The application of bovine in vitro embryo production technology to the rescue of Valdostana Castana breed

The Aosta Valley breed brown (Valdostana Castana) is a native rustic breed of alpine zone with a good attitude to the production of milk and meat. Excellent grazer, easily exploits even the high-altitude pastures, a characteristic that has led her to develop a defensive behaviour of its territory and an instinct for pugnacity. This propensity has been selected over the centuries and has turned the breed into "sporting animals” to be presented in local competitions and increasing the interest toward the most performing individuals. Moreover the Valdostana Castana counts a limited number of about 5000 animals giving further incentive to apply modern biotechnologies to preserve the breed. Sporadic attempts to apply superovulation have met with inconsistent results most likely due to the lack of a reliable stimulation protocol for the breed. Therefore over the last three years the ovaries of 15 donor cows have been referred to our laboratory for embryo production by in vitro technologies. The oocytes were aspirated from ovarian follicles larger than 2mm and those with a non atretic cumulus cells were matured in medium TCM199 supplemented with 10% FCS, ITS (insulin, transferrin, sodium selenite, Sigma), FSH and LH (1:1, Menopur, Ferring) for 24h. Frozen semen was used for IVF following separation on a Redigrad gradient composed of two fractions: 45% and 90%. The motile fraction was resuspended in medium SOF-heparin (1µg/ml) at a concentration ranging from 0.3 to 1 million sperm/ml depending on the bull. The day after IVF (day 0) the presumptive zygotes were transferred in 500 microl of medium SOF1 plus aminoacids. On Day 4 and Day 6 half of the medium was changed with SOF2 plus aminoacids. On day 7 and 8 of culture the G1 embryos (IETS grading) that had reached the full/expanded blastocyst stage were frozen in 1.5M ethylene glycol. The freezing curve was seeding at -6°C and cooling at 0.5°C/min down to -32°C and plunging in liquid nitrogen. The total number of oocytes recovered was 694 and the average number per donor was 46.27, ranging from 15 to 88. In total 8 different bulls were used for IVF and cleavage rate was between 25% and 89% with an average of 66.28%. The number of transferrable embryos was 141 ranging from 0 (1 cow) to 30 with an average of 9.4 per cow. The number of freezable embryos was 112 ranging from 0 (1 cow) to 26 with an average of 7.47 per cow. The percentage of transferable and freezable embryos was 30.65% and 24.35% of cleaved respectively. All the grade 1 embryos (112) were frozen in ethylene glycol. None of the non freezable embryos (29) was transferred as fresh. Sixty-three frozen-thawed embryos were transferred into recipient heifers obtaining 21 pregnancies (33% pregnancy rate). At present 14 calves have been born from 17 pregnancies gone to term and 3 pregnancies were lost. These results confirm that the in vitro embryo production technology can contribute to the preservation of special cattle breeds by generating valuable calves from donors destined to the abattoir.