209 EXPRESSION OF GENES RELATED TO OVULATORY CAPACITY (LHR AND AGTR2) IN GRANULOSA CELLS FROM SUPERSTIMULATED OR NOT SUPERSTIMULATED ANGUS COWS

Abstract

Multiple ovulation and embryo transfer (MOET) has contributed to genetic improvement of Brazilian cattle and the P36 superstimulatory treatment protocol has been used successfully in MOET programs. Recently, increased mRNA levels of LH receptor (LHR) and angiotensin II receptor type 2 (AGTR2) have been reported in granulosa cells (GC) of superstimulated Nelore cows using P36 protocol and the efficacy of the P36 protocol has been investigated with the addition of a LH stimulus (eCG or LH), on the last day of treatment. We aimed to evaluate mRNA abundance of AGTR2 and LHR, related to ovulatory capacity, in GC of superstimulated Angus cows by different variations of the P36 protocol. Multiparous Angus cows (n = 37) were randomly assigned to four groups: control, P36, P36+eCG, and P36/FSH+LH. The control group (non-superstimulated cows, n = 7) received an intravaginal device containing progesterone (1.0 g; Primer®; Agener Animal Health, São Paulo, SP, Brazil) and 2.5 mg of oestradiol benzoate (EB; RIC-BE®; Agener Animal Health; IM) on a random day of the oestrous cycle (Day 0). After 8 days (Day 8) cows were treated with PGF2α (150 mg d-cloprostenol; Prolise®; Agener Animal Health; IM; 7:00 a.m.) and the intravaginal device was removed (7:00 p.m.). Cows from P36 group (n = 10) received the intravaginal device and 2.5 mg of EB at the beginning of the protocol (Day 0). Starting 5 days later (Day 5), 200 mg pFSH (Folltropin®; Bioniche Animal Health, Belleville, ON, Canada) was administered twice daily by IM injections over 4 days; on Day 7, PGF2α was administered and the intravaginal device was removed 36 h later (Day 8, 7:00 p.m.). Groups with the addition of a LH stimulus (eCG or LH administration) were submitted to the P36 protocol with replacement of the last two FSH doses by 200 IU eCG (P36+eCG; Novormon®, MSD Animal Health, São Paulo, SP, Brazil, IM; n = 10) or 2 mg pLH concomitant with the last two doses of FSH (P36/FSH+LH; Lutropin®, Bioniche Animal Health, IM; n = 10). Cows in all groups were slaughtered on Day 9, 12 h after progesterone-device removal and ovaries were transported on ice to the laboratory immediately after slaughter. Non-superstimulated dominant follicles and superstimulated follicles were dissected from the ovaries to obtain GC and total RNA was extracted. Amplification of housekeeping (peptidyl-prolyl isomerase A; PPIA) and target genes was performed by real-time RT–PCR according to the Sybr Green protocol. Relative gene expression was determined by Pfaffl's equation. The mRNA abundance was compared by Tukey's test and the significance level was P < 0.05. In GC, the mRNA abundance (mean ± s.e.m.) of LHR and AGTR2 was higher in the control group (1.02 ± 0.24 and 3.76 ± 1.07, respectively; P < 0.05) when compared with the superstimulated groups: P36 (0.33 ± 0.24 and 1.02 ± 0.21), P36+eCG (0.42 ± 0.10 and 1.40 ± 0.33) and P36/FSH+LH (0.38 ± 0.07 and 1.45 ± 0.49). In conclusion, the superstimulatory treatments decrease mRNA levels of LHR and AGTR2. We acknowledge CAPES for E. Lucacin's scholarship and FAPESP–São Paulo Research Foundation for grant number 2011/50593-2.