Chickens are animals that are sensitive to thermal stress, which may decrease their production level in terms that it affects feed intake and thus, decreasing body weight gain. The Heat Shock Factors (HSF) and Heat Shock Proteins (HSP) genes are involved in the key cellular defense mechanisms during exposure in hot environments. Aimed with this study to analyze the expression of HSF1, HSF3, HSP70 and HSP90 genes in two local breeds (Peloco and Caneluda) and a commercial broiler line (Cobb 500^®) to verify differences in resistance of these chicken to Heat stress treatment. Chicken were submitted to heat stress under an average temperature of 39°C ± 1. Under stress environment, the HSP70 and HSP90 genes were more expressed in backyard chickens than in broiler. There was a difference in HSP70 and HSP90 expression between Caneluda and Cobb and between Peloco and Cobb under stress and comfort environment respectively. HSP70 expression is higher in local breeds during heat stress than in a commercial broiler line. No significant differences were observed in the expression of HSF1 and HSF3 genes between breeds or environments. HSP70 and HSP90 genes are highly expressed, HSF1 and HSF3 genes did not have high expression in all genetic groups. HSP70 and HSP90 are highly expressed in Peloco and Caneluda within heat stress, these breeds proved to be very resistant to high temperature.

Quantitative RT-PCR is an important technique for assessing gene expression. However, a proper normalization of reference genes prior to expression analyses of target genes is necessary. The best normalizer is that gene which remains stable in all samples from different treatments. The aim of this study was to identify stable reference genes for normalization of target genes in muscle tissue from three genetically divergent chickens groups (Peloco, Cobb 500^® and Caneluda) under environmental (heat stress and comfort) and sex influence. Expressions of ten reference genes were tested for stability in breast muscular tissue (Pectoralis major muscle). Samples were obtained from 36 males and females of two backyard breeds (Caneluda and Peloco) and one commercial line (Cobb 500^®) under two environments. The heat stress and comfort temperature were 39 and 23°C, respectively. Animals were housed in the Animal Science Department at Universidade Estadual do Sudoeste da Bahia. We analyzed the expression data by four statistical tools (SLqPCR, NormFinder, Bestkeeper and Comparative CT). According to these tools, genes stability varied according to sex, genetic group and environment, however, some genes remained stable in all analyzes. There was no difference between the most stable genes for sex effect, being MRPS27 more stable for both males and females. In general, MRPS27 was the most stable gene. Within the three genetic groups, the most stable genes were RPL5, HMBS and EEF1 to Cobb 500^®, Peloco and Caneluda, respectively. Within the environment, the most stable gene under comfort and heat stress conditions was HMBS and MRPS27, respectively. BestKeeper and Comparative Ct were less correlated (28%) and SLqPCR and NormFinder were the most correlated (98%). MRPS27, RPL5 and MRPS30 genes were considered stable according the overall ranking and can be used as normalizer of relative expression of target genes in muscle tissue of chickens under heat stress.

Background: Five hundred 8-d old male broilers Cobb500 were randomly allotted into 10 treatments in factorial arrangement with 5 Cu levels (0, 4, 8, 12, and 16 mg/kg), and 2 sources (Cu proteinate, CuPro and Cu sulphate, CuSO4.5H2O) for a 10-d-experiment. Results: Feed conversion ratio (FCR) was better (P < 0.05) in CuPro fed chicks compared with CuSO4.5H2O group. Average daily feed intake (ADFI) decreased linearly (P < 0.05) as dietary Cu increased. A quadratic response (P < 0.05) to Cu levels was found for FCR, being optimized at 9.87 and 8.84 mg Cu/kg in CuPro and CuSO4.5H2O diets, respectively. Copper supplementation linearly increased liver Cu content (P < 0.05) and tended to linearly increase (P = 0.07) phosphorus (P) and copper in tibia. Manganese and zinc were higher (P < 0.05) in tibia of CuPro fed birds. Broilers fed CuPro exhibited lower liver iron (P < 0.05) content, lower activities of Cu, Zn superoxide dismutase (CuZnSOD) in breast muscle and liver, and glutathione peroxidase in liver. Glutathione peroxidase reduced linearly (P < 0.05) with CuPro levels and increased linearly (P < 0.05) with CuSO4.5H2O levels and were lower (P < 0.05) in all CuPro levels in breast muscle. Breast muscle malondialdehyde concentration tended to be higher (P = 0.08) in broilers fed CuSO4.5H2O. Copper levels linearly increased (P < 0.05) metallothionein (MT) and malate dehydrogenase (MDH) expression in liver, and six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) in the intestine. Copper elicited a quadratic response (P < 0.050) in AKT-1 and mammalian target of rapamycin (mTOR) in breast muscle, CuZnSOD in liver and antioxidant 1 copper chaperone (ATOX 1) in intestine. Broilers fed CuPro exhibited higher mRNA expression of mTOR in muscle breast and lower CuZnSOD in liver and ATOX 1 in intestine. Interaction (P < 0.05) between levels and sources was found in mRNA expression for GSK-3β, MT, and CuZnSOD in breast muscle, FAS and LPL in liver and MT and CTR1 in intestine. Conclusions: CuPro showed beneficial effects on feed conversion and bone mineralization. Organic and inorganic Cu requirements are 9.87 and 8.84 mg Cu/kg, respectively.

Sexual dimorphism is a relevant factor in animal science, since it can affect the gene expression of economically important traits. Eventually, the interest in the prenatal phase in a transcriptome study may not comprise the period of development in which male and female conceptuses are phenotypically divergent. Therefore, it would be interesting if sex differentiation could be performed using transcriptome data, with no need for extra techniques. In this study, the sex of pig conceptuses (embryos at 25 days-old and fetuses at 35 days-old) was determined by reads counts per million (CPM) of Y chromosome-linked genes that were discrepant among samples. Thus, ten genes were used: DDX3Y, KDM5D, ZFY, EIF2S3Y, EIF1AY, LOC110255320, LOC110257894, LOC396706, LOC100625207, and LOC110255257. Conceptuses that presented reads CPM sum for these genes (ΣCPM_chrY) greater than 400 were classified as males and those with ΣCPM_chrY below 2 were classified as females. It was demonstrated that the sex identification can be performed at early stages of pig development from RNA-sequencing analysis of genes mapped on Y chromosome. Additionally, these results reinforce that sex determination is a mechanism conserved across mammals, highlighting the importance of using pigs as an animal model to study sex determination during human prenatal development.

Backfat is an important trait in pork production, and it has been included in the breeding objectives of genetic companies for decades. Although adipose tissue is a good energy storage, excessive fat results in reduced efficiency and economical losses. A large QTL for backfat thickness on chromosome 5 is still segregating in different commercial pig breeds. We fine mapped this QTL region using a genome-wide association analysis (GWAS) with 133,358 genotyped animals from five commercial populations (Landrace, Pietrain, Large White, Synthetic, and Duroc) imputed to the porcine 660K SNP chip. The lead SNP was located at 5:66103958 (G/A) within the third intron of theCCND2gene, with the G allele associated with more backfat, while the A allele is associated with less backfat. We further phased the QTL region to discover a core haplotype of five SNPs associated with low backfat across three breeds. Linkage disequilibrium analysis using whole-genome sequence data revealed three candidate causal variants within intronic regions and downstream of theCCND2gene, including the lead SNP. We evaluated the association of the lead SNP with the expression of the genes in the QTL region (includingCCND2) in a large cohort of 100 crossbred samples, sequenced in four different tissues (lung, spleen, liver, muscle). Results show that the A allele increases the expression ofCCND2in an additive way in three out of four tissues. Our findings indicate that the causal variant for this QTL region is a regulatory variant within the third intron of theCCND2gene affecting the expression ofCCND2.

Hernia is one of the most common defects in pigs. The most prevalent are the scrotal (SH), inguinal (IH) and umbilical (UH) hernias. We compared the inguinal ring transcriptome of normal and SH-affected pigs with the umbilical ring transcriptome of normal and UH-affected pigs to discover genes and pathways involved with the development of both types of hernia. A total of 13,307 transcripts was expressed in the inguinal and 13,302 in the umbilical ring tissues with 94.91% of them present in both tissues. From those, 35 genes were differentially expressed in both groups, participating in 108 biological processes. A total of 67 polymorphisms was identified in the inguinal ring and 76 in the umbilical ring tissue, of which 11 and 14 were novel, respectively. A single nucleotide polymorphism (SNP) with deleterious function was identified in the integrin α M (ITGAM) gene. The microtubule associated protein 1 light chain 3 γ (MAP1LC3C), vitrin (VIT), aggrecan (ACAN), alkaline ceramidase 2 (ACER2), potassium calcium-activated channel subfamily M α 1 (KCNMA1) and synaptopodin 2 (SYNPO2) genes are highlighted as candidates to trigger both types of hernia. We generated the first comparative study of the pig umbilical and inguinal ring transcriptomes, contributing to the understanding of the genetic mechanism involved with these two types of hernia in pigs and probably in other mammals.