Active immunization against GnRH as an alternative therapeutic approach for the management of Bos indicus oocyte donors diagnosed with chronic cystic ovarian disease.

The aim of this study was to evaluate the effect of active immunization against GnRH in Nelore (Bos indicus) cows repeatedly used as oocyte donors that developed chronic cystic ovarian disease (COD). In the first experiment, ovarian and uterine features were first compared between COD cows (n = 15) and healthy cows (n = 22, cycling control group) from the same breed and herd. Cows with COD had a greater number of large (P < 0.0001) and medium follicles (P < 0.01) but lesser small follicles (P < 0.05) than cycling controls. Mucometra was diagnosed in 73.3% of COD cows, but in none of the controls. No difference in average thickness of the endometrium was detected between groups; however, endometrial thickness and mucometra score were negatively correlated (R = -0.73, P = 0.0029) in COD cows. In the second experiment, COD cows were randomly allocated into two experimental groups, which received two 1.0 mL SC injections of either an anti-GnRH vaccine (COD immunized group, n = 8) or saline (COD control group, n = 7), given 28 days apart. Cows were examined weekly by transrectal ultrasonography during nine consecutive weeks after the first injection to evaluate the number and distribution of follicles among size classes, endometrial thickness, and presence of clinical mucometra. Vaccination against GnRH resulted in a progressive suppression of follicle growth and a reduction in the average size of the largest follicle, as well as in the number of large follicles (P < 0.01) in COD immunized cows compared with COD controls from week 7 onwards. Conversely, the number of small follicles in the COD immunized group increased after week 5 and was greater (P = 0.0023) than controls on week 9. Endometrial thickness and mucometra score were not affected (P > 0.05) by immunization against GnRH. In the third experiment, the COD immunized cows with effective suppression of follicle growth four weeks after the 2nd injection (n = 6) were submitted to three consecutive ovum pick-up (OPU) sessions (weeks 10, 11, and 12) for in vitro embryo production (IVEP). Cumulus-oocyte complexes (COC) collected from slaughterhouse ovaries were used as controls for IVEP. COD cows with produced 25.0 ± 3.8 COC per OPU session with no apparent detrimental effect of anti-GnRH vaccine on oocyte developmental potential in vitro, i.e., we observed similar cleavage rate (P = 0.5914) and greater blastocyst rate (P = 0.0177) in immunized cows compared with COC from slaughterhouse controls. Finally, in the fourth experiment wave emergence and follicular dynamics after follicle ablation were compared between COD immunized cows with effective suppression of follicle growth and a subset (n = 6) of the cycling, control group. No follicles grew beyond 4 mm diameter after follicle ablation in the COD immunized group, whereas a normal follicular wave emergence occurred in cycling controls. Antral follicle count was similar between cycling controls and COD immunized groups at 24 h and 96 h post-follicle ablation (P > 0.05), but greater in cycling controls at 48 h and 72 h post-follicle ablation (P < 0.05). In summary, our results suggest that active immunization against GnRH is effective to induce the regression of follicular cysts as well as increase the number of small follicles growing on the ovaries, in oocyte donors diagnosed with chronic COD, with no apparent negative effect on oocyte developmental potential in vitro.

Effect of delipidant agents during in vitro culture on the development, lipid content, gene expression and cryotolerance of bovine embryos.

In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high-lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L-carnitine and the trans-10 cis-12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n = 616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L-carnitine (n = 648), SOF medium with 5% FBS and 0.6 mg/ml of L-carnitine; T3: CLA (n = 627), SOF medium with 5% FBS and 100 µM trans-10 cis-12 CLA; and T4: L-carnitine + CLA: (n = 597), SOF medium with 5% FBS plus 0.6 mg/ml L-carnitine and 100 µM trans-10 cis-12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p < .05) blastocyst rate on D7 (T1 = 49 ± 3.5; T2 = 39 ± 3.0; T3 = 42 ± 3.9 and T4 = 39 ± 3.9), but did not affected gene expression (p > .05). Although embryos cultured in the presence of L-carnitine contained fewer (p < .05) lipid droplets than the control embryos, they showed a lower re-expansion rate 24 hr post-thaw than those (p < .05). In conclusion, although L-carnitine reduced the amount of lipids in cultured embryos, the use of L-carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.