Superovulation and recovery of zygotes suitable for microinjection in different breeds of sheep

This study investigated the appropriateness of different superovulatory protocols in various breeds of sheep for obtaining a maximum of zygotes suitable for microinjection. Animals were mated either once or two to three times to fertile rams. In Experiment 1, a 24 h interval between a two to three times mating and egg recovery resulted in 42.2% suitable zygotes whereas with single mating only 10.4% fertilized eggs were obtained. The extension of the interval to 40 h associated with a two to three times mating resulted in a recovery of 42.9% fertilized eggs but most (70%) of these were already at the two-cell stage. In Experiment 2, eCG resulted in similar superovulatory responses in Merino ewes as the more labour requiring FSH treatment (8.1 ± 4.5 versus 7.5 ± 4.1 corpora lutea (CL); 6.3 ± 3.0 versus 6.8 ± 4.0 oocytes/ zygotes; 39.4% versus 40.6% fertilization rate). In Experiment 3, following superovulation with pFSH (Folltropin®) the number of CL was not different among Merino, Finn, Crossbreds (Blackface X Finn) and Texel sheep (8.6 ± 5.2; 10.3 ± 4.5; 8.5 ± 3.8; 8.2 ± 2.8, respectively) as was the number of recovered oocytes/ zygotes (7.4 ± 5.6; 9.8 ± 4.3; 7.3 ± 3.8; 6.4 ± 2.9, respectively). However, the number of unfertilized ova was higher (P < 0.05) in Finn sheep as compared with Crossbreds and Texel sheep (5.0 ± 3.3 versus 2.2 ± 2.3 and 1.9 ± 2.6). Similarly, the fertilization rate was higher (P < 0.05) in Crossbreds and Texel sheep (64.4% and 65.5%) as compared with Finn and Merino sheep (38.3% and 42.5%). In Experiment 4, it was shown that in Merino sheep purified FSH supplemented with 68.6% LH resulted in lower (P < 0.05) superovulatory responses as compared with purified FSH supplemented with 133.1% LH or Folltropin (LH contamination 0.1%) (4.7 ± 3.3 versus 8.8 ± 3.8 and 8.6 ± 5.2 CL; 3.8 ± 2.5 versus 7.4 ± 3.6 and 7.4 ± 5.6 oocytes/zygotes, respectively). A three times repeated superovulatory treatment and oviductal flush per animal at monthly intervals did reduce (P < 0.05) the number of CL, but had no deleterious effect on zygote yields and the percentage of microinjectable zygotes. We conclude that (1) at least a two to three times mating is required to obtain acceptable fertilization rates; (2) the interval between mating and recovery should be 24–26 h in order to obtain zygotes; (3) eCG results in similar superovulatory responses as FSH; (4) Folltropin® is a suitable drug to induce superovulation in sheep; (5) the LH content of the FSH preparation plays a significant role in the superovulatory response of sheep; (6) superovulation and embryo recoveries can be repeated at least three times per animal without decrease in efficiency.