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.

Features of the daily rhythms of blood melatonin and glucose in goats during different natural photoperiod.

The scope of the present study was to explore the features of the daily rhythms of plasma melatonin and blood glucose in goats during different natural photoperiod imposed by the four seasons. Five Sarda breed pluriparus female goats, 2 years old, with a mean body weight 40.5 ± 2.3 kg, lead to a natural pasture, were cannulated the day before the start of the study for sample collection performed during spring and autumn equinoxes, and winter and summer solstices for a 48-h period. On blood samples, melatonin and glucose concentrations were assessed. Our results indicate a daily rhythm of both parameters studied which characteristics were influenced by the different photoperiod due to season. During the experimental period, the two parameters showed a negative correlation, despite the onset of melatonin concentration was linked to the sunset, whereas the onset and offset of glucose were not linked to the photoperiod. In conclusion, we can claim that the two parameters are driven by different entrainable systems, but further studies are necessary to establish their correlation.

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.

Melatonin treatment in spring and reproductive recovery in sheep with different body condition score and age.

With the aim to evaluate the effect of melatonin treatment on the advanced reproductive recovery in sheep with different body condition score (BCS) and age, 800 ewes were selected from two farms. These ewes (3-6 years old, multiparous and with BCS 2.5-4.0) were subdivided into two Groups (200 M and 200 C), balanced on their BCS and age. On 20 March, Group M was treated with one melatonin implant (18 mg). Group C was untreated. Males were introduced to the groups 35 days after treatment. Gestation was diagnosed between day 45 and 90 after mating by transabdominal ultrasonography. From day 150 to 190 after rams introduction, lambing date and newborns' number were recorded. The average time in days between male introduction and lambing resulted shorter in treated than in control ewes (166.4 ± 0.48 vs. 172.5 ± 0.50) (P < 0.05). At day 160 and 170 from ram introduction the fertility rate was higher in Group M than in C (P < 0.05). The overall fertility at day 190 from rams introduction showed no differences between Group M and C (337 and 339, respectively). At day 170 from male introduction the number of the 5-6 years-old lambed ewes were 2-fold higher than the youngers (P < 0.05). The animals with a BCS 3.5-4.0 had a faster response to male effect, and a shorter mean distance in days from rams introduction to lambing, compared to those scored 2.5-3.0 (166.1 ± 0.48 vs. 174.8 ± 0.51) (P < 0.05). We concluded that the ewes with BCS 3.5-4.0 and aged 5-6 years showed a better response to melatonin treatment in spring.

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.

Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding.

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.

Characterization of the Mediterranean Italian buffaloes melatonin receptor 1A (MTNR1A) gene and its association with reproductive seasonality.

The aim of this study was to examine the polymorphism in MTNR1A gene and its relation to reproductive seasonality in Mediterranean Italian buffaloes reared in Sardinia. The mating period and calving of 100 multiparous buffalo-cows were recorded for three years (2005-2008). Genomic DNA was subjected to PCR for the amplification of the exon II, then 40 amplicons were sequenced. The obtained sequence was deposited in GeneBank database (accession number GU817415). PCR products were checked for the presence of HpaI restriction sites and assigned to genotypes "C/C", "C/T" or "T/T". Allelic frequency of C and T alleles was 0.44 and 0.56 and genotypic frequency was 26% for genotype C/C, 40% for C/T and 34% for T/T. In the three observed years the animals with C/C genotype showed the highest number of mating in the semester between August and January and their calving mainly occurred from August to September. On the other hand animals with T/T genotype showed mating mostly in the semester between February and July and calving occurred largely from March to May in all the three years. Heterozygous, in all the three years, showed about the same number of animals mated within each six-month period. The results of the present study provide for the first time a partial sequence as well as one polymorphic site of the MTNR1A receptor gene from buffaloes. Moreover our data showed an association between Single Nucleotide Polymorphism and seasonal reproductive activity in these animals.

Effect of genotype at the MTNR1A locus and melatonin treatment on first conception in Sarda ewe lambs.

A sample of 423 Sarda ewe lambs from three different farms was used to evaluate the effect of one or two melatonin implants on the time of first conception. On each farm, 141 animals were divided into three groups. On June 30 these animals received either no treatment (Group C), 18 mg melatonin (Group M1, one implant), or 18 + 18 mg melatonin (Group M2, two implants). Thirty-five days after treatment, rams were introduced in the ewe lambs flock and subsequently removed after 40 days. Lambing dates were recorded between 150 and 190 days from the first day of male introduction. Genotyping and sequencing of the MT1 exon 2 were carried out to analyze the structure and the possible influence of the MT1 receptor gene on reproductive response to melatonin treatment. Melatonin-treated animals had a higher rate of pregnancy (P < 0.05) and lambed earlier (P < 0.05) compared with untreated animals. Single nucleotide polymorphisms were found in exon II of MT1 gene at positions C606T and G612A leading to genotypes C/C, C/T or T/T and +/+, +/- and -/-, respectively. Melatonin-treated animals of +/+ genotype showed a higher number of pregnancies (P < 0.05) and lambed earlier (P < 0.05) compared to untreated animals of the same genotype. Melatonin treatment did not affect reproductive activity in any other genotype analyzed. No correlation between genotype and the time of first conception was found in untreated animals. Concluding data revealed the positive effect of melatonin treatment on the time of first conception in ewe lambs and highlighted that +/+ genotype is able to influence reproductive response to melatonin treatment.

Polymorphism of the melatonin receptor MT1 gene and its relationship with seasonal reproductive activity in the Sarda sheep breed.

The aim was to study the polymorphisms of the melatonin receptor 1A gene (MTNR1A) and its relationship with seasonal reproduction in the Sarda sheep breed. Four-thousand multiparous ewes reared under natural photoperiod were randomly chosen. Genomic DNA was extracted and subjected to PCR for the amplification of the main part of exon II of the ovine MTNR1A gene (GenBank U14109). PCR products were subjected to restriction enzymes MnlI and RsaI and placed into +/+, +/- or -/- group for MnlI and C/C, C/T or T/T group for RsaI. Samples were cloned and sequenced. The sequences were aligned with the U14109 sequence of GenBank. Data were subjected to allelic frequency analysis and to the chi(2) test in order to evaluate the link between genotype and reproductive activity. After MnlI digestion, allelic frequency was 0.78 for allele +and 0.22 for allele -; genotype frequency of the +/+ homozygote was 68%, 20.5% for +/- and 11.5% for -/-. After RsaI, allelic frequency was 0.66 for allele C and 0.34 for allele T; genotype frequency of the C/C homozygote was 53.5%, 26% for C/T and 20.5% for T/T. The population was in Hardy-Weinberg disequilibrium both for the MnlI and RsaI. Lambing frequency of +/+ genotype ewes was higher in the period September-December while for -/- genotype in January-April (P<0.01). Lambing of C/C genotype ewes showed a higher frequency in September-December while for T/T genotype in January-April (P<0.01). Results confirmed that the polymorphism of the MTNR1A locus was also present in the Sarda with a higher incidence of the +/+ and C/C genotypes. The animals that carried one of these two gene isoforms showed a not seasonal reproductive activity with the lambing period in September-December.

Relationship between MTNR1A melatonin receptor gene polymorphism and seasonal reproduction in different goat breeds.

The reproductive activity of goats bred in temperate latitude follows a seasonal pattern, influenced by annual variation in day length. Daily variation in pineal melatonin secretion is the neuroendocrine signal recognized by animals through the link between this hormone and melatonin receptor 1a (MTNR1A). A total of 345 goats of different breeds (225 Sarda, 30 Saanen, 30 Chamois Coloured, 30 Maltese and 30 Nubian) with a kidding period in October-December or January-March were analysed to verify if a link exists between the structure of the receptor gene and reproductive activity. The main part of exon II of MTNR1A gene was amplified by PCR and then digested with MnlI and RsaI to prove the presence of restriction sites. Sequencing of 20 cloned samples and 20 purified samples permitted comparison with previously published sequences. No polymorphism was found using MnlI enzyme, as all 345 samples showed the cleavage site in position 605 and all the goats were MM genotype. However, using RsaI enzyme, some Sarda goats, showed a polymorphic site in position 53. Nine Sarda goats were R/r genotype, lacking this cleavage site only in one allele, while the other animals, both Sarda and the other breeds, presented the cleavage site in both the alleles and were thus R/R genotype. No r/r genotype was found in any of the breeds. In Sarda goats the allelic frequency was 0.98 for R allele and 0.02 for r allele; genotypic frequency was 96.00% for R/R genotype and 4.00% for R/r genotype. A strong link emerged from statistical analysis (P<0.001) between R/r genotype and reproductive activity, which was strongly influenced by photoperiod. Sequencing indicated six nucleotide changes that did not induce any amino acid change. Data showed that polymorphism was present and that it influences reproductive activity only in the Sarda breed.

Productive performance and meat quality of Mouflon×Sarda and Sarda×Sarda suckling lambs.

The objective of this study was to compare slaughtering performance, carcass characteristics, and meat quality in lambs of two different genotypes. Sixteen crossbred Mouflon×Sarda (M×S) and sixteen pure Sarda breed (S×S) suckling lambs were analysed. Chemical and fatty acid composition were determined on semitendinosus and longissimus dorsi muscles and perirenal and pelvic fats. Dressing percentage was higher (P<0.01) in M×S group and after histological dissection M×S carcasses had more muscle tissue (P<0.01) and less separable fat (P<0.05). Muscles of M×S had less cholesterol than the S×S lambs (P<0.01). Polyunsaturated/saturated and n-6/n-3 fatty acids ratios of the intramuscular lipids were optimal in both groups. Indexes of atherogenicity and of thrombogenicity in muscles varied between 0.9 and 1.1. The results indicated several good qualities in both the genotypes, but M×S lambs have some traits which could provide a higher market price.