130 In vitro embryo production using frozen semen from cloned and non-cloned Bos indicus bulls

Abstract

Reproductive biotechnology has continued to evolve rapidly, allowing the development of techniques to increase reproductive efficiency and contribute to the genetic improvement of cattle. Some of these techniques include the in vitro maturation and IVF of oocytes, sperm sexing and cloning. These modern assisted reproductive techniques can help produce offspring of desired genetic characteristics and of a pre-determined sex. However, studies of the bull’s contribution to in vitro reproductive performance are scarce in the Brahman breed. The aim of this study was to compare oocyte maturation and embryo production in vitro using frozen semen from 5 Brahman bulls (Bos indicus), cloned (n = 1) and non-cloned (n = 4), with characteristics and genetics of high commercial value. The age of the bulls at the time of semen collection and cryopreservation ranged from 2 to 7 years. The oocytes were obtained on 2 different dates (45 days between collections) using pooled oocytes collected by ovum pickup at random stages of the oestrous cycle, from a total of 15 Brahman donor cows. Oocytes were transported to a laboratory in the State of Chiapas, Mexico (Genemex Internacional). The oocytes were cultured in maturation medium for 24 h. For IVF, conventional semen was used from one bull (B1) and his clone (B1²), the grandson of B1 (B2), and 2 non-related bulls (B3 and B4). The gametes were co-incubated for 22 h and afterward placed in medium for embryo development and cultured for 7 days in a humid atmosphere with 5% CO₂ in air. Of the matured oocytes, 36/43 (84%), 16/32 (50%), 101/143 (70%), 46/67 (68%) and 53/65 (81%) were fertilized using semen from B1, B1², B2, B3 and B4, respectively. Of the fertilized oocytes, 15/30 (50%), 8/16 (50%), 45/101 (44%), 21/46 (45%) and 18/53 (34%) resulted in transferrable embryos, corresponding to semen from the same bulls, respectively. This would appear to be the first scientific report in Mexico about the use of semen from a cloned bull for in vitro embryo production. In IVF, similar results were observed between B1 and a non-related bull (B4). Similar results in transferrable embryos were observed between B1 and B1² but also similar to a related bull (B3) and a non-related bull (B4). A Fisher’s exact test of the IVF results comparing B1 and B1² found a significantly (P < 0.05) higher number of fertilized oocytes for B1. However, a significant difference was not found (P > 0.05) concerning the number of transferrable embryos produced by these two bulls. In conclusion, the Brahman bulls in this study differ in their contribution to IVF and embryo production. Further studies are required to determine the factors responsible for such effects, e.g. age differences or clone versus non-clone mosaicism. Results from this research contribute to the study and development of assisted reproductive techniques for increasing in vitro production efficiency in Zebu cattle.

We thank the Rosales family, from El Herradero Ranch, in the State of Campeche, Mexico, for allowing the use of their cattle for this project.