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

Wild small ruminants living at temperate latitudes reproduce during decreasing photoperiods, resulting in spring lambing and thus a greater possibility of newborn survival. This process depends on the nocturnal pineal secretion of melatonin, which plays an essential role in the control of seasonal reproduction as a humoral encoding of photoperiodic information. Melatonin exerts its seasonal effect by acting on specific receptors. Of these receptors, MT1 seems to be the only one involved in the regulation of reproductive activity. Some polymorphisms have been found in the MT1 gene in sheep. In particular, using MnlI endonuclease, the homozygous genotype for the absence of the cleavage site at position 328 of MT1 exon II was associated with the seasonal anovulatory activity in ewes (Pelletier et al., 2000). 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. Blood samples from 80 mouflon (35 females and 15 males living wild in restocking areas on land belonging to Sardinia’s Regional Forestry Commission and another 25 females and 5 males kept in a fenced outdoor environment) were collected and used for genomic DNA extraction to allow amplification of the main part of exon II of the MT1 gene (Messer et al., 1997). The amplicons from 30 samples were randomly chosen, cloned and sequenced, and then aligned to the ovine MT1 sequence (GenBank accession number U14109) to evaluate any difference to the sheep sequence (Carcangiu et al., 2009). All 80 samples were genotyped using MnlI endonuclease which identifies anA (allele, cleavage site absent) to aG(þallele, cleavage site present) substitution at position 328 (GenBank FJ801038). Allele and genotype / frequency (0.66 and 0.50, respectively) were predominant compared to allele þ and genotype þ/þ (0.34 and 0.18, respectively). Sequencing permitted the characterization of MT1 exon II in mouflon (GenBank FJ801038). Five mutations were shown: four silent (T169G, C322T, A328G, T607C) and one leading to an amino acid substitution (A422G!Ile141Val). The presence of an A at position 328 was always found to be associated with the presence of a T at positions 169 and 607 and an A at position 422. Lambing dates of the 25 females kept in the outdoor environment were recorded. The comparison of reproductive activity with genotype in this latter group showed that the five animals with þ/þ genotype lambed earlier than the 12 with / genotype (P<0.05) (Table 1). In conclusion, the present research provides the partial sequence of theMT1exon II in the wild sheep mouflon and shows that / genotype is more frequent than the others. This genotype’s link to reproductive activity strongly influenced by photoperiod results in late lambing. This presumably contributes to a greater possibility of newborn survival as suggested by the high frequency of the / genotype.