Identification of protein-damaging mutations in 10 swine taste receptors and 191 appetite-reward genes.

BACKGROUND: Taste receptors (TASRs) are essential for the body's recognition of chemical compounds. In the tongue, TASRs sense the sweet and umami and the toxin-related bitter taste thus promoting a particular eating behaviour. Moreover, their relevance in other organs is now becoming evident. In the intestine, they regulate nutrient absorption and gut motility. Upon ligand binding, TASRs activate the appetite-reward circuitry to signal the nervous system and keep body homeostasis. With the aim to identify genetic variation in the swine TASRs and in the genes from the appetite and the reward pathways, we have sequenced the exons of 201 TASRs and appetite-reward genes from 304 pigs belonging to ten breeds, wild boars and to two phenotypically extreme groups from a F2 resource with data on growth and fat deposition. RESULTS: We identified 2,766 coding variants 395 of which were predicted to have a strong impact on protein sequence and function. 334 variants were present in only one breed and at predicted alternative allele frequency (pAAF) ≥ 0.1. The Asian pigs and the wild boars showed the largest proportion of breed specific variants. We also compared the pAAF of the two F2 groups and found that variants in TAS2R39 and CD36 display significant differences suggesting that these genes could influence growth and fat deposition. We developed a 128-variant genotyping assay and confirmed 57 of these variants. CONCLUSIONS: We have identified thousands of variants affecting TASRs as well as genes involved in the appetite and the reward mechanisms. Some of these genes have been already associated to taste preferences, appetite or behaviour in humans and mouse. We have also detected indications of a potential relationship of some of these genes with growth and fat deposition, which could have been caused by changes in taste preferences, appetite or reward and ultimately impact on food intake. A genotyping array with 57 variants in 31 of these genes is now available for genotyping and start elucidating the impact of genetic variation in these genes on pig biology and breeding.

Identification of genetic variation in the swine toll-like receptors and development of a porcine TLR genotyping array.

BACKGROUND: Toll-like receptors (TLR) are crucial in innate immunity for the recognition of a broad range of microbial pathogens and are expressed in multiple cell types. There are 10 TLR genes described in the pig genome. RESULTS: With a twofold objective i.e. to catalogue genetic variants in porcine TLR genes and develop a genotyping array for genetic association studies on immune-related traits, we combined targeted sub-genome enrichment and high-throughput sequencing to sequence the 10 porcine TLR genes in 266 pigs from 10 breeds and wild boars using a DNA-pooling strategy. We identified 306 single nucleotide variants across the 10 TLR and 11 populations, 87 of which were novel. One hundred and forty-seven positions i.e. six stop-gains and 141 non-synonymous substitutions were predicted to alter the protein sequence. Three positions were unique to a single breed with alternative allele frequencies equal to or higher than 0.5. We designed a genotyping array for future applications in genetic association studies, with a selection of 126 variants based on their predicted impact on protein sequence. Since TLR4, TLR7 and TLR9 were underrepresented in this selection, we also included three variants that were located in the 3'UTR of these genes. We tested the array by genotyping 214 of the 266 sequenced pigs. We found that 93 variants that involved the 10 TLR genes were polymorphic in these animals. Twelve of these variants were novel. Furthermore, seven known variants that are associated with immune-related phenotypes are present on the array and can thus be used to test such associations in additional populations. CONCLUSIONS: We identified genetic variations that potentially have an impact on the protein sequence of porcine TLR. A genotyping array with 80 non-synonymous, 10 synonymous and three 3'UTR polymorphisms in the 10 TLR genes is now available for association studies in swine populations with measures on immune-related traits.

Regional differences in recombination hotspots between two chicken populations.

BACKGROUND: Although several genetic linkage maps of the chicken genome have been published, the resolution of these maps is limited and does not allow the precise identification of recombination hotspots. The availability of more than 3.2 million SNPs in the chicken genome and the recent advances in high throughput genotyping techniques enabled us to increase marker density for the construction of a high-resolution linkage map of the chicken genome. This high-resolution linkage map allowed us to study recombination hotspots across the genome between two chicken populations: a purebred broiler line and a broiler x broiler cross. In total, 1,619 animals from the two different broiler populations were genotyped with 17,790 SNPs. RESULTS: The resulting linkage map comprises 13,340 SNPs. Although 360 polymorphic SNPs that had not been assigned to a known chromosome on chicken genome build WASHUC2 were included in this study, no new linkage groups were found. The resulting linkage map is composed of 31 linkage groups, with a total length of 3,054 cM for the sex-average map of the combined population. The sex-average linkage map of the purebred broiler line is 686 cM smaller than the linkage map of the broiler x broiler cross. CONCLUSIONS: In this study, we present a linkage map of the chicken genome at a substantially higher resolution than previously published linkage maps. Regional differences in recombination hotspots between the two mapping populations were observed in several chromosomes near the telomere of the p arm; the sex-specific analysis revealed that these regional differences were mainly caused by female-specific recombination hotspots in the broiler x broiler cross.

Single-Step Methodology for Genomic Evaluation in Turkeys (Meleagris gallopavo).

Genomic information can contribute significantly to the increase in accuracy of genetic predictions compared to using pedigree relationships alone. The main objective of this study was to compare the prediction ability of pedigree-based best linear unbiased prediction (PBLUP) and single-step genomic BLUP (ssGBLUP) models. Turkey records of feed conversion ratio, residual feed intake, body weight, breast meat yield, and walking ability were provided by Hybrid Turkeys, Kitchener, Canada. This data was analyzed using pedigree-based and single-step genomic models. The genomic relationship matrix was calculated either using observed allele frequencies, all allele frequencies equal to 0.5 or with a different scaling. To avoid potential problems with inversion, three different weighting factors were applied to combine the genomic and pedigree matrices. Across the studied traits, ssGBLUP had higher heritability estimates and significantly outperformed PBLUP in terms of accuracy. Walking ability was genetically negatively correlated to body weight and breast meat yield; however, it was not correlated to feed conversion ratio (FCR) or residual feed intake (RFI). Body weight showed a moderate positive genetic correlation to feed conversion ratio, residual feed intake and breast meat yield. Feed conversion ratio was strongly correlated to residual feed intake (0.68 ± 0.06). There was almost no genetic correlation between breast meat yield and feed efficiency traits. Larger differences in accuracy between PBLUP and ssGBLUP were observed for traits with lower heritability. Results of the three weighting factors showed only slight differences and an increase in accuracy of prediction compared to PBLUP. Slightly different levels of bias were observed across the models, but were higher among the traits; BMY was the only trait that had a regression coefficient higher than 1 (1.38 to 1.41). We show that incorporating genomic information increases the prediction accuracy for preselection of young candidate turkeys for the five traits investigated. Single-step genomic prediction showed substantially higher accuracy estimates than the pedigree-based model, and only slight differences in bias were observed across the alternate models.

Hypothalamic control of the thyroidal axis in the chicken: over the boundaries of the classical hormonal axes.

The pituitary gland, occupying a central position in the hypothalamo-pituitary thyroidal axis, produces thyrotropin (TSH), which is known to stimulate the thyroid gland to synthetize and release its products, thyroid hormones. TSH is produced by a specific cell population in the pituitary, the so-called thyrotropes. Their secretory activity is controlled by the hypothalamus, releasing both stimulatory and inhibitory factors that reach the pituitary through a portal system of blood vessels. Based on early experiments in mammals, thyrotropin-releasing hormone (TRH) is generally mentioned as the main stimulator of the thyrotropes. During the past few decades, it has become clear that the hypophysiotropic function of the hypothalamus is more complex, with different hormonal axes interacting with each other. In the chicken, it was found that not only TRH, but also corticotropin-releasing hormone (CRH), the main stimulator of corticotropin release, is a potent stimulator of TSH secretion. Somatostatin (SRIH), a hypothalamic factor known for its inhibitory effect on growth hormone secretion, was demonstrated to blunt the TSH response to TRH and CRH. In this review we summarize the latest studies concerning the "interaxial" hypothalamic control of TSH release in the chicken, with a special emphasis on the molecular components of these control mechanisms. It remains to be demonstrated if these findings could also be extrapolated to other species or classes of vertebrates.

Developmental endocrinology of the reproductive axis in the chicken embryo.

In mammals, the phenotype of the homogametic sex develops in the (relative) absence of steroids and the phenotype of the heterogametic sex is imposed by the early action of steroids. In contrast, the heterogametic sex in avian species is the female and the presence of estrogens and their receptors plays a crucial role in female sexual differentiation. The time- and sex-dependent expression of enzymes involved in steroidogenesis which determine the ratio of androgens/estrogens produced by the gonads has been extensively investigated during the last 5-6 years. These results all show that the lack of estrogen synthesis in the male appears to be due to the extremely low levels of 17beta-hydroxysteroid dehydrogenase and P450aromatase expression. In females, extensive expression of the aromatase gene (around day 5-6 of incubation), leading to estrogen synthesis, and specific expression of the estrogen receptor-mRNA in the left gonad results in the development of a functional left ovary. Other sex differences can be found in the expression of the inhibin subunit genes in gonads of chicken embryos and in circulating concentrations of inhibin, follicle stimulating hormone (FSH) and steroids. Sex reversal attempts have been made by varying incubation temperatures, by using anti-estrogens, androgens, aromatase inhibitors and synthetic steroids. In ovo administration of a sex steroid hormone or an inhibitor of endogenous sex steroid synthesis can cause phenotypical sex reversal. All these experiments show that the development of gonads in birds is very sensitive to changes in the embryonic hormonal environment, sometimes resulting in changes of postnatal reproduction and even growth.

Pre- and postprandial changes in plasma hormone and metabolite levels and hepatic deiodinase activities in meal-fed broiler chickens.

The present study aimed to study the effects of food deprivation and subsequent postprandial changes in plasma somatotrophic and thyrotrophic hormone levels and focused on the inter-relationships between these hormonal axes and representative metabolites of the intermediary metabolism of meal-fed broiler chickens. Male broiler chickens (2 weeks old) were fed a meal of 40-45 g/bird per d for two consecutive weeks (food-restricted (FR) treatment). The daily allowance was consumed in about 30 min. At 4 weeks of age, FR chickens were killed at several time intervals (ten per sampling time) in relation to the daily food allowance: before feeding (about 23.5 h of food deprivation), and at 10, 20, 30 (end of feeding), 40, 50, 60, 90, 120 and 200 min after initiation of feeding. Birds fed ad libitum served as controls (ad-libitum (AL) treatment). Liver tissue was collected for deiodinase type I and type III activity measurements and blood samples for analysis of growth hormone (GH), insulin-like growth factor (IGF)-I, thyroxine (T4), 3,3',5-triiodothyronine (T3), glucose, non-esterified fatty acids (NEFA), uric acid, triacylglycerol (TG) and lactate levels. Food deprivation caused a shift from lipogenesis to lipolysis and increased fatty acid turnover, a reduction in protein anabolism and reduced metabolic rate. Food intake was followed immediately by a pronounced increase in metabolic rate, initially mainly based on anaerobic mechanisms. Refeeding gradually reversed the fasting-induced alterations in plasma hormone and metabolite levels, but the time course of changes differed between metabolites, which clearly preceded those of the hormones investigated. The order of responsiveness after food provision were glucose>uric acid>or=NEFA>lactate>TG for the plasma metabolites and for hormones. Based on these different postprandial time courses, several functional relationships are proposed. Glucose is believed to be the primary trigger for the normalisation of the effects of fasting on these plasma variables by restoring hepatic GH receptor capacity, as well as decreasing deiodinase type III activity.

Cold-induced enhancement of avian uncoupling protein expression, heat production, and triiodothyronine concentrations in broiler chicks.

The relationships among avian uncoupling protein (avUCP) mRNA expression, heat production, and thyroid hormone metabolism were investigated in 7-14-day-old broiler chicks (Gallus gallus) exposed to a low temperature (cold-exposed chicks, CE) or a thermoneutral temperature (TN). After 7 days of exposure, CE chicks exhibited higher heat production (+83%, P<0.01), avUCP mRNA expression (+20%, P<0.01), and circulating triiodothyronine (T(3)) levels (+104%, P=0.07) for non-statistically different body weights and feed intake between 3 and 7 days of exposure as compared to TN chicks. Plasma thyroxine (T(4)) concentration was clearly decreased in CE chicks (-33%, P=0.06). The lower hepatic inner-ring deiodination activity (-47%) and the higher renal outer-ring deiodination activity (+75%) measured in CE compared to TN chicks could partly account for their higher plasma T(3) concentrations. This study describes for the first time the induction of avUCP mRNA expression by low temperature in chickens, as it has been previously shown in ducklings, and supports the possible involvement of avUCP in avian thermogenesis.

Effects of dietary macronutrient content on energy metabolism and uncoupling protein mRNA expression in broiler chickens.

The objective of the present study was to investigate the effects of dietary macronutrient ratio on energy metabolism and on skeletal muscle mRNA expression of avian uncoupling protein (UCP), thought to be implicated in thermogenesis in birds. Broiler chickens from 2 to 6 weeks of age received one of three isoenergetic diets containing different macronutrient ratios (low-lipid (LL) 30 v. 77 g lipid/kg; low-protein (LP) 125 v. 197 g crude protein (Nx6.25)/kg; low-carbohydrate (LC) 440 v. 520 g carbohydrate/kg). LP chickens were characterised by significantly lower body weights and food intakes compared with LL and LC chickens (-47 and -38 % respectively) but similar heat production/kg metabolic body weight, as measured by indirect calorimetry, in the three groups. However, heat production/g food ingested was higher in animals receiving the LP diet (+41 %, P<0.05). These chickens also deposited 57 % less energy as protein (P<0.05) and 33 % more as fat. No significant differences in energy and N balances were detected between LL and LC chickens. The diets with the higher fat contents (i.e. the LP and LC diets) induced slightly but significantly higher relative expressions of avian UCP mRNA in gastrocnemius muscle, measured by reverse transcription-polymerase chain reaction, than the LL diet (88 and 90 v. 78 % glyceraldehyde-3-phosphate dehydrogenase respectively, P<0.05). Our present results are consistent with the recent view that UCP homologues could be involved in the regulation of lipid utilisation as fuel substrate and provide evidence that the macronutrient content of the diet regulates energy metabolism and especially protein and fat deposition.

Thyroid status, but not insulin status, affects expression of avian uncoupling protein mRNA in chicken.

The aim of this study was to investigate the hormonal regulation of the avian homolog of mammalian uncoupling protein (avUCP) by studying the impact of thyroid hormones and insulin on avUCP mRNA expression in chickens (Gallus gallus). For 3 wk, chicks received either a standard diet (control group), or a standard diet supplemented with triiodothyronine (T(3); T3 group) or with the thyroid gland inhibitor methimazole (MMI group). A fourth group received injections of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th wk of age, all animals received two daily injections of either human insulin or saline solution. The results indicate a twofold overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a clear downregulation (-74%) in MMI chickens compared with control chickens. Insulin injections had no significant effect on avUCP mRNA expression in chickens. This study describes for the first time induction of avUCP mRNA expression by the thermogenic hormone T(3) in chickens and supports a possible involvement of avUCP in avian thermogenesis.