In vitro embryo production and semen cryopreservation in sheep

Abstract

Two trials were conducted at Agricultural Research Council (Irene – Pretoria), between March (autumn) and October (spring), 2008. The first trial evaluated the effect of the oocyte harvesting technique on the quantity and quality of ovine oocytes recovered, as well as the effect of the culture media used on embryonic development. The second trial evaluated the effect of breed and semen cryopreservation on the embryonic development following in vitro fertilization (IVF). For the first trial, ovine ovaries were collected from slaughtered animals at the Boekenhout abattoir near Pretoria. All ovaries were immediately transported to the laboratory for further processing and use in the trials. On reaching the laboratory, ovaries were processed, and oocytes were harvested, either by slicing (140 ovaries), or follicular aspiration (167 ovaries). The oocytes collected were matured, fertilized with fresh ram semen and cultured in order to produce embryos. The average total number of ovine oocytes recovered per ovary, and the total number of oocytes collected were higher (P<0.05) when using the slicing (5.2±0.4 oocytes/ovary and 75.3±12.9 total oocytes) technique, compared to the aspiration technique (2.6±0.5 oocytes/ovary and total of 40.3±9.1 oocytes). The number of acceptable quality (intact cumulus layers) oocytes was also higher (P<0.05) following the slicing (46.7±10.3 oocytes), compared to aspiration (10.3±2.0 oocytes) technique. However, the number of poor quality oocytes did not differ between the 2 oocyte harvesting techniques. The acceptable oocytes were then matured in vitro for 24h and no difference (P > 0.05) in oocyte maturation rate between the oocytes recovered using the slicing or aspiration technique was recorded. A comparison of the 3 culture media (Potassium simplex optimization medium - KSOM, Synthetic oviductal fluid - SOF and Charles Rosenkrans medium - CR1) used for maintaining subsequent embryonic development was then evaluated. All oocytes were further matured, using the maturation medium - TCM 199 containing FSH, LH and E2, supplemented with 10% FBS. After maturation, the oocytes were fertilized (using fresh ram semen) and incubated for a period of 18h. At the end of 18h fertilization period, oocytes were vortexed in an Eppendorf tube containing 100μl M199 + 10% FBS for 1.5min. The vortexing was performed to remove the cumulus cells surrounding the zygote. A total of 1405 presumptive zygotes were randomly allocated to the 3 different culture media (481, 461 and 463 zygotes in KSOM, SOF and CR1, respectively). The zygotes were then cultured for a period of 7 days. No significant difference between all 3 culture media was recorded regarding cleavage rates, showing that culture media had no effect on the subsequent cleavage. However, the percentages of embryos decreased with an advancement of the embryonic developmental stages.