Sex and PRNP genotype determination in preimplantation caprine embryos

in Reproduction in Domestic Animals (2011), 46(4), 656-663

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of ... [more ▼]

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. [less ▲]

Determination of sex and scrapie resistance genotype in preimplantation ovine embryos.

in Molecular Reproduction and Development (2009), 76(2), 183-190

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of ... [more ▼]

The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P = 0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo [less ▲]

Improved vitrification method allowing direct transfer of goat embryos

in Theriogenology (2006), 66(4), 1004-1011

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep ... [more ▼]

The aim of this study was to design a vitrification method suited to field embryo transfer experiments in goat. In a first experiment, a standard vitrification protocol, previously designed for sheep embryos was compared to slow freezing of goat embryos. No significant difference was observed on kidding rate (48% versus 69%, respectively), nor on embryo survival rate (35% versus 45%). Second experiment: all embryos were vitrified. After warming, embryos were either transferred directly (direct transfer), or after in vitro dilution of the cryoprotectants (conventional transfer). The kidding rate was not affected by the transfer method (38% versus 23%, respectively). However, embryo survival rate tended to be higher after direct transfer (26% versus 14%). Third experiment: OPS vitrification was compared to standard vitrification. The kidding rate was not affected (22% versus 39%, respectively), but the embryo survival rate was lower after OPS (14% versus 28%). Fourth experiment: 0.4 M sucrose was added with cryoprotectants in vitrification. The kidding rate after direct transfer was significantly enhanced after addition of sucrose (56% versus 27%, respectively), whereas embryo survival rate was not significantly affected (32% versus 18%). Fifth experiment: vitrification with sucrose supplementation was compared to slow freezing. No significant difference was observed after direct transfer on kidding rate (52% versus 31%, respectively), but embryo survival rate tended to be higher after vitrification (34% versus 2 1 %). In conclusion, our results indicate that addition of 0.4 M sucrose in association with direct transfer improves significantly the viability of goat vitrified embryos. [less ▲]

In vitro survival of vitrified goat embryos: comparison of two vitrification methods

in Proceedings: 17th Annual Meeting AETE (2001)

Amélioration des méthodes de cryopréservation et de transfert d’embryons chez les petits ruminants

in Proceedings: 8e Rencontres autour des Recherches sur les Ruminants (2001)

ln small ruminants, the costs of embryo transfer is a main limiting factor to the use of this method. The use of ultra rapid techniques such as embryo vitrification and direct transfer may contribute to ... [more ▼]

ln small ruminants, the costs of embryo transfer is a main limiting factor to the use of this method. The use of ultra rapid techniques such as embryo vitrification and direct transfer may contribute to reduce a part of the costs and increase the use of embryo transfer in sheep and goats. ln order to evaluate the efficiency of these techniques, two experiments were performed. ln a first experiment the viability of vitrified/thawed embryos was compared to results obtained after transfer of fresh embryos in ewes or frozen embryos (with slow freezing method) in goats. The pregnancy raté at term as weIl as embryo survival rate did not differ significantly according to embryo treatment (in ewes : 72% and 60% for fresh embryos vs 72% and 50% for vitrified embryos; in goats : 69% and 55% for frozen embryos vs 48% and 39 % for vitrified embryos). ln a second experiment, the possibility to transfer the vitrified embryos or frozen embryos directly after thawing (without cryoprotectant removal and evaluation of the morphological status of the embryos) was tested by comparison with the standard technique of transfer of vitrified or frozen/thawed embryos (removal of cryoprotectant and morphological evaluation). No significant effect of the transfer method was observed on the pregnancy rate at term and embryo survival rate (in ewes/vitrified embryos : 67% and 49% for traditional transfer vs 75% and 53% for direct transfer ; in goats/ vitrified embryos : 23% and 15% for traditional transfer vs 38% and 26% for direct transfer ; in goats/frozen embryos : 74% and 45% for traditional transfer vs 71% and 57% for direct transfer) [less ▲]