Sebastiano Luridiana

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.

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.

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.

A polymorphism at the melatonin receptor 1A (MTNR1A) gene in Sarda ewes affects fertility after AI in the spring

The effect of MTNR1A gene polymorphisms on the fertility rate after AI in Sarda sheep was evaluated in 600 lactating adult ewes. Genomic DNA was subjected to amplification of the MTNR1A gene exon II. Amplicons were digested with restriction endonuclease MnlI. Ten samples from each genotype were sequenced. A polymorphism was detected (A612G) and ewes were determined to be +/+, +/- or -/- for the allele. Allelic frequency was 0.77 for the+allele and 0.23 for the - allele. The frequency of the +/+, +/- and -/- genotypes was 68, 19 and 13%, respectively. On 16 May 2009, 60 ewes from each genotype group were synchronised using intravaginal sponges containing 40 mg fluorogestone acetate for 14 days. At sponge removal, the ewes were administered 350 IU pregnant mares serum gonadotropin and were then inseminated, 54-56 h later, with 400 × 10(6) spermatozoa. Pregnancies were confirmed 50 days after AI using transabdominal ultrasonography. Lambing dates and the number of newborn lambs were recorded within 155 days after AI. Conception and lambing rate were higher for ewes with the +/+ and +/- genotypes compared with those with the -/- genotype (P<0.01). In conclusion, there was a positive correlation between MTNR1A allele polymorphisms the reproductive response following synchronisation and AI in the spring.

Effect of CSN1S1 gene polymorphism and stage of lactation on milk yield and composition of extensively reared goats.

The effect of CSN1S1 genotype and lactation stage on milk yield and composition were investigated in 80 extensively reared goats. Milk yield was recorded in early, mid and late lactation and individual milk samples were collected to determine: fat, protein, lactose and casein content, pH, freezing point, somatic cell count (SCC) and total microbic mesophilic count (TMC). Relative casein composition and amino acid profile were quantified by HPLC. Fatty acid profile was measured by gas-chromatography. Genotype did not affect milk yield, while this trait was significantly affected by lactation stage (P < 0.01). CSN1S1 BB goats produced significantly higher protein and casein percentages (P < 0.05). αs1-casein (CN) was significantly higher in BB and AB goats than AF and BF, showing intermediate values in AA goats (P < 0.01). The protein percentage and the αs1 and αs2-CN fractions were not affected by lactation stage, while the casein content and the β and κ-CN significantly increased throughout lactation (P < 0.01). C4 : 0 and C6 : 0 were not affected by genotype, while C8 : 0 and C10 : 0 were higher in the AA goats than BB; most of the long chain FA were higher in BB than AA goats. MUFA and PUFA increased in late lactation. In addition, BB goats showed higher essential amino acids, resulting in an optimal composition from the nutritional point of view, when compared with AA goats. The increase of MUFA, PUFA, essential and cis-FA in late lactation indicate that the lipid composition of goats milk, with the progress of lactation, tends to improve its nutritional value.

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.

Daily rhythm of blood melatonin concentrations in sheep of different ages

The aim of the study was to investigate the daily pattern of melatonin during short photoperiods in sheep of different age. Thirty Sarda sheep kept under natural light conditions and divided into three equal groups were used: Group 1 (12–18 months); Group 2 (3–4 years); Group 3 (8–9 years). Blood samples were collected every 2 h for 22 h, starting at 21 December (12:00 h) until 22 December (10:00 h) for plasma melatonin radioimmunoassay determination. Statistical significant effect of time of day and age on melatonin concentration was registered. Mesor and amplitude values were statistically lower in Group 3 than in Groups 1 and 2. This study provides the first characterization of the relationship between age and daily pattern of melatonin in sheep, highlighting a decrease in melatonin production in the older animals.

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 melatonin receptor gene MT1 in mouflon (Ovis Gmelini Musimon) and its relationship with reproductive activity.

The goals of the present research were to characterize the MT1 gene in mouflon, identify possible polymorphisms, and facilitate a better understanding of their relationship with reproductive activity.

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

Summary 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.