115 RELATIONSHIP BETWEEN IN VIVO FERTILITY OF MANCHEGA EWES LAPAROSCOPICALLY INSEMINATED WITH FROZEN–THAWED RAM SEMEN AND HETEROLOGOUS CALF IN VITRO FERTILIZATION

Abstract

Cryopreservation may damage the fertilizing ability of spermatozoa. For this reason, it is important to assess the sperm before artificial insemination. In vitro fertilization (IVF) tests are the most suitable for assessing overall sperm function during fertilization. The aim of this work was to study whether a relationship exists between heterologous calf in vitro fertilization and in vivo fertility in Manchega sheep, with the purpose of predicting in vivo fertility of males before using their semen. Frozen–thawed sperm from 4 Manchega rams was used to laparoscopically inseminate 481 ewes of the same breed. Sperm was cryopreserved in a TRIS-yolk-glycerol extender. A minimum of 10 females were laparoscopicly inseminated per ram. These same straws were used for IVF (4 replicates per male). Domestic calf ovaries were collected at a slaughterhouse. Inmature cumulus–oocyte complexes (COCs) were aspirated and matured in vitro in TCM-199 with 10 ng mL^-1 EGF and 100 µM cysteamine. After 24 h, zona-intact mature oocytes were incubated in synthetic oviduct fluid supplemented with 10% estrous sheep serum. Thawed spermatozoa were co-incubated with the oocytes (one million per well) for 40 h, and the cleavage rate was asessed. A regresion analysis was performed. The in vivo fertility ranged from 25.00 to 62.50%. Two rams had a fertility under 30.00% and the other ones over 55.00%. The in vitro fertility ranged from 42.50 to 58.50%. The in vivo fertility was not related to the in vitro fertility (P = 0.17). This could be due to the low number of males used in this work. Heterologous calf in vitro fertilization tests cannot be used to predict in vivo fertility of ram semen since no relationship was found between both variables. Nevertheless, these results are preliminary and we are working with more rams and replicates to obtain more information.

This work was funded by INIA and Consejeria de Agricultura de Castilla-La Mancha. Garcia-Alvarez enjoyed a studentship from the INIA