Influences of melatonin treatment, melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) gene polymorphisms on first conception in Sarda ewe lambs.

In order to investigate if the melatonin receptor 1A (MTNR1A) and kisspeptin (KiSS-1) genes influence the reproductive response to melatonin treatment, 510 Sarda ewe lambs were divided into groups C (control) and M; Group M received one melatonin implant (18mg). After 35 days rams were introduced for 40 days and subsequent lambing dates and number of newborns were recorded. The MTNR1A gene Exon II and KiSS-1 gene Exon I were amplified and genotyped by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism analysis. Two single nucleotide polymorphisms (SNPs; C606T and G612A) in MTNR1A and one (G1035A) in KiSS-1 were found. The most frequent genotypes were G/G (63%) and C/C (53%) for MTNR1A and G/G (92%) for KiSS-1. Treated animals showed a higher lambing rate (P<0.05) and an advanced lambing date (P<0.05) compared with controls. The three SNPs did not influence the onset of reproductive activity. The majority of the G/G animals of Group M lambed before 190 days after ram introduction (P<0.05), while in Group C a higher number of G/G animals lambed after this date. Data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that the G/G genotype of the MTNR1A gene is able to influence the reproductive response to melatonin treatment.

Association between melatonin receptor 1A (MTNR1A) gene polymorphism and the reproductive performance of Mediterranean Italian buffaloes.

A melatonin receptor 1A (MTNR1A) gene polymorphism in adult buffaloes has been reported to affect reproductive seasonality. Consequently, the aim of the present study was to assess whether this polymorphism can affect age at first conception and the interval between first and second calving in Mediterranean Italian buffaloes. The allelic frequency of the C and T alleles was 0.44 and 0.56, respectively, whereas the genotypic frequency was 26% for C/C, 40% for C/T and 34% for T/T. The average age at first mating was approximately 20 months, whereas that at calving was approximately 32 months. The largest number of calvings of primiparous buffaloes was recorded between June and October. No associations between genotype, first mating and subsequent calving date were found. The duration from first to second calving was longer in buffaloes with the C/C genotype compared with those with the T/T and C/T genotypes (P<0.01). The period of calving for buffaloes with the C/C genotype was mainly from July to September, whereas that for buffaloes with the T/T genotype was largely from March to May. The MTNR1A gene had no effect on the age of first conception in Mediterranean Italian buffaloes. Rather, the association between the T/T genotype and reproductive activity during days with a long photoperiod indicates that this polymorphism may be considered a genetic marker to identify buffaloes that are able to reproduce out of the breeding season.

MTNR1A gene polymorphisms and reproductive recovery after seasonal anoestrus in different Mediterranean sheep breeds.

The aims of this study were 1) to investigate the effect of MTNR1A gene polymorphisms on reproductive performance in ewes of one Italian and two Slovenian dairy sheep breeds (Sarda, Istrian Premenka and Boska, respectively) which were located at different latitudes, and 2) to highlight if the different season of the male placement with females that was utilized in the different breeding systems in Sardinia (Italy) and Slovenia resulted in different effects of these polymorphisms on reproductive functions. Reproductively mature ewes (n = 100) from each breed were utilized to conduct the study. To evaluate the reproductive efficiency, lambing dates and number of lambs born were recorded per ewe; additionally, the duration in days from ram placement with ewes to lambing (DRPEL), litter size and the fertility rate were determined based on lambing dates. In each breed, there were eight nucleotide variations within the MTNR1A gene exon II, two of which (g.17355358 and g.17355171), respectively, resulted in a valine to isoleucine, and alanine to aspartic acid substitution, in amino acid sequence. The SNPs at position g.17355452 and g.17355458 were determined to have effects on reproductive performance. Genotypes C/C and C/T at g.17355452 in Bovska and Sarda and genotype A/A at g.17355458 in Istrian Pramenka were associated with a greater fertility and a lesser duration in days from ram placement with ewes to lambing. These findings confirmed that the nucleotide sequences of the MTNR1A gene could affect reproductive functions of Mediterranean sheep.

Improving dairy performance through molecular characterization of SREBP-1 gene in Sarda sheep breed.

This research has two aims: (i) to characterize the coding sequence of the SREBP-1 gene in dairy sheep in order to investigate possible relationships between single nucleotide polymorphisms (SNPs) and milk traits; and (ii) to investigate possible relationship between SREBP-1 gene expression and nucleotide variation. Four hundred adult and multiparous lactating Sarda breed ewes were selected from two farms. Milk samples were collected from Day 30 to Day 150 of lactation to determine the mean yield, somatic cell count, lactose, fat, and protein content of the milk. RNA was extracted from the milk samples, after which the SREBP-1 gene coding regions were amplified and sequenced to scan mutations. Whilst eight SNPs were identified, none had statistically significant association with the analysed milk traits. Moreover, the identified expression patterns were not affected by the SNP or combined genotypes. High SREBP-1 gene expression levels were found to be correlated with high milk fat content (P < 0.01), indicating the crucial role of this gene in the milk fat synthesis. In conclusion, the polymorphisms found within SREBP-1 gene exhibited no significant associations with milk traits or with individual SREBP-1 mRNA expression patterns. The findings thus suggest that this small genetic variability may derive from the selection carried out in Sarda breed to improve milk yield.

Reproductive performance in three Slovenian sheep breeds with different alleles for the MTNR1A gene.

The aim of the research was to describe the polymorphisms of the MTNR1A gene in three Slovenian sheep breeds, and determine whether these polymorphisms affect reproductive performance. A total of 100 Bovska, 110 Istrian Pramenka, and 108 Jezersko-Solcavska ewes, were used. The rams were placed with the ewes on 10 October (Bovska), and 20 September (Istrian Pramenka). At each farm, the lambing date and number of newborn lambs were recorded to evaluate the fertility rate, number of days from ram placement with ewes to lambing (DRIL), and litter size. The Jezersko-Solcavska rams were maintained with the ewes of the flock throughout the year and data related to lambing during the entire year were recorded. The DNA sequences indicated there were eight nucleotide variations and that in the position g.17355358 was always associated with that in the position g.17355452. For the Bovska breed, there was an association between the G/G genotype in the position g.17355452, and relatively greater fertility (P < 0.05) and a shorter DIRL (P < 0.05). For the Istrian Pramenka breed, the T/T genotype in the position g.17355458 was positively associated with a greater fertility (P < 0.05) and shorter DRIL (P < 0.05). For the Jezersko-Solcavska breed, ewes with the G/G genotype in the position g.17355452 lambed more when there was a decreasing photoperiod whereas the ewes with the A/A genotype lambed more (P < 0.05) during the period when there were increasing photoperiods. The different polymorphisms studied were associated with reproductive performance in the three Slovenian breeds.

Melatonin treatment and male replacement every week on the reproductive performance in Sarda sheep breed.

The aim of this study was to highlight whether the combination of melatonin treatment and weekly male replacement could influence reproductive performances in Sarda sheep breed. In a preselected farm, on 5th March 2016, 400 lactating ewes were identified, aged 3-5 years, with body condition score (BCS) 2.5-4.0, who lambed between 20th October and 1st December 2015. The 400 chosen animals were subsequently divided into 4 groups with 100 animals in each group (M, MR, C and CR). On 20th March 2016 the animals of Group M and MR (consisting of 100 ewes each) were treated with melatonin implants and on 24th April 2016 five entire rams were introduced into each group. In groups MR and CR, males were replaced each week with other males whereas in groups M and C the introduced males were not replaced. The MR group showed the highest fertility (number of lambed ewes) compared to the other three groups (P < 0.01). Both the melatonin treated groups (M and MR) exhibited an increase in fertility greater than the controls groups C and CR (P < 0.01). However, when the two groups treated with melatonin were compared, the MR group showed a higher fertility than the M group (P < 0.01). The same can be said for the two control groups, of which the CR group showed a greater increase in fertility than the C group (P < 0.01). The mean interval of days from male introduction to lambing was lower in the treated than in the control groups (P < 0.05). The MR group presented the lowest mean interval of days from male introduction to lambing amongst all of the groups (P < 0.05). A similar trend was recorded for group CR when compared to group C (P < 0.05). In the MR and M groups the lambing peak was recorded close to 170 days after the males introduction, whereas the CR group and the C group were reported to be around 180 days and 190 days, respectively. At the 170th day from the male introduction the number of ewes lambed in the MR group was greater than that of the M group (P < 0.05) (60 vs. 42), and more than double of that of the C and CR groups (P < 0.01). In conclusion, weekly male replacement improved the reproductive activity and strengthened the effect of the pineal hormone on reproductive efficiency. Therefore male replacement either in untreated animals or in association with the melatonin implants, can be straightforwardly applied to guarantee a more efficient reproduction in sheep breeding.

Influence of melatonin receptor 1A gene polymorphisms on seasonal reproduction in Sarda ewes with different body condition scores and ages.

In several species, circadian changes in melatonin concentrations play a key role in the photoperiodic control of seasonality. In sheep, two silent mutations in the melatonin receptor 1A gene (MTNR1A) at positions 606 and 612 of the exon II are associated with seasonal reproduction. However, in some sheep breeds, no relationships have been found between MTNR1A polymorphisms and reproductive seasonality. This lack of relationship could be due to effects of breed, body condition, age, and/or environmental conditions. Thus, the present study was conducted with the Sarda sheep breed with the aim of documenting the effect of MTNR1A gene polymorphisms on reproductive resumption and to evaluate whether such this effect was modified by differences in body condition score (BCS) and age. Six hundred three- to six-year-old multiparous ewes with BCSs between 2.5 and 3.5 were selected. Genomic DNA was extracted and subjected to PCR to amplify the ovine exon II of the MTNR1A gene. The amplicons were subjected to digestion with the restriction enzymes RsaI and MnlI to detect the T606C and A612G polymorphisms, respectively. Ewes carrying the G/G, G/A, C/C, and C/T genotypes exhibited higher fertility rates (P<0.05) and fewer numbers of days between the introduction of rams and parturition (P<0.05) than did the A/A and T/T genotypes. The data revealed that the MTNR1A gene polymorphisms influenced spring reproductive resumption in the Sarda sheep breed. Moreover, the data also indicated that, over the limited ranges evaluated in this study, BCS and age had no significant influence on reproductive activity.