The LEPR gene is associated with reproductive seasonality traits in Rasa Aragonesa sheep

The aim of this study was to characterize and identify causative polymorphisms in the leptin receptor (LEPR) gene responsible for the seasonal variation of reproductive traits in sheep. Three reproductive seasonality traits were studied: the total days of anoestrous (TDA), the progesterone cycling months (P4CM) and the oestrous cycling months (OCM). In total, 18 SNPs were detected in 33 ewes with extreme values for TDA and OCM. Six SNPs were non-synonymous substitutions and two of them were predicted in silico as deleterious: rs596133197 and rs403578195. These polymorphisms were then validated in 239 ewes. The SNP rs403578195, located in exon 8 and leading to a change of alanine to glycine (Ala284Gly) in the extracellular domain of the protein, was associated with the OCM trait, being the G allele associated with a decrease of 12 percent of the OCM trait. Haplotype analyses also suggested the involvement of other non-synonymous SNP located in exon 20 (rs405459906). This SNP also produces an amino acid change (Lys1069Glu) in the intracellular domain of the protein and segregates independently of rs403578195. These results confirm for the first time the role of the LEPR gene in sheep reproductive seasonality

SNP rs403212791 in exon 2 of the MTNR1A gene is associated with reproductive seasonality in the Rasa aragonesa sheep breed

The aim of this study was to characterize and identify causative SNPs in the MTNR1A gene responsible for the reproductive seasonality traits in the Rasa aragonesa sheep breed. A total of 290 ewes (155, 84 and 51 mature, young and ewe lambs, respectively) from one flock were controlled from January to August. The following three reproductive seasonality traits were considered: the total days of anoestrus (TDA) and the progesterone cycling months (P4CM); both ovarian function seasonality traits based on blood progesterone levels; and the oestrus cycling months (OCM) based on oestrous detection, which indicate behavioural signs of oestrous. We have sequenced the total coding region plus 733 and 251 bp from the promoter and 3'-UTR regions, respectively, from the gene in 268 ewes. We found 9 and 4 SNPs associated with seasonality traits in the promoter (for TDA and P4CM) and exon 2 (for the three traits), respectively. The SNPs located in the gene promoter modify the putative binding sites for various trans-acting factors. In exon 2, two synonymous SNPs affect RFLP sites, rs406779174/RsaI (for the three traits) and rs430181568/MnlI (for OCM), and they have been related with seasonal reproductive activity in previous association studies with other breeds. SNP rs400830807, which is located in the 3'-UTR, was associated with the three traits, but this did not modify the putative target sites for ovine miRNAs according to in silico predictions. Finally, the SNP rs403212791 (NW_014639035.1: g.15099004G > A), which is also associated with the three seasonality phenotypes, was the most significant SNP detected in this study and was a non-synonymous polymorphism, leading a change from an Arginine to a Cysteine (R336C). Haplotype analyses confirmed the association results and showed that the effects found for the seasonality traits were caused by the SNPs located in exon 2. We have demonstrated that the T allele in the SNP rs403212791 in the MNTR1A gene is associated with a lower TDA and higher P4CM and OCM values in the Rasa Aragonesa breed

Effects of passive immunization against testosterone in "Rasa Aragonesa" ewes

International audienc

Relationship between plasma AMH at prepuberty and adulthood in sheep

International audienceThe high between-animal variability in the ovarian response to exogenous stimulation treatments is one of the main reasons responsible for the low efficiency of LOPU-IVP in sheep. At present, Anti-Müllerian Hormone (AMH), which is produced by granulosa cells of preantral and small antral follicles, is a great predictive endocrine marker of the number of available follicles in response to ovarian stimulatory treatments in many species. Recently, we also extended its ability to adult sheep (Reprod. Domest. Anim. 2012, 47(s4):492). While the use of AMH in adulthood is of great interest, it would be even more interesting if it could be determined earlier in life, allowing for a precocious selection of the best future donors for embryo biotechnologies. Therefore, our objective was to define plasma AMH before puberty and to relate it with AMH levels and ovarian responses to FSH in the adulthood in the same animals. With this purpose, ten Rasa Aragonesa ewe lambs were blood sampled when aged 3, 4.5 and 6 months, and later at the adult age (19 months), at the first session of four repeated LOPU sessions. Lithium heparinized tubes were used and plasma was stored at -20 ºC until assayed for AMH (GenII ELISA kit; Beckman Coulter, France). Before puberty, plasma AMH concentrations increased from 3 to 4.5 months, and then declined at 6 months up to levels similar to those observed later in adults: 370.4 ± 77.7, 519.2 ± 153.3 and 171.1 ± 29.7 pg/ml, respectively (Mean ± SEM; P<0.1). At these early ages, strong between-animal variability in AMH was found, with concentrations ranging from 20.4 to 1370.8 pg/ml. Within-animal repeatability in AMH at 3, 4.5 and 6 months was found to be very low (0.21; NS). At the adult age, mean plasma AMH concentration was 201.9 ± 24.1 pg/ml. Within-animal repeatability of AMH at 3, 4.5 or 6 months with AMH at 19 months was close to zero. Relationships between AMH before puberty and AMH or follicle yield after FSH stimulation at LOPUs in the adult age were not significant. In conclusion, plasma AMH before puberty showed a great variability between individuals and between ages, apparently reflecting chronological differences between animals on ovarian maturity further related with sexual precocity, as we proposed in a previous work. Therefore, a direct relationship with AMH levels at the adult age seems to be uncertain and could not be established for now, as it seems to reflect different physiological situations which require more studies. While in adult sheep circulating AMH determination by a single blood sample prior to FSH treatment was a suitable method for selecting the best oocyte donors, the precocious selection of such animals by AMH sampling at their prepubertal age would not be feasible

Transferencia de embriones en la oveja Rasa Aragonesa

International audienc

The use of plasma anti-müllerian hormone in sheep as an endocrine marker of the ovarian response to follicle-stimulating hormone in multiple-ovulation embryo transfer programs

National audienc

Cryopreservation of human ovarian tissue by direct plunging into liquid nitrogen: Negative effect of disaccharides in vitrification solution

Given that it has been possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen, this study was designed to establish the future direction to be taken in this line of research. Bovine oviductal epithelial fragments (as a tissue model) and large biopsy fragments (~2.0 mm3) of human ovarian tissue were used for cryopreservation. Two protocols were tested: with permeable cryoprotectants (dimethyl sulphoxide, propylene glycol, glycerol) + egg yolk + sucrose or trehalose + a synthetic blocker of ice nucleation, Supercool® X-1000; and using only permeable cryoprotectants (glycerol and ethylene glycol) + egg yolk + Supercool® X-1000. The cryopreserved tissue specimens were subsequently thawed and the cryoprotectants removed by dilution in graded sucrose solutions. Both the dynamic growth and hormonal activity of the ovarian tissue pieces, vitrified using only permeable cryoprotectants, were greater than after vitrification in a mixture of permeable cryoprotectants and sucrose. Unlike the case for other reproductive tissue (spermatozoa, oocytes, embryos), these findings suggest that the cryopreservation of ovarian tissue by direct plunging into liquid nitrogen must be achieved by vitrification using only permeable cryoprotectants and agents that prevention ice formation

Reproducción asistida aplicada a la recuperación de la raza ovina ansotana

Resumen de la comunicación presentada al III Congreso Ibérico sobre Recursos Genéticos Animale