Discrepancies between Genetic and Visual Coat Color Assignment in Sarcidano Horse.

This study aimed to evaluate the discrepancies between genetic and visual coat color assignment in the Sarcidano Horse and to elucidate potential reasons. Individual DNA from 90 Sarcidano Horses was used for genetic assignment of coat color to explore the correspondence with individual forms containing phenotypical traits. The MC1R exon 1 and ASIP exon 3 have been genotyped and sequenced to obtain a picture of the coat color distribution in this breed. Surprisingly, once we compared the genetic results with the individual forms reporting the phenotypic data for each subject, a certain degree of non-correspondence between the phenotypic and genetic data in relation to coat color emerged. From the genetic analysis, Chestnuts (n = 58) resulted the most common Sarcidano Horse (n = 58), followed by a quite large number of Blacks (n = 28) and a very small number of Bays (n = 4), whereas phenotypic distribution resulted in 38 Chestnuts, 40 Bays, only 2 Blacks, and 10 Grays (without the possibility of recognizing the true color they carried). Chestnut resulted a very representative coat color, while many horses that visually identified as Bays were genetically Blacks. This discrepancy, that could be due to a variety of individual and external factors, including age, time of year, living situation and dietary condition, suggesting the importance of accurate coat color identification to ensure adequate features registration and reliable prediction of offspring's coat color.

Polymorphism of the MTNR1A Melatonin Receptor Gene in Goat Breeds of Northern Italy.

Melatonin receptor MT1, encoded by the MTNR1A gene, is the main one involved in the seasonal regulation of reproductive activity. A correlation between this gene polymorphism and reproductive performance has been demonstrated in sheep. To date, no data about MTNR1A gene polymorphism are available regarding Italian goat breeds other than the Sarda goat. This study aimed to detect any PCR-RFLP polymorphic sites of MTNR1A using MnlI and RsaI enzymes in Northern Italian goat breeds, which are characterized by a pronounced reproductive seasonality. One-hundred-eight adult female goats belonging to four different breeds were included in the study (i.e., Frisa Valtellinese, n = 29; Orobica, n = 23; Lariana, n = 29; Camosciata delle Alpi, n = 27). Blood was sampled from each goat. Genomic DNA was extracted from each sample and the main part of exon II of MTNR1A gene was amplified by PCR and digested with MnlI and RsaI enzymes. Unexpectedly, none of the fragments were found to be polymorphic. The absence of polymorphism may be linked to the macro group of goat breeds that evolved during human migrations. Breeds of the Alpine-European strain would appear to show no polymorphism, as confirmed by our study, whereas breeds belonging to the Mediterranean-African or Asian-Middle Eastern strains do.

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.

Characterization of the Sarcidano Horse Coat Color Genes.

The goal of this study was to contribute to the general knowledge of the Sarcidano Horse, both by the identification of the genetic basis of the coat color and by updating the exact locations of the genotyping sites, based on the current EquCab3.0 genome assembly version. One-hundred Sarcidano Horses, living in semi-feral condition, have been captured to perform health and biometric checks. From that total number, 70 individual samples of whole blood were used for DNA extraction, aimed to characterize the genetic basis of the coat color. By genotyping and sequencing analyses of the MC1R Exon 1 and ASIP Exon 3, a real image of the coat color distribution in the studied population has been obtained. Chestnut and Black resulted in the most representative coat colors both from a phenotypic and genotypic point of view, that is suggestive of no human domestication or crossbreeding with domestic breed. Due to its ancient origin and genetic isolation, an active regional plan for the conservation of this breed would be desirable, focused on maintenance of resident genotypes and genetic resources. Collection and management of DNA, sperm, embryos, with the involvement of research centers and Universities, could be a valid enhancing strategy.

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.

Ovine insulin induced-gene-2: molecular characterization, polymorphisms and association with milk traits.

The aim was to characterize the INSIG-2 gene in Sarda sheep and to highlight associations between polymorphisms and milk traits. Two-hundred ewes, in their third or fourth lactation who lambed a single lamb between 20th and 30th of November, were chosen. Monthly individual milk yield was recorded and from each ewe a sample of milk was taken to analyze fat and protein content. PCR-RFLP and DNA sequencing were carried out to detect polymorphisms. Five exons have been characterized and five mutations have been found G88A, 436TCAGdel, A471G, C1071T and T1737G all in the intronic regions. The ovine sequence and related variations were deposited in GenBank with accession number JX843812.1. The animals carrying AA genotype at position 88 showed a lower milk fat concentration than those with the AG or GG genotype (P < 0.05). A lower milk fat concentration was registered also in the animals with the TCAG deletion in position 436 (P < 0.05) and in the animals carrying AA genotype at position 471 compared to those with the AG or GG genotype (P < 0.05). Moreover, the animals carrying CC genotype at position 1071 had a greater milk yield than those with CT or TT genotype (P < 0.05) while ewes with TT genotype showed a higher milk protein concentration compared to the others (P < 0.05). A total of 11 haplotypes were detected but no significant associations with milk traits were found. In conclusion for the first time the complete coding sequence of INSIG-2 gene and its association with milk trait has been reported in this study.

Association between SREBP-1 gene expression in mammary gland and milk fat yield in Sarda breed sheep.

The aim of this study was to examine the expression patterns of SREBP-1 gene in milk somatic cells and its association with milk fat yield during early lactation in Sarda breed sheep. A sample of 20 Sarda ewes, aged between 4 and 5 years, in their third to fourth lactation were chosen. From each ewe 28 days after lambing milk yield was measured, and a 160 ml milk sample for the RNA extraction and to test somatic cells count and lactose, fat and protein contents were collected. From the obtained RNA, total cDNA was synthesized and the quantitative PCR was performed. The fat, proteins and lactose content showed many differences among the animals, but these variations were no correlated with the milk yield. The SREBP-1 gene expression resulted higher in the high milk fat producing ewes. The correlation analysis showed that the SREBP-1 expression level is directly related to the amount of milk fat (g/die) (P < 0.001), while the total RNA obtained from each sample was found to be related to the somatic cells number (P < 0.001). Instead the expression of this gene showed no relations with the concentration of fat in milk. Our data highlight that in sheep SREBP-1 gene is expressed in the mammary gland during early lactation. Moreover, the positive relationship between SREBP-1 gene expression and the milk fat yield suggests that SREBP-1 gene is required for the lipid synthesis in the sheep mammary gland.