Reproductive Resumption in Winter and Spring Related to MTNR1A Gene Polymorphisms in Sarda Sheep.

The aim of the present research was to evaluate the association between the SNPs rs430181568 and rs407388227 located in the MTNR1A gene with the reproductive recovery of Sarda sheep in different months of ram introduction in the flock (February, March, April and May). In order to address this research gap, we selected two farms, each of which consisted of approximately 1000 animals; a total of 800 ewes (400 for each farm) were genotyped for the two single nucleotide polymorphisms rs430181568 and rs407388227 located in the exon 2 of the MTNR1A. These SNPs are completely linked; thus, each genotype of rs430181568 corresponded to the same genotype for rs407388227. Among the genotyped animals, 240 individuals were selected and divided into four homogeneous groups (A, B, C and D) of 60 subjects, each group based on age (range 3−6 years old), body condition score (BCS) (range 2.0−4.0) and genotype (20 ewes carrying CC/CC, 20 CT/CT and 20 TT/TT genotype). The dates of the ram introduction in each group were 15 February, 15 March, 15 April and 15 May, respectively. In all groups, the lambing date and the number of lambs born from 150 to 220 days after the ram introduction were recorded. In all the groups, the genotypes CC/CC and CT/CT of the polymorphism (rs430181568 and rs407388227) showed the greatest fertility (the ratio between the number of lambed ewes and the ewes exposed to the rams) (p < 0.01) and the shortest distance between ram introduction to lambing (p < 0.01), compared with the TT/TT genotype. In conclusion, we determined that the polymorphisms rs430181568 and rs407388227 were associated with reproductive recovery, after ram introduction, even in February and March, months subsequent to the photorefractoriness period.

Effect of Body Condition Score, Treatment Period and Month of the Previous Lambing on the Reproductive Resumption of Melatonin-Treated Sarda Breed Sheep during Spring.

Stakeholders place great emphasis upon rationalizing the management and rearing techniques which are utilized within sheep farms. The present study aimed to investigate factors which may improve the reproductive performance of melatonin-treated Sardinian sheep via a series of three trials. The first trial (n = 100) investigated the effect of melatonin treatment alongside body condition score (BCS), the second trial (n = 150) investigated the effect of treatment alongside the date of treatment (treatment period) and the third trial (n = 150) investigated the effect of treatment alongside the previous lambing of the ewes. The findings indicated that melatonin is an effective tool for anticipating and improving the reproductive activity of in Sarda breed sheep during the springtime. Furthermore, to obtain optional results, melatonin implantation should be conducted in April, in ewes that have a BCS of >2.5 and that have passed their third month of lactation.

Melatonin Treatment in Rams and Their Replacement with Novel Treated Rams Advance First Lambing and Increase Fertility in Sarda Ewe Lambs.

This study aims to find reliable strategies for advancing first mating and shortening the period from weaning to first lambing in ewe lambs. Sarda ewe lambs (n = 400) were selected from two farms and allocated into four separated groups of 100, all of which were exposed to fertile, adult rams over the course of a 50-day breeding period. The first treatment group (RMR) was exposed to four melatonin-treated rams which were replaced every ten days, whilst the second treatment group (RM) was exposed to four melatonin-treated rams which were not replaced. Alternatively, the first control group (RCR) was exposed to four untreated rams which were replaced every ten days, whilst the second control group (RC) was exposed to four untreated rams which were not replaced. In each group, lambing dates, fertility rate, litter size, and distance in days from ram introduction to lambing (DRIL) were recorded. The highest fertility rate was recorded in the RMR group (p ≤ 0.05). Shorter DRIL (p ≤ 0.01) and higher lambing concentrations were recorded in the RM and RMR groups as compared to the controls. The findings indicate that melatonin treatment of rams and their replacement at 10-day intervals results in earlier onset of first mating, increased fertility rate in ewe lambs, and a higher number of ewes that lambs in a shorter time frame.

Detection of Polymorphisms in the MTNR1A Gene and Their Association with Reproductive Performance in Awassi Ewes.

The economy in Mediterranean areas is tightly linked to the evolution of the sheep-farming system; therefore, improvement in ewe's reproductive performance is essential in the developing countries of this area. MTNR1A is the gene coding for Melatonin receptor 1 (MT1), and it is considered to be involved in the reproductive activity in sheep. The aims of this study were: (1) identifying the polymorphisms from the entire MTNR1A coding region and promoter in Lebanese Awassi sheep flocks, and (2) investigating the association between the found polymorphisms and the reproductive performance, assessed as lambing rate, litter size, and days to lambing (DTL). The study was conducted in two districts of Lebanon, where 165 lactating ewes, aged 5.2 ± 1.5 years, with body condition score (BCS) 3.3 ± 0.4, were chosen and exposed to adult and fertile rams. From 150 to 220 days after ram introduction, lambing dates and litter sizes were registered. This study provided the entire coding region of the MTNR1A receptor gene in the Awassi sheep breed. Thirty-one single nucleotide polymorphisms (SNPs) were detected, five of which were missense mutations. The H2, H3, and H4 haplotypes were associated with lower DTL (p < 0.05), as well as the SNPs rs430181568 and rs40738822721, named from now on SNP20 and SNP21, respectively. These SNPs were totally linked and can be considered as a single marker. The ewes carrying the C allele at both these polymorphic sites advanced their reproductive recovery (p < 0.05). These results are essential for improving reproductive management and obtaining advanced lambing in Awassi ewes.

New polymorphisms at MTNR1A gene and their association with reproductive resumption in sarda breed sheep.

The aim of this study was to characterize the MTNR1A locus in Sarda sheep breed, in order to identify potential single nucleotide polymorphisms (SNPs) associated with reproductive resumption. The reproductive performance of 200 lactating ewes, aged 3-5 years, with body condition score (BCS) 2.5-4.0, at least at their third lambing were monitored for two consecutive years. In both year the enrolled ewes were exposed for 100 days to 10 adult, fertile rams. Mating, pregnancy and lambing for each ewe were recorded in order to evaluate differences in reproductive performance according to the analysed genotypes. From individual blood samples, DNA was extracted to amplify and to sequence promotor, the coding region, a part of intron and of 3' Untranslated region (3' UTR) of the MTNR1A gene. A total number of 29 SNPs were found (named SNP1 to SNP29), five of which caused also amino acid changes. The polymorphic sites found at positions g.17355452C > T (SNP16, rs430181568) and g.17355358C > T (SNP17, rs407388227) were linked (D' = 1 and r2 = 1) and showed a significant association to DRIL trait (distance in days from ram introduction to lambing). In both years, the ewes carrying C/C genotype in both these polymorphic sites showed the lowest DRIL compared to the other genotypes (P < 0.05). The ewes carrying C/C and T/C genotype exhibited the lambing peak at 170 days, and approximately 60% of the total lambing at 180 days from the ram introduction. Instead, ewes carrying T/T genotype showed the lambing peak around 200 days after ram introduction. Six haplotypes have been identified and the most frequent haplotype was also associated with lower DRIL (P < 0.05). Litter size displayed no statistical significance either among genotypes or among haplotypes. This study provided the major part of the MTNR1A gene in Sarda sheep breed and evidenced that SNP17 is associated with a shorter DRIL. The obtained results underlined the role of this polymorphism in improving reproductive efficiency in Sarda sheep and provides a suitable information for improving genetic selection.

Reproductive response to male joining with ewes with different allelic variants of the MTNR1A gene.

The aims were to evaluate the reproductive response to ram placement with Sarda ewes with different allelic variants at position g.15099485 A > G of the MTNR1 A gene. Ram placements occurred between the early and late spring and there was analysis of whether this polymorphism is associated with other nucleotide substitutions. In each of the eight farms where the study was conducted (named F1-F8), 150 ewes (50 with A/A, A/G and G/G genotypes) were selected. In each group of 150 ewes, eight males were joined with ewes on the following dates: 25 March (T1) for F1-F2, 15 April (T2) for F3-F4, 5 May (T3) for F5-F6, and 1 June (T4) for F7-F8. The lambing dates and number of new born lambs were recorded until 220 days after joining rams with ewes. The ewes with G/G or A/G genotypes had a greater fertility rate compared with those with A/A in T1, T2 (P < 0.01), and in T3 and T4 (P < 0.05). The duration of time in days from the time of ram joining with ewes to lambing was less in the ewes with G/G and A/G compared with those with A/A (P < 0.01). The g.15099485A>G variation was always associated with that at position g.15099391G>A. Results indicate there is a positive effect of the g.15099485A>G variant on reproduction when males were joined with ewes in March or April. The association that was ascertained in this study between the g.15099391G>A and g.15099485A>G polymorphisms for the MTNR1 A gene, could provide new insights to clarify the mechanism of action of melatonin.

Melatonin treatment in winter and spring and reproductive recovery in Sarda breed sheep.

This study aimed to evaluate the effect of melatonin treatment carried out between late winter and early spring on reproductive response in Sarda breed sheep and whether the photo-refractoriness can influence this reproductive response. The study was conducted on 3200 adult ewes, aged 3-6years old, with body condition score (BCS) 2.5-4.0, from 16 commercial sheep farms in Northern Sardinia. In each farm 200 animals were enrolled and subdivided into 2 groups (n=100 each): Group M (treated with one 18mg melatonin implant), and group C (untreated). The melatonin treatments were performed on 10th February; 10th March; 10th April and on 10th May each time in 4 different randomly selected farms. Adult males, treated with 3 melatonin implants 1 week before females, were introduced in each flock 35days after ewes' treatment, and removed after 45days of cohabitation with females. Pregnancy was determined by transabdominal ultrasonography examination between 45th and 90th day after ram introduction. Data showed that melatonin treatment increased the fertility rate significantly (P<0.05), with the higher fertility rate in the ewes treated in April and May. The average time in days from male introduction to lambing was shorter in treated than in control ewes (P<0.05). Further, at the 160th and 170th day after male introduction the group M showed a higher number of lambed ewes compared to C (P<0.01). This effect was observed at 180th and 190th days after ram introduction, also, but with lower significance (P<0.05). In conclusion, melatonin treatment improved reproductive efficiency and advanced breeding season in Sarda sheep, especially when ewes were treated in spring.

Production of Farmstead Lactose-Free Pecorino di Osilo and Ricotta Cheeses from Sheep's Milk.

The present work was aimed to define and validate farmstead production of lactose-free Pecorino di Osilo cheese, fresh ricotta cheese, and salted and smoked ricotta cheese (Ricotta mustia). The enzymatic activity of the commercial preparation containing lactase (1.1 g/mL), preliminarily tested using a spectrophotometric titration, showed activity equal to 4950±40 neutral lactase unit/g. The amount of lactase required to obtain the lactose-free milk was then established in triplicate laboratory trials, by adding the enzyme at concentrations of 0.7, 0.9 and 1.1 g/L in flasks containing 160 mL of raw sheep's milk. Samples were incubated under conditions expected during milk storage and cheese-making. The residual lactose content in milk was determined by enzymatic method. The addition of lactase at concentration of 1.1 g/L of milk reduced the lactose concentration below the limit of detection (LOD) of 0.06 g/L. The procedure was validated at a dairy farm, using three different batches of bulk raw sheep's lactose-free milk that were transformed into Pecorino di Osilo cheese. The resulting whey was used to produce fresh ricotta and Ricotta mustia cheese. Raw milk and whey samples were always below lactose detection limit. The residual lactose was measured in Pecorino di Osilo cheese, after 24 hours and 30 days from production; in fresh ricotta cheese, after 48 hours; in Ricotta mustia cheese after 7 days. The determination of lactose content in cheese samples was conducted by a gas chromatography-flame ionization detection method, which showed a LOD and limit of quantification respectively of 1.8 and 5.6 mg/kg for cheese, and 1.35 and 4.2 mg/kg for both ricotta cheeses. The lactose concentration was always below the relevant LOD values in all samples. The mean concentration of galactose and glucose were respectively 13,000±2000 and 11,000±2000 mg/kg in fresh Pecorino di Osilo, 1100±300 and 1200±300 mg/kg in fresh ricotta, and 950±400 and 750±250 mg/kg in Ricotta mustia. The results of the present study showed that the production of farmstead lactose-free Pecorino di Osilo cheese and ricotta cheeses from raw sheep's milk is easily achievable. The main issue for farmstead production of artisanal lactose-free products is the implementation of permanent procedures based on hazard-analysis and critical control principles aimed at guaranteeing the effectiveness of the process and at acquiring analytical evidences to demonstrate the fulfilment of law requirements for labelling.