A review of recent studies on the enrichment of eggs and poultry meat with omega-3 polyunsaturated fatty acids: novel findings and unanswered questions.

Studies from our laboratory over the past decade have yielded new information with regard to the dietary enrichment of eggs and poultry meat with omega-3 (n-3) polyunsaturated fatty acids (PUFA) but have also generated a number of unanswered questions. In this review, we summarize the novel findings from this work, identify knowledge gaps, and offer possible explanations for some perplexing observations. Specifically discussed are: 1) Why feeding laying hens and broilers an oil rich in stearidonic acid (SDA; 18:4 n-3), which theoretically bypasses the putative rate-limiting step in the hepatic n-3 PUFA biosynthetic pathway, does not enrich egg yolks and tissues with very long-chain (VLC; ≥20 C) n-3 PUFA to the same degree as obtained by feeding birds oils rich in preformed VLC n-3 PUFA; 2) Why in hens fed an SDA-rich oil, SDA fails to accumulate in egg yolk but is readily incorporated into adipose tissue; 3) How oils rich in oleic acid (OA; 18:1 n-9), when co-fed with various sources of n-3 PUFA, attenuates egg and tissue n-3 PUFA contents or rescues egg production when co-fed with a level of docosahexaenoic acid (DHA; 22:6 n-3) that causes severe hypotriglyceridemia; and 4) Why the efficiency of VLC n-3 PUFA deposition into eggs and poultry meat is inversely related to the dietary content of α-linolenic acid (ALA; 18:3 n-3), SDA, or DHA.

Comparison of Ahiflower oil containing stearidonic acid to a high-alpha-linolenic acid flaxseed oil at two dietary levels on omega-3 enrichment of egg yolk and tissues in laying hens.

Enrichment of egg yolks with very long chain omega-3 fatty acids (VLCn-3 FA) is of interest because of their beneficial effects on human health. The ability of Ahiflower® oil (AHI; Buglossoides arvensis), which is naturally rich in stearidonic acid (SDA), and a high-alpha-linolenic acid (ALA) flaxseed (FLAX) oil to enrich eggs and tissues of laying hens with VLCn-3 FA was investigated. Forty 54-week-old Hy-Line W-36 White Leghorn hens were fed a diet that contained soybean oil (control; CON) or AHI or FLAX oils at 7.5 or 22.5 g/kg of the diet in substitution for the soybean oil for 28 days. Dietary treatments had no effects on egg number or components or follicle development. Total VLCn-3 FA contents of egg yolk, liver, breast, thigh, and adipose tissue were greater in the n-3 treatments compared to CON, with the greatest increase observed at the higher oil level, especially for AHI oil which had the greater VLCn-3 enrichment than FLAX in yolk (p < 0.001). Efficiency of VLCn-3 enrichment of egg yolks was decreased with n-3 oils and by increasing oil level with lowest efficiency at 22.5 g/kg FLAX. In conclusion, both SDA-rich (AHI) and ALA-rich (FLAX) oils increased VLCn-3 FA deposition into egg yolks and hens' tissues, but dietary AHI oil promoted a greater enrichment than comparative amounts of FLAX oil, especially in liver and egg yolks.

Feeding laying hens docosa hexaenoic acid-rich microalgae oil at 40 g/kg diet causes hypotriglyceridemia, depresses egg production, and attenuates expression of key genes affecting hepatic triglyceride synthesis and secretion, but is rescued by dietary co-supplementation of high-oleic sunflower oil.

The primary goal of this study was to investigate the effect of feeding White Leghorn hens graded levels of a docosahexaenoic acid (DHA)-rich microalgae oil (MAO) on productive performance and enrichment of eggs with very long-chain (VLC) omega-3 (n-3) polyunsaturated fatty acids (PUFA). Forty-nine-week-old hens (8 per diet) were fed the following diets for 28 d: 1) A corn-soybean meal-based diet with no supplemental oil (CON); 2) CON + 10 g/kg MAO; 3) CON + 20 g/kg MAO; 4) CON + 30 g/kg MAO; 5) CON + 40 g/kg MAO; 6) CON + 40 g/kg MAO + 20 g/kg high-oleic sunflower oil (HOSO); and 7) CON + 40 g/kg MAO + 40 g/kg HOSO. Diets 6 and 7 were included because we previously reported that co-feeding high-oleic acid oils with n-3 PUFA-containing oils attenuated egg yolk n-3 PUFA contents vs. feeding hens the n-3 oils alone. All data were collected on an individual hen basis. Egg VLC n-3 PUFA enrichment plateaued, in terms of statistical significance, at the 30 g/kg MAO level (266 mg/yolk). Hens fed 40 g/kg MAO had greatly attenuated measures of hen performance, marked liver enlargement, an altered ovarian follicle hierarchy, greatly lowered circulating triglyceride levels, and depressed hepatic expression of key genes involved in triglyceride synthesis and secretion. As compared to hens fed 40 g/kg MAO alone, feeding hens 40 g/kg MAO co-supplemented with HOSO (Diets 6 and 7) restored egg production, ovarian morphology, and all other measures of hen productive performance to CON levels, elevated plasma triglyceride levels, prevented liver enlargement, and increased the hepatic expression of key genes involved in triglyceride synthesis and secretion. In conclusion, MAO can greatly enrich hens' eggs with VLC n-3 PUFA, but its recommended dietary inclusion should not exceed 20 g/kg. This would allow for near-maximal yolk VLC n-3 PUFA enrichment without impairing hen productive performance, altering the ovarian follicle hierarchy or, based on the work of others, presumably imparting off-flavors in the egg.

Comparison of Ahiflower oil containing stearidonic acid to a high-alpha-linolenic acid flaxseed oil at two levels on tissue omega-3 enrichment in broilers.

Enrichment of broiler meat with very long-chain omega-3 fatty acids (VLCn-3 FA) is of interest because of their beneficial effects on human health. The ability of Ahiflower® (AHI) oil (Buglossoides arvensis), which naturally contains stearidonic acid (SDA), and a high-alpha-linolenic acid (ALA) flaxseed (FLAX) oil to enrich VLCn-3 FA contents in broilers tissues was investigated. Fifty-five Cobb 500 chicks were fed from days 12 to 35 of life either a control (CON) diet that contained 27.9 g/kg soybean oil or AHI or FLAX oils, each individually at 7.5 or 22.5 g/kg of the diet in substitution for soybean oil (all on an as fed basis). Total VLCn-3 FA contents were greater in breast, thigh, liver, adipose tissue, and plasma of all n-3 treatments compared to CON, with the greatest increase observed at the highest level of AHI and FLAX oils (p < 0.001). AHI oil at 7.5 g/kg promoted the most efficient synthesis and deposition of VLCn-3 in broiler tissues measured as deposition of VLCn-3 FA in tissues relative to intake of n3 FA. In conclusion, both ALA and SDA oils increased VLCn-3 FA deposition in tissues, but there were diminishing returns when increasing dietary levels of the oils.

Supplemental dietary oils rich in oleic acid or linoleic acid attenuate egg yolk and tissue n-3 polyunsaturated fatty acid contents in laying hens co-fed oils enriched in either stearidonic acid or α-linolenic acid.

We previously reported that when laying hens were fed diets supplemented with oils enriched in α-linolenic acid (ALA) and oleic acid (OA), the deposition of n-3 PUFA in egg yolk was attenuated as compared to feeding hens a diet supplemented with the ALA-rich oil alone. The present work extends those findings to another n-3 PUFA-rich oil (stearidonic acid [SDA]-enriched soybean oil) and two other high-OA oils, suggesting that the effect is not plant oil-specific. Feeding hens a supplemental linoleic acid (LA)-rich oil plus an oil rich in either SDA or ALA also attenuated egg yolk ALA and SDA contents (Experiment 1), or egg yolk and liver ALA contents (Experiment 2), respectively, as compared to feeding the SDA- or ALA-rich oils alone. Future work should focus on the lack of neutrality of OA and LA in relation to n-3 PUFA nutrition.

Dietary High-Oleic Acid Soybean Oil Dose Dependently Attenuates Egg Yolk Content of n-3 Polyunsaturated Fatty Acids in Laying Hens Fed Supplemental Flaxseed Oil.

Chickens can hepatically synthesize eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) from α-linolenic acid (ALA; 18:3 n-3); however, the process is inefficient and competitively inhibited by dietary linoleic acid (LNA; 18:2 n-6). In the present study, the influence of dietary high-oleic acid (OLA; 18:1 n-9) soybean oil (HOSO) on egg and tissue deposition of ALA and n-3 polyunsaturated fatty acids (PUFA) synthesized from dietary ALA was investigated in laying hens fed a reduced-LNA base diet supplemented with high-ALA flaxseed oil (FLAX). We hypothesized that reducing the dietary level of LNA would promote greater hepatic conversion of ALA to very long-chain (VLC; >20C) n-3 PUFA, while supplemental dietary HOSO would simultaneously further enrich eggs with OLA without influencing egg n-3 PUFA contents. Nine 51-week-old hens each were fed 0, 10, 20, or 40 g HOSO/kg diet for 12 weeks. Within each group, supplemental dietary FLAX was increased every 3 weeks from 0 to 10 to 20 to 40 g/kg diet. Compared to controls, dietary FLAX maximally enriched the total n-3 and VLC n-3 PUFA contents in egg yolk by 9.4-fold and 2.2-fold, respectively, while feeding hens 40 g HOSO/kg diet maximally attenuated the yolk deposition of ALA, VLC n-3 PUFA, and total n-3 PUFA by 37, 15, and 32%, respectively. These results suggest that dietary OLA is not neutral with regard to the overall process by which dietary ALA is absorbed, metabolized, and deposited into egg yolk, either intact or in the form of longer-chain/more unsaturated n-3 PUFA derivatives.

Feeding laying hens stearidonic acid-enriched soybean oil, as compared to flaxseed oil, more efficiently enriches eggs with very long-chain n-3 polyunsaturated fatty acids.

The desaturation of α-linolenic acid (ALA) to stearidonic acid (SDA) is considered to be rate-limiting for the hepatic conversion of ALA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in humans, rodents, and chickens. Thus, we hypothesized that feeding laying hens SDA, as a component of the oil derived from the genetic modification of the soybean, would bypass this inefficient metabolic step and result in the enrichment of eggs with EPA and DHA at amounts comparable to that achieved by direct supplementation of hens' diet with these very long-chain (VLC) n-3 polyunsaturated fatty acids (PUFAs). In a 28-d study, laying hens incorporated 0.132 mg, 0.041 mg, or 0.075 mg of VLC n-3 PUFAs into egg yolk for each milligram of ingested dietary ALA derived primarily from conventional soybean oil (CON), dietary ALA derived primarily from flaxseed oil (FLAX), or dietary SDA derived from SDA-enriched soybean oil, respectively. Moreover, the amounts of total yolk VLC n-3 PUFAs in eggs from hens fed the CON (51 mg), FLAX (91 mg), or SDA (125 mg) oils were markedly less than the 305 mg found in eggs from fish oil-fed hens. Unexpectedly, SDA appeared to be more readily incorporated into adipose tissue than into egg yolk. Since egg yolk FAs typically reflect the hens' dietary pattern, these tissue-specific differences suggest the existence of an alternate pathway for the hepatic secretion and transport of SDA in the laying hen.

Characterization of ascites-derived ovarian tumor cells from spontaneously occurring ovarian tumors of the chicken: evidence for E-cadherin upregulation.

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, ßA, ßB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-ß, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.

The restricted ovulator chicken: a unique animal model for investigating the etiology of ovarian cancer.

OBJECTIVE: The main goal of this study was to compare the incidence of ovarian cancer (OC) in 2 genetically different lines of hens--one that generally fails to lay eggs (the mutant "restricted ovulator" [RO] strain) and the other consisting of the wild-type (WT) siblings of the mutant RO hens. METHODS: Individual egg production data were obtained over a 972-day period for 31 RO hens and 33 WT hens. At 38 months of age, hens were killed, and their abdominal cavities were examined for any gross evidence of tumors. Samples of ovarian tissue were processed and assessed for histopathology and protein expression of ovalbumin. Plasma estradiol concentrations were also determined. RESULTS: Only 1 (3%) of the 31 RO hens was diagnosed with OC as compared with 9 (27%) of the 33 WT hens (P G 0.05). Wild-type hens laid more eggs than did RO hens during the 31-month collection period (average of 422 vs 28, respectively; P < 0.05). Although there was no difference in overall rate of ovulation between hens with and without OC, WT hens diagnosed with OC laid a greater percentage of their total number of eggs in the first year of production. Plasma estradiol concentrations were higher (P < 0.01) in RO versus WT hens. CONCLUSIONS: The results of this study strongly suggest that the number of ovulatory events is directly related to the incidence of OC in chickens. Clearly, other factors modify the risk of OC because there was no difference in ovulation rate between WT hens with and without OC. The mutant RO hen represents a valuable animal model for studying the etiology of OC.

Pituitary progesterone receptor expression and plasma gonadotrophin concentrations in the reproductively dysfunctional mutant restricted ovulator chicken.

Female mutant restricted ovulator (RO) chickens of the White Leghorn strain carry a naturally occurring single nucleotide mutation in the very low density lipoprotein receptor (VLDLR) gene. Due to this mutation, RO hens fail to express a functional VLDLR protein on the oocyte membrane, which results in an impaired uptake of circulating yolk precursor macromolecules. Mutant RO hens subsequently develop hyperlipidemia and generally fail to lay eggs due to follicular atresia. Since RO hens also reportedly have three-fold higher basal plasma estrogen concentrations, combined with four-fold lower levels of circulating progesterone as compared to wild-type (WT) hens, we hypothesized that RO hens would have an increased abundance of pituitary progesterone receptor (PR) mRNA and PR isoforms A and B as well as alterations in circulating gonadotrophin levels. Quantitative PCR assays revealed significantly greater (P

Effects of atorvastatin on lipid metabolism in normolipidemic and hereditary hyperlipidemic, non-laying hens.

As a result of a hereditable point mutation in the oocyte very low density lipoprotein (VLDL) receptor, sexually mature restricted ovulator (RO) female chickens (Gallus gallus), first described as a non-laying strain, exhibit endogenous hyperlipidemia and develop atherosclerotic lesions. In a 20-day study, RO hens and their normolipidemic (NL) siblings were fed either a control diet, or the control diet supplemented with 0.06% atorvastatin (AT), a potent 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) inhibitor. Compared to NL hens, RO birds exhibited greatly elevated baseline plasma total cholesterol (CHOL) and triglyceride (TG) concentrations (1.56 vs. 4.55 g/l and 30.7 vs. 138.4 g/l, respectively). AT attenuated plasma CHOL and TG concentrations by 60.3% and 70.1%, respectively, in NL hens and by 45.1% and 34.3%, respectively, in RO hens. Messenger RNA levels of several key genes involved in hepatic VLDL assembly were suppressed in RO vs. NL hens, but were unaffected by AT. In contrast, AT elevated liver HMGR mRNA levels in NL and RO birds, but only NL hens exhibited an AT-associated increase in hepatic HMGR immunoreactive protein levels. Down-regulation of HMGR gene expression due to higher baseline levels of circulating CHOL may explain why RO birds responded less robustly than NL hens to AT administration.

Assessment of egg nutrient compositional changes and residue in eggs, tissues, and excreta following oral administration of atorvastatin to laying hens.

Laying hens were fed a control diet alone or with 0.06 g of atorvastatin, a synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, per 100 g of diet for 20 days. Compared to controls, egg yolks from treated hens contained greater amounts of amino acids and reduced levels of total fatty acids and cholesterol. In contrast, egg albumen amino acid contents were unaffected by dietary treatments. In a residue study, seven hens each received a single oral dose of approximately 20 microCi of [(14)C]atorvastatin. Approximately 71% of the radioactivity was recovered in the excreta and liver, whereas virtually no radioactivity was detected in kidney, heart, muscle, bile, plasma, or egg albumen at 15 days postdosing. Yolk radioactivity peaked at 4 days postdosing in six of the seven birds and was absent in eggs laid after day 10. Reminiscent of that of certain antibiotic drugs, the atorvastatin egg residue pattern appeared to coincide with the physiological pattern of daily yolk accretion within the ovary.

Nutritional value of a highly digestible sorghum cultivar for meat-type chickens.

The nutritional value of a newly discovered sorghum mutant cultivar (P851171), with high in vitro protein digestibility, was compared to those of corn and two normal sorghums (P721N and 611Y) in two chick feeding trials. Although 8-20 day protein efficiency ratios and net protein ratios of all three sorghums were inferior to those of corn, P851171 and 611Y had markedly greater mean true amino acid digestibilities (TAAD) than either corn or P721N. In a subsequent 42-day experiment, all three sorghums supported weight gains equal to those of the corn-fed chicks. Feeding suboptimal levels of dietary protein resulted in reduced weight gains and no observed benefits of P851171 or 611Y. Furthermore, chicks fed P851171 exhibited poorer feed/gain values as compared to those fed the other cereals. It is possible that the starch content/carbohydrate profile of P851171 was inferior to that of the other sorghums, which offset its superior TAAD and resulted in poorer broiler performance.