Effects of ovarian synchronization associated with a two-step IVM strategy on in vitro embryo production in cattle

Current strategies for in vitro embryo production (IVP) include OPU on random day of estrous cycle. Oocytes recovered are heterogeneous in chromatin configuration, oocyte-cumulus coupling, transcriptional activity and developmental competence. Since oocyte in vitro maturation (IVM) should promote synchrony between nuclear and cytoplasmic maturation, this heterogeneity may underlie the low efficiency of current IVP strategies. This study aimed to characterize oocyte population obtained by OPU and to develop a protocol to synchronize oocytes destined to IVP in Nellore. In experiment 1, 14 Nellore cows were subjected to two treatments in crossover design: 1) OPU at random day (Control); 2) aspiration of follicles >2mm at random day (D0), two IM injections of FSH (Folltropin; 24+16mg) 10h apart on D2, OPU from follicles >2mm on D5 (ASP/FSH-40). In experiment 2, 12 Nellore cows were subjected to the same treatments as in experiment 1, except for reduction of FSH dose (Folltropin; 12+8mg) (ASP-FSH/20). At OPU, antral follicles were counted and classified as small (3-5mm), medium (6-8mm) and large (>8mm). Oocytes were counted, fixed and stained with Hoechst 33342 then examined by fluorescence microscopy to be classified according with chromatin condensation and germinal vesicle configuration in GV0, GV1, GV2, GV3 and GVBD (Lodde et al., 2007). Data were arcsine transformed and groups compared by paired T test (P<0.05). Total number of follicles aspirated at OPU was not affected by treatment in any experiments. In experiment 1, 285 follicles were aspirated in Control group and 306 in Asp/FSH-40 group, while in experiment 2, total numbers 468 and 463 follicles were aspirated in Control and ASP-FSH/20 groups, respectively. However, in experiment 1, ASP-FSH/40 treatment reduced percentage of small follicles (P=0.02). Percentages of small, medium and large follicles were 67.6*, 24.1 and 8.3 in Control, and 50*, 35.4 and 14.6 in ASP-FSH/40, respectively. Distribution of oocytes in different categories of chromatin compaction was only affected by treatment in experiment 2, with ASP-FSH/20 reducing the percentage of GV3 oocytes (P=0.01). In experiment 1, percentages of GV0, GV1, GV2, GV3, GVBD and degenerated oocytes in Control group were 1.34, 0.25, 39.06, 46.37, 0.12 and 12.86 respectively (n=177), while they were 0, 4.14, 40.49, 38.94, 0 and 16.42 in ASP-FSH/40 respectively (n=195). In experiment 2, percentages of GV0, GV1, GV2, GV3, GVBD and degenerated oocytes were 0, 1.95, 47.55, 40.47*, 0 and 10.03 in Control group respectively (n=267), while they were 0, 4.22, 49.53, 30.50*, 0 and 15.75 in ASP-FSH/40 respectively (n=265). In conclusion, oocytes recovered by OPU at a random day in Nellore were mostly at intermediate (GV2) or advanced (GV3) stage of chromatin compaction. Since these stages have been associated with higher developmental competence, this finding may help to explain the suitability of the Nellore breed to IVP. Protocols combining antral follicle aspiration and low doses of FSH to stimulate follicle recruitment may further homogenize the oocyte population for OPU in Nellore. However, further improvement of ASP-FSH protocols and fine-tuning with IVM culture system are needed before their real impact on IVP outcomes can be determined. FAPESP, CAPES.