In vitro maturation of Nelore breed (Bos taurus indicus) oocytes using either purified porcine FSH (pFSH) or recombinant human FSH (rhFSH): results from a large-scale in vitro embryo production routine

Leticia Prates Martins¹Luany Alves Galvao Martinhao¹Joao Gabriel Viana Grazia²Otavio Augusto Costa Faria¹Ricardo Alamino Figueiredo³Joao Henrique Moreira Viana^1,3*

• ¹University of Brasilia, Brasilia, Distrito Federal, Brazil
• ²FIVX Apoyar Biotech, Juiz de Fora, Brazil
• ³Brazilian Agricultural Research Corporation (EMBRAPA), Brasília, Brazil

This study evaluated whether porcine FSH (pFSH) could be replaced by follitropin-alpha, a recombinant human FSH (rhFSH), during in vitro maturation (IVM) of cumulus-oocyte complexes (COC) recovered from Nelore (Bos taurus indicus) cattle in a large-scale in vitro embryo production (IVP) program. We performed three experiments, all using grade I COC (n=10,208) submitted to IVM in TCM199 without FSH (-FSH, negative control) or supplemented with either 0.5 μg/mL pFSH (Folltropin-V) or 0.1 IU rhFSH (Gonal-F). The remaining procedures, media, and culture conditions for in vitro embryo production (IVP) were similar. In Exp. 1, COC (n=2,791) were IVM in the absence of FSH (-FSH) or with pFSH or rhFSH, and underwent IVP. Blastocysts were assessed for hatching as fresh or after vitrification and warming. Blastocyst rates were greater (P<0.05) in groups treated with than without FSH (39.3% and 40.6% vs 34.8% for pFSH, rhFSH and -FSH, respectively; P=0.0244). However, there was no effect of treatment on hatching rates of fresh (74.5%, 79.0% and 70.1% for -FSH, pFSH, and rhFSH, respectively; P=0.2621) or vitrified blastocysts (75.0%, 69.6% and 73.2% for -FSH, pFSH, and rhFSH, respectively; P=0.7623). In Exp.2, COC (n=720) were IVM in groups -FSH, pFSH, or rhFSH, and the expanded blastocysts produced were stained with Hoechst 33342 and propidium iodide. Blastocysts from the rhFSH group presented more cells in the trophoblast (110.3±5.6 vs 80.0±5.7 and 878.4±5.1 for rhFSH, -FSH, and pFSH, respectively; P=0.0002), but not in the ICM (P=0.3231), when compared with pFSH and -FSH groups. In Exp. 3, we used the same experimental design as in Exp. 1 but in a commercial IVP routine, which included the use of COC (n=6,697) recovered both by OPU and from slaughterhouse, Y-sorted semen, and the transfer of part of the blastocysts produced. Both pFSH and rhFSH increased cleavage (76.0% and 75.1% vs 71.6%, P=0.0035) and blastocyst rates (38.1% and 39.5% vs 34.3%, P=0.0102), but not pregnancy rate (43.8% and 33.3% vs 56.0%, P=0.1080), compared with -FSH. In summary, follitropin-alpha is an alternative to pFSH as a medium supplement for IVM of bovine COC.