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The aim of this study was to determine whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and influence the out-of-season sexual activity of adult rams. In experiment 1, 24 Rasa Aragonesa ram-lambs born in September were genotyped for their RsaI and MnlI allelic variants of the MTNR1A gene, and the date of their first mounting with ejaculation after a period of semen collection training was documented. In experiment 2, the reproductive behavior, testicle size, and plasma testosterone concentrations of 18 adult rams (6 rams for each RsaI genotype) were recorded at the beginning (March) and end (May) of the seasonal anestrus. The number of days of training to achieve the first mating with ejaculation in T/T (C/C: 85.17 ± 12.08 C/T: 86.60 ± 18.87; T/T; 26.50 ± 24.50 d; P < 0.05), and G/G ram-lambs (G/G: 51.57 ± 14.99; A/G: 95.58 ± 10.95 d; P < 0.05) was significantly fewer than it was in the other genotypes. Likewise, for the RsaI genotype, 55% of the vulva-sniffing (P < 0.001), 48% of the approaches (P < 0.01), 48% of the mountings (P < 0.05) and 49% total activities (P < 0.001) were performed by T/T rams in March, and 50% of the sexual events in May (P < 0.001). For the Mnll variant, G/G rams performed a significantly (P < 0.001) larger proportion of the vulva-sniffing (41%), approaches (46%) and total activities (40%) in March, and 52% of the vulva-sniffing (P < 0.001), 43%, of the approaches (P < 0.001), 46% of the mountings (P < 0.05), and 47% of the total activities (P < 0.001) in May. Scrotal circumference, testicular volume, and plasma testosterone concentrations did not differ significantly among genotypes. Results confirmed that the polymorphisms of the MTNR1A gene sequence can influence reproductive performance in young and adult rams. Autumn-born ram-lambs that carried the T/T or G/G genotype had an advanced ability to reproduce, and T/T or G/G adult rams exhibited the most intense reproductive behavior. Genotyping might be a useful procedure for identifying the correct and rational use of rams in modern sheep farming. • The melatonin receptor 1A (MTNR1A) gene exhibits several polymorphic sites in sheep. • This gene has two allelic variants (RsaI and MnlI), associated with seasonal activity in the ewe. • Autumn-born ram-lambs that carried the T/T or G/G genotype had an earlier ability to reproduce. • T/T or G/G adult rams exhibited the a most intense reproductive behavior in spring. • Genotyping might be a useful procedure for identifying the correct and rational use of rams. [ABSTRACT FROM AUTHOR]

Contents The effect of melatonin implants administered during non-breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer-assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona-pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen-thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non-breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non-breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained. [ABSTRACT FROM AUTHOR]

• P4 and E2 induce changes in P-Tyr location in ram sperm. • P4, but not E2, increments ROS and Ca2+ levels in ram sperm. • P4 and E2 did not induce hyperactivation in ram sperm. • Neither P4 nor E2 seem to modulate AR in ram sperm by the cAMP-PKA pathway. Steroid hormones progesterone (P4) and 17β-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 μM in the media induced an increase in Ca2+ i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H 2 DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification. [ABSTRACT FROM AUTHOR]