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High ambient temperature exhibits a retrograde effect on buffalo reproduction because of heat stress. Moreover, melatonin is known to regulate reproductive changes in seasonally reproductive animals by binding to high affinity, G protein-coupled receptors. The MTNR1A gene is a prime receptor, mediating the effect of melatonin at the neuroendocrine level to control seasonal reproduction. In sheep, the role of melatonin is well known; however, studies have not been conducted in buffalo to determine its effect during favorable and unfavorable breeding seasons. Therefore, the present study aimed to (1) determine the expression of MTNR1A, ERα, ERβ, and PR gene transcripts in the ovarian follicles of buffalo during the summer and winter seasons and (2) analyze melatonin, 17β-estradiol, and progesterone concentrations in the follicular fluid of buffalo during both seasons. Murrah buffalo ovaries were collected during both the summer (May-June) and winter (December-January) seasons. All visible ovarian follicles were allocated into one of three groups: (1) small (8-9.9 mm); (2) medium (10-11.9 mm); and (3) large (12-14 mm). Follicular fluid was aspirated from each group of follicles for hormone analyses. The granulosa cells were processed for RNA extraction. Furthermore, they were subjected to real-time quantitative PCR to analyze the expression (relative quantification) of MTNR1A, ERα, ERβ, and PR in each follicular group. The expression of MTNR1A gene transcript decreased with the increasing size of the follicle and intrafollicular melatonin concentration. Expression of ERα and PR remained unaffected by the season and was similar (P > 0.05) in all groups. Expression of ERβ was higher (P < 0.05) in summer than winter; nevertheless, small-sized follicles from the summer exhibited higher (P < 0.05) expressions than medium- and large-sized follicles. The overall intrafollicular melatonin concentration was positively correlated (P < 0.05) with 17β-estradiol and progesterone concentrations. In conclusion, the decreased expression of MTNR1A and increased concentration of intrafollicular melatonin with the increasing size of the follicle indicates a probable role in folliculogenesis and ovulation in buffalo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim of this study was to characterize and identify causative SNPs in the MTNR1A gene responsible for the reproductive seasonality traits in the Rasa aragonesa sheep breed. A total of 290 ewes (155, 84 and 51 mature, young and ewe lambs, respectively) from one flock were controlled from January to August. The following three reproductive seasonality traits were considered: the total days of anoestrus (TDA) and the progesterone cycling months (P4CM); both ovarian function seasonality traits based on blood progesterone levels; and the oestrus cycling months (OCM) based on oestrous detection, which indicate behavioural signs of oestrous. We have sequenced the total coding region plus 733 and 251 bp from the promoter and 3'-UTR regions, respectively, from the gene in 268 ewes. We found 9 and 4 SNPs associated with seasonality traits in the promoter (for TDA and P4CM) and exon 2 (for the three traits), respectively. The SNPs located in the gene promoter modify the putative binding sites for various trans-acting factors. In exon 2, two synonymous SNPs affect RFLP sites, rs406779174/RsaI (for the three traits) and rs430181568/MnlI (for OCM), and they have been related with seasonal reproductive activity in previous association studies with other breeds. SNP rs400830807, which is located in the 3'-UTR, was associated with the three traits, but this did not modify the putative target sites for ovine miRNAs according to in silico predictions. Finally, the SNP rs403212791 (NW&#95;014639035.1: g.15099004G > A), which is also associated with the three seasonality phenotypes, was the most significant SNP detected in this study and was a non-synonymous polymorphism, leading a change from an Arginine to a Cysteine (R336C). Haplotype analyses confirmed the association results and showed that the effects found for the seasonality traits were caused by the SNPs located in exon 2. We have demonstrated that the T allele in the SNP rs403212791 in the MNTR1A gene is associated with a lower TDA and higher P4CM and OCM values in the Rasa Aragonesa breed., (Copyright © 2018 Elsevier Inc. All rights reserved.)