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.

Metformin improves ovarian function and increases egg production in broiler breeder hens.

In brief: The pathophysiology of the ovarian dysfunction encountered in broiler breeder hens remains poorly understood but is similar to a condition in women known as polycystic ovary syndrome. This study reveals that metformin may provide a cheap and effective method of improving ovarian function in broiler breeder hens. Abstract: Broiler breeder hens, the parent stock of commercial broiler chickens, have poor reproductive efficiency associated with aberrant and excessive recruitment of ovarian follicles which results in sub-optimal egg production, fertility, and hatchability. The reproductive dysfunction observed in these hens resembles polycystic ovary syndrome in women, a condition wherein metformin is prescribed as a treatment. The main objectives of this study were to determine the effect of metformin on body weight, abdominal fat pad weight, ovarian function, and plasma steroid hormone concentrations. Broiler breeder hens were treated with 0, 25, 50, or 75 mg/kg body weight of metformin mixed in the diet for 40 weeks (n = 45 hens/treatment; 2565 weeks of age). At 65 weeks of age, hens that received the highest dose of metformin had significantly lower body and abdominal fat pad weights (P < 0.05) than the control. Metformin treatment, at all levels, normalized the preovulatory and prehierarchical ovarian follicular hierarchy. Metformin (50 or 75 mg/kg body weight) significantly increased the total number of eggs laid per hen during the entire production period and these hens had significantly greater fertility and hatchability at 65 weeks of age compared to the control (P < 0.05). Metformin treatment at all levels altered the plasma profile of reproductive hormones, with significantly lower plasma testosterone concentrations and a decreased testosterone to androstenedione ratio in hens that received metformin (P < 0.05). Future studies should focus on the mechanisms underlying the beneficial effects of metformin in improving the reproductive efficiency of broiler breeder hens.

Metformin modulates the gut microbiome in broiler breeder hens.

Broiler breeder hens, the parent stock of commercial broiler chickens, are genetically selected for rapid growth. Due to a longer production period and the focus of genetic selection on superior carcass traits in their progeny, these hens have the propensity to gain excess adipose tissue and exhibit severe ovarian dysfunction, a phenotype that is similar to human polycystic ovary syndrome (PCOS). Metformin is an antihyperglycemic drug approved for type 2 diabetes that is prescribed off-label for PCOS with benefits on metabolic and reproductive health. An additional effect of metformin treatments in humans is modulation of gut microbiome composition, hypothesized to benefit glucose sensitivity and systemic inflammation. The effects of dietary metformin supplementation in broiler breeder hens have not been investigated, thus we hypothesized that dietary metformin supplementation would alter the gut microbiome of broiler breeder hens. Broiler breeder hens were supplemented with metformin at four different levels (0, 25, 50, and 75 mg/kg body weight) from 25 to 65 weeks of age, and a subset of hens (n = 8-10 per treatment group) was randomly selected to undergo longitudinal microbiome profiling with 16S rRNA sequencing. Metformin impacted the microbial community composition in 75 mg/kg metformin compared to controls (adjusted PERMANOVA p = 0.0006) and an additional dose-dependent difference was observed between 25 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) and between 50 mg/kg and 75 mg/kg (adjusted PERMANOVA p = 0.001) but not between 25 mg/kg and 50 mg/kg (adjusted PERMANOVA p = 0.863). There were few differences in the microbiome attributed to hen age, and metformin supplementation did not alter alpha diversity. Bacteria that were identified as differentially relatively abundant between 75 mg/kg metformin treatment and the control, and between metformin doses, included Ruminococcus and members of the Clostridia family that have been previously identified in human trials of PCOS. These results demonstrate that metformin impacts the microbiome of broiler breeder hens in a dose-dependent manner and several findings were consistent with PCOS in humans and with metformin treatment in type 2 diabetes. Metformin supplementation is a potentially promising option to improve gut health and reproductive efficiency in broiler breeder hens.

Metformin attenuates steroidogenesis in ovarian follicles of the broiler breeder hen.

The follicular hierarchy in broiler breeder chicken ovary is often deranged due to excessive ovarian follicular recruitment, resulting in a condition that resembles polycystic ovary syndrome (PCOS) in women. Metformin is widely prescribed to correct PCOS and has been shown to affect granulosa cell functions in humans and rodent models. The objectives of this study are to determine the effects of metformin on signal transduction pathways, gene expression related to steroidogenesis, and progesterone secretion from granulosa cells isolated from the most recently recruited preovulatory and prehierarchical follicles of broiler breeder chickens. Granulosa cells were treated with 0, 1, 10, or 20 mM of metformin in the presence of FSH. The abundance of pAMPK, pACC, pERK, and pAkt was determined by Western blotting. The expression of genes related to progesterone biosynthesis was quantified by qPCR. Progesterone concentrations in culture media were quantified by ELISA. Metformin treatment did not have an effect on the abundance of pAMPK and pACC in prehierarchical follicles but significantly decreased the abundance of pERK and pAkt in a dose-dependent manner in preovulatory and prehierarchical follicles. The expression of genes related to steroidogenesis such as FSHR, STAR, CYP11A1, HSD3B, and progesterone secretion was significantly decreased in response to metformin treatment in a dose-dependent manner. Our data suggest that metformin treatment attenuates progesterone secretion via AMPK-independent pathways in granulosa cells of prehierarchical and preovulatory follicles of broiler breeder hens. Further studies are required to determine if metformin administration could ameliorate ovarian dysfunction in obese broiler breeder hens.

Short Chain Fatty Acids Enhance Aryl Hydrocarbon (Ah) Responsiveness in Mouse Colonocytes and Caco-2 Human Colon Cancer Cells.

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.

Editor's Highlight: Microbial-Derived 1,4-Dihydroxy-2-naphthoic Acid and Related Compounds as Aryl Hydrocarbon Receptor Agonists/Antagonists: Structure-Activity Relationships and Receptor Modeling.

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.