214 EFFECT OF OVARIAN SUPERSTIMULATION ON EXPRESSION OF GENES ASSOCIATED WITH THE OOCYTE DEVELOPMENTAL COMPETENCE OF NELORE COWS

Abstract

The P36 protocol has contributed to the genetic improvement of Brazilian herd through its successful use in embryo transfer programs. We aimed to investigate the effect of P36 protocol on embryo yield and mRNA expression of genes correlated with the competence of cumulus–oocyte complex (COC): receptors of FSH (FSHR), EGF (EGFR), and pentraxin 3 (PTX3) in cumulus cells; receptors of LH (LHR) and angiotensin 2 (AT2) in granulosa cells; and GDF9, BMP15, and histone H2A (H2A) in oocytes. Multiparous Nelore cows were allocated in control and P36 groups. Control group (non-superovulated, n = 15) received a progesterone intravaginal device (P4, 1.0 g, Primer®, Tecnopec, Sao Paulo, Brazil) and 2.5 mg of oestradiol benzoate (EB, IM, BER-BE®, Syntex, Buenos Aires, Argentina) at a random day of the oestrous cycle (Day 0). A PGF2α analogue (150 mg d-cloprostenol, IM, Prolise®, RARS SRL) was administered (Day 8) and Primer® was removed. The P36 group (n = 10) received a Primer® and 2.0 mg of EB (Day 0). The FSH treatment (160 mg Folltropin®, Bioniche Animal Health, Ontario, Canada) was initiated at decreasing doses: 40, 30, 20, and 10% of the total dose twice daily for 4 days (Day 5). The PGF2α analogue was administered (Day 8) and after 36 h primer was removed. Animal slaughter to ovary collection was performed 12 h after Primer® removal (Day 9). Some of the oocytes were matured (TCM199), fertilized with Nelore semen (n = 6), and cultured (SOF-synthetic oviduct fluid) to the blastocyst stage. Embryos were removed from culture (Day 6), allocated in 5 pools with 5 embryos in each group, and subjected to RNA extraction. Remaining oocytes were denuded from cumulus and zona pellucida (vortex and Protease®, Sigma-Aldrich, St. Louis, MO, USA). Pools of 20 oocytes and of their respective cumulus cells (n = 6 pools; control group and n = 4 pools, P36 group) were subjected to RNA extraction (RNeasy kit, Qiagen, Valencia, CA, USA). Gene expression was performed by real-time RT-PCR using oligo-dT in reverse transcription and bovine-specific primers. Expression of cyclophilin A was used as endogenous control. Change to developmental rates to the blastocyst stage and transcript abundance were compared by t-test and significance was considered when P < 0.05. Blastocyst rates were also similar (P > 0.05) in groups P36 (40/99; 40%) and control (16/43; 37%). Expression of H2A, EGFR, FSHR, and PTX3 in cumulus cells did not differ (P > 0.05) among treatment groups. The expression of GDF9 and BMP15 in cumulus cells was higher (P < 0.05) in the P36 group, but in oocytes these transcripts were more expressed in the control group (P < 0.05). Although important genes (GDF9 and BMP15) were less expressed in oocytes from superstimulated cows, the maintenance of H2A in oocytes, as well as PTX3, EGFR, and FSHR, and the increases in GDF9 and BMP15 expression in cumulus cells do not seem to affect oocyte competence due to the similar embryo yield of both groups. Supported by FAPESP.