Francis Ectors

Recent progress in cryopreservation of cattle embryos

Abstract Two cryopreservation methods adapted to field use were investigated with cow embryos. Firstly, a freezing method combining 1.36 M glycerol and 0.25 M sucrose in PBS. The use of sucrose, by its osmotic properties, predehydrates the embryo allowing slow cooling at 0.3°C/min to be interrupted at −25°C. Direct transfer, after thawing from liquid nitrogen, gave a pregnancy rate of 51.8% ( 14 27 ). Secondly, a vitrification method involving a mixture of glycerol and 1,2 propanediol in PBS at concentrations giving an amorphous state on plunging in liquid nitrogen. Pregnancy rate after transfer of vitrified late morulae very early blastocysts was 39.1% ( 9 23 ).

Determination of porcine plasma follitropin levels during superovulation treatment in cows

Porcine follicle stimulating hormone (pFSH) and porcine luteinizing hormone (pLH), are widely used to induce superovulation in cows. An advantage of this treatment is that the LH:FSH ratio can be varied to optimize the growth of the ovarian follicles. However, due to the relatively short half-life of FSH, the superovulatory treatment requires numerous injections. A performant radioimmunoassay system (sensitivity=0.2 ng/ml plasma) was used to determine plasma pFSH levels in cows that were superovulated with 2 daily injections of 4 Armour Units (A.U.) of pFSH for 4 d. From plasma profiles, the half-life and the disappearance of pFSH were estimated at 5 h and at 10 to 12 h, respectively, confirming the necessity of using two daily injections.

Some factors affecting successful vitrification of mouse blastocysts.

The effects of temperature and exposure time to vitrification solutions on In vitro survival of mouse blastocysts were investigated. Blastocysts were first exposed for 10 min to vitrification Solution 1 (VS1) containing 10% glycerol-20% 1,2 propanediol in phosphate buffered saline (PBS), then to vitrification Solution 2 (VS2) with 25 % glycerol-25% 1,2 propanediol for various periods either at room temperature or at 4 degrees C. At room temperature survival dropped quickly, while at 4 degrees C an increase in survival was observed. It is concluded that the viability of mouse blastocyts after vitrification is dependent on the temperature and duration of equilibration in vitrification solutions.

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.

Fast freezing of cow embryos in French straws with an automatic program.

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.

Fractionation and partial characterization of proteins extracted from the bovine fallopian tube: Preparation of reagents for further purifications

Abstract Described in the present paper is a combined biochemical and immunological approach to study oviductal proteins in the bovine. Antisera were raised against semi-purified proteins extracted from bovine tubal mucosal tissue and were characterized. These antisera are available to monitor purifications of specific oviductal proteins in the future. Oviducts from 170 cyclic cows were collected at a slaughterhouse, and high amounts of mucosal proteins were extracted. The proteins were fractionated after precipitation with ammonium sulfate, anti-bovine serum albumin (bSA) and anti-bovine immunoglobulins (bIg) affinity chromatography and ion exchange chromatography. Each of the 12 fractions obtained after ion exchange chromatography was used to immunize a rabbit. Conditioned media were recovered from bovine oviduct cell monolayers cultured without serum to confirm the oviductal origin of the extracted proteins. After Western blot analysis, 15 proteins were detected in the bovine oviductal extracts, and their molecular weights and isoelectric points were determined by 2 dimensional electrophoresis. Among these 15 proteins, 11 were also detected in conditioned media of bovine oviductal cells. These results demonstrate an oviductal origin of the 11 detected proteins and strongly suggest their secretion by the oviductal cells.