High-ALA diet post-hatch had higher levels of total n−3 PUFA, ALA and n−3 LCPUFA, and lower levels of MUFA and SFA compared with those fed on the control diet, independent of the treatment the chicks had been exposed to in ovo (Figs 4 and 5). The percentage of SFA in the tissues ranged from ~28 to ~38% across the different treatments, and was higher in progeny fed the Control diet compared with those fed the High-ALA diet (P<0.0001). Tissue levels of n−9 and n−7 MUFA were lower in broilers fed the High-ALA diet post-hatch compared with broilers fed a control diet, independent of in ovo diet exposure (P<0.0001). The n−6 PUFA content of the meat was not different between broilers fed the Control and High-ALA diets. The n−6:n−3 ratio therefore reflected the variation in n−3 PUFA content, and was lower in broilers fed the High-ALA diet (0.8–1.4) compared with those fed the Control diet (4.7–6.4) (Table 2).
Fig. 4 Fatty acid profile in breast meat of 42-day-old broilers received three maternal by two progeny diets (n=3±s.e.m.).
Fig. 5 Omega-3 distribution in meat tissues of 42–day-old broilers received three maternal by two progeny diets (n=3±s.e.m.).
Table 2 P values at different levels of interaction of three experimental factors (maternal by progeny diets by gender) on 6-week-old broilers (n=3/group)
aG, gender. bM, maternal diets (birds received 3 different diets). cP, progeny (birds received two different progeny diets). dSFA, saturated fatty acid. en−9 MUFA, omega-9 monounsaturated fatty acid. fn−7 MUFA, omega-7 monounsaturated fatty acid. gn−6 PUFA, omega-6 polyunsaturated fatty acid. hn−3 PUFA, omega-3 polyunsaturated fatty acid. iALA, α-linolenic acid. jn−3 LCPUFA=omega-3 long-chain polyunsaturated fatty acid. kns, not significant (P>0.05).
Broilers that were exposed to the Fish-Oil treatment in ovo had higher ALA levels in the breast meat compared with those exposed in ovo to the Control and High-ALA treatments, independent of the diet fed post-hatch, however, the magnitude of this difference was small (Table 2 and Fig. 5, P<0.05). This was accompanied by a reduction in the EPA content of the meat from these broilers. There were no other differences in fatty acid composition of either the breast or leg meat between in ovo treatment groups (Table 2 and Fig. 5). There was, however, an interaction between in ovo dietary exposure and post-hatch diet on tissue n−3 PUFA concentrations (Table 2, P<0.05), such progeny exposed in ovo to the Fish-Oil diet that were fed the High-ALA diet post-hatch had significantly higher levels of ALA in breast meat compared with other two maternal treatments fed this same diet (Fig. 5).
The fatty acid profiles of the leg meat tissue of progeny at 42 days of age were largely consistent with the breast meat with the exception that males had higher level of n−3 PUFA in their leg meat compared with females (Table 2, P<0.01) and maternal High-ALA diet reduced ALA content only in breast meat (Table 2 and Fig. 5, P<0.05).
Discussion
The reduced number of chicks in the Fish-Oil group appeared to be a result of reduced laying rate in the breeder hens fed the Fish-Oil diet, whereas the proportion of eggs that were laid that produced live chicks did not appear to be adversely affected. It is important to note, however, that the study was not powered to investigate differences in production characteristics between treatments and thus further studies are required to confirm if there are any adverse effects of the Fish-Oil diet on laying performance.
Although our study was not specifically designed to investigate differences in growth in the progeny, there are no suggestions of adverse effects of any of the diets on the productivity of the chicks. The broilers from all treatments grew at a normal rate, and BW, FCR and mortality in the progeny across the different treatments were within the normal standards for this strain 29 and in agreement with our previous findings.Reference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9
Our finding that feeding breeder hens a High-ALA diet increased not only the ALA content, but also the n−3 LCPUFA content of the eggs, supports the capacity of chickens for ALA conversion to n−3 LCPUFA, and confirms that ALA-supplementation of feed is an effective strategy for increasing the n−3 LCPUFA content of eggs.Reference Goldberg, Ryland, Aliani and House 20 , Reference Cherian and Sim 22 , Reference Cherian 30 – Reference Cherian, Bautista-Ortega and Goeger 32 This is likely to be due to the combined effects of the higher amount of ALA, and lower amount of n−6 PUFA in the feed, as the n−3 and n−6 PUFA compete with each other for both metabolic conversion and accumulation into tissues.Reference Qi, Chen, Zhao, Zheng and Wen 33
Interestingly, although supplementing the breeder hens directly with n−3 LCPUFA, in the form of fish oil, led to the greatest increase in n−3 LCPUFA (five-fold of the control eggs), the n−3 LCPUFA content of the eggs (8%), was still only half of that of the feed (16.1%). This observation suggests there is a maximum level to which n−3 LCPUFA can be incorporated into egg yolk, which may be due to structural limitation of triglycerides and phospholipids. In contrast, the n−3 LCPUFA percentage was relatively increased in eggs of the other two treatments in comparison with the levels in the diets. This observation indicates the importance of optimizing the dietary content of n−3 LCPUFA in broiler feed, as the incorporation of these fatty acids into eggs appears to follow a curvilinear, rather than linear, pattern and reach a plateau at high n−3 LCPUFA intakes.
Previous studies have established that >80% of lipids deposited in the egg yolk are consumed by the developing embryo before hatch, and therefore represent a major source of nutrition for supporting chick growth and development.Reference Noble and Cocchi 34 In addition, ~50% of egg total fatty acids are incorporated into the newly hatched chick with embryonic preference to incorporate PUFA at the expense of MUFA.Reference Lin, Connor and Anderson 35 Consistent with the findings of the current study, previous studiesReference Cherian and Sim 22 , Reference Cherian, Bautista-Ortega and Goeger 32 have demonstrated that the relationship between fatty acid content of the egg yolk and post-hatch chick were closest for the essential fatty acids (n−3 and n−6 PUFA), compared with MUFA and SFA. Our finding that the n−3 and n−6 PUFA content in all groups were relatively higher in the meat of the newly hatched chicks than in the eggs provides evidence of continuous synthesis and accumulation of both of PUFA types in the muscle tissue in ovo. Hence, the ratio of n−6:n−3 PUFA in the meat tissues of chicks (especially in the high n−3 PUFA treatments) did not shift as much as the levels of the individual PUFA. This was important for the second stage of the experiment, since it ensured that the capacity of the chicks for converting ALA to the n−3 LCPUFA was unlikely to be limited by the presence of excessive amounts of n−6 PUFA in the tissues.
Interestingly, the contribution of n−3 LCPUFA to the total n−3 PUFA pool in day-old chicks exposed to the High-ALA diet in ovo was ~37% greater than in eggs from High-ALA hens, suggesting that these chicks had the capacity for ALA conversion to LCPUFA during embryogenesis. As with the eggs, however, the ability of the chicks to accumulate n−3 LCPUFA appeared to be limited at higher concentrations, as the n−3 LCPUFA content of meat tissues in chicks exposed to the Fish-Oil diet in ovo was about half that in the yolk. Lin et al.Reference Lin, Connor and Anderson 35 found a linear relationship in n−3 and n−6 PUFA levels between eggs and chicks. However, these authors also suggested that the synthesizing of n−3 LCPUFA from ALA was suppressed at higher levels of dietary n−3 LCPUFA.
The growth of the 42-day-old broilers (data not shown) in the current study agreed with our previous studyReference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9 and was not affected by dietary fat exposure either in ovo or post-hatch.Reference Ajuyah, Cherian, Wang, Sunwoo and Sim 24 , Reference Koppenol, Delezie, Buyse and Everaert 36 At 6 weeks of age, the fatty acid profile of broilers was found to be mainly affected by the post-hatch diet and sex, with minimal influences of dietary exposure in ovo. As the post-hatch control diet contained more SFA, trans, n−9 and n−7 MUFA, these fatty acids were predominant in broilers fed this diet. On the other hand, n−3 and n−6 PUFA were the predominant fatty acid groups in broilers fed High-ALA diet, consistent with our previous findings.Reference Kanakri, Carragher, Hughes, Muhlhausler and Gibson 9 Although the High-ALA diet was relatively higher than the Control diet in n−6 PUFA content, this did not affect the n−6 PUFA level in broiler meat. This is probably related to the preferential utilization of n−3 PUFA substrates (ALA) by the enzymes involved in the metabolic conversion of shorter-chain fatty acids to their long-chain derivatives and preferential incorporation of n−3 PUFA into tissues.Reference Kartikasari, Hughes, Geier, Makrides and Gibson 10 , Reference Jing, Gakhar, Gibson and House 15
The major finding of this study was that exposure to either a high n−3 PUFA (ALA) or n−3 LCPUFA (Fish-Oil) diet in ovo had very little impact on the capacity of the progeny for converting ALA to the n−3 LCPUFA. Indeed, the only effect observed was an apparently inhibitory effect of in ovo exposure to maternal fish oil supplementation on ALA conversion to n−3 LCPUFA. One possibility is that in ovo exposure to a high dietary n−3 LCPUFA content may have acted to suppress the expression and/or activity of genes involved n−3 PUFA metabolism, specifically the desaturase and elongase enzymes required for the conversion of ALA to n−3 LCPUFA. This is supported by previous studies showing that DHA supplementation suppresses endogenous synthesis of n−3 LCPUFA from ALA in human subjectsReference Emken, Adlof, Duval and Nelson 37 and that feeding chickens a diet enriched in fish oil-based diet resulted in an increase in the percentage of both of ALA and linolenic acid in the chicken meat, suggesting reduced conversion.Reference Lopez-Ferrer, Baucells, Barroeta and Grashorn 38 Similarly, in chicken, Ajuyah et al.Reference Ajuyah, Cherian, Wang, Sunwoo and Sim 24 reported no effect of a reserve of yolk n−3 LCPUFA from maternal Fish-Oil diet on the n−3 PUFA in the offspring cardiac tissue, but showed an adverse effect on the EPA percentage. In contrast, in ovo exposure to the High-ALA diet had no effect on the subsequent capacity of the chickens for ALA conversion. Haug et al.Reference Haug, Nyquist, Thomassen, Hostmark and Ostbye 39 reported that the concentration of dietary ALA does not affect the gene expression of the elongation and desaturation enzymes in adult chickens, and the results of the current study suggest that this may also be the case during the embryonic stage. Thus, exposing embryos to either High-ALA or High-n−3 LCPUFA environments do not enhance their subsequent capacity for depositing more n−3 LCPUFA after hatch.
Although there were relatively few differences between the male and female chickens in their response to the diet, we did identify that male broilers fed the High-ALA diet post-hatch accumulated more total n−3 PUFA in the leg meat, independent of their in ovo exposure. In addition, despite no differences in fatty acid profile of male and female chicks at 1-day post-hatch (Control and High-ALA groups), there were sex differences in the levels of all fatty acids (except n−6 PUFA) in the 42-day-old broilers. Thus, tissues from male chickens contained relatively more n−3 PUFA and lower n−3 LCPUFA, which indicates their lower ALA conversion efficiency, a finding is consistent with our previous study.Reference Carragher, Mühlhäusler and Geier 14
In summary, we have shown that exposing broiler chickens to elevated levels of ALA or n−3 LCPUFA in ovo was effective in increasing n−3 LCPUFA deposition into their meat tissues during embryonic development. However, neither of these strategies were effective at increasing the subsequent capacity of the chickens for accumulating n−3 LCPUFA in their meat tissues when fed a High-ALA diet post-hatch. In fact, increased in ovo n−3 LCPUFA exposure appeared to be associated with an impaired capability of the broilers to convert ALA to n−3 LPCUFA.
Conclusion
In ovo n−3 LCPUFA exposure appeared to be associated with an impaired capability of the broilers to convert ALA to n−3 LPCUFA. Manipulation of dietary fatty acids can affect yolk composition, but on its own it is not an appropriate strategy for enhancing n-3 LCPUFA content in the offspring at market age.
Acknowledgments
The authors would like to thank Derek Schultz, Mandy Bowling, Kylee Swanson, Nicole Heberle, Natasha Edwards, Saad Gilani, Dr Carolyn Dekoning and Dr Reza Barekatain from Roseworthy campus for their assistance in sampling the animals and Dr Liu Ge from Waite campus, University of Adelaide for his assistance in the laboratory work.
Financial Support
K.K. received Research Training Program (RTP) from The Australian Government Research Training Program Scholarship and full PhD scholarship from the Faculty of Sciences, University of Adelaide. The experimental work was financed by a grant from the South Australian Department of Further Education, Employment, Science and Technology (DFEEST), Australia. B.M. is supported by a Career Development Fellowship from the National Health and Medical Research Council of Australia (NHMRC). R.G. is supported by a NHMRC Senior Research Fellowship.
Conflicts of Interest
None.
Ethical Standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national guides on the care and use of animals. This study was approved by the Animal Ethics Committee of the University of Adelaide (approval S-2013-152) and the Department of Primary Industries and Regions South Australia, Australia (approval 15/13).
