145 Inhibitory effects of gossypol on bovine in vitro embryo production

Abstract

Cottonseed and its derivatives are frequently used in cattle feed as an effective dietary fibre supply and high protein and energy food source. However, the cotton plant contains gossypol, which in its free form induces male and female infertility. Therefore, this study aimed to evaluate the effect of gossypol supplementation on bovine in vitro embryo production. Ovaries were retrieved from slaughterhouses, and cumulus-oocyte complexes (COC) were recovered by follicular puncture. Based on free gossypol concentration present on the in vitro maturation, sperm capacitation, IVF and in vitro culture media, grades I, II and III COC (n=646) were divided in 3 treatments: 0μg mL−1 (control), 5μg mL−1 (G5) and 10μg mL−1 (G10). The COC were matured under a humidified atmosphere of 5% CO2 in air at 38.5°C for 24h in 90-μL droplets containing TCM-199 supplemented with 10% FCS, 0.2mM sodium pyruvate, LH, FSH, 75μg mL−1 amikacin, 17β-oestradiol. Each droplet corresponded to one replicate (n=14) and contained 15 to 18 COC. Matured COC and sperm were co-incubated in droplets (8-13 COC per 90μL) of TALP-IVF media supplemented with 6mg mL−1 BSA, 0.2mM sodium pyruvate, 30μg mL−1 heparin, 20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine, 75μg mL−1 amikacin under a 5% CO2 humidified atmosphere at 38.5°C, for 20h. For IVF, non-sexed frozen-thawed semen was selected with Percoll® gradient. The resulting pellet was subjectively evaluated for motility and concentration and then diluted to final concentration of sperm mL−1 with fertilization medium. Presumptive zygotes were then cultured in 90-μL droplets of SOFaaci medium supplemented with 2.7mM myo-inosytol, 0.2mM pyruvate, 2.5% FCS (v/v), 5mg mL−1 BSA, 75μg mL−1 amikacin, and maintained for 8 days at 38.5°C in a humidified atmosphere with 5% CO2 in air. Cleavage, blastocysts production and hatching rates were evaluated at Days 3, 7 and 8, respectively. Data were submitted to ANOVA for parametric data and Wilcoxon test for non-parametric variables using the SAS software (SAS Institute Inc., Cary, NC, USA). Significance level was set at 5%. Cleavage rates of the control (81.05%) and G5 (71.85%) were higher compared with G10 (19.64%; P < 0.0001). Blastocyst production was lower in G5 (12.18%) compared with control (30.35%), and the addition of 10μg mL−1 of free gossypol (G10) completely inhibited embryo development (0%; P < 0.0001). As for the percentage of hatched blastocysts, the control (66.75%) had greater values compared with G5 (34.52%; P < 0.0001). Thus, the addition of 5 and 10μg mL−1 of free gossypol are extremely hazardous for in vitro bovine embryo development. Whether these deleterious effects take place in a similar fashion during in vivo embryo production remains to be investigated.