306 EFFECT OF DIFFERENT IN VITRO MATURATION MEDIA ON DEVELOPMENTAL POTENTIAL OF GOAT OOCYTES ALREADY FOUND DENUDED AT COLLECTION

Abstract

A grade classification (I, II, and III) based on the number of cumulus layers and oocyte morphology is currently used by many laboratories. Oocytes found denuded at collection (grade III) are considered not suitable and routinely discarded. Thus, if a particular strategy could be applied to their use in labour-intensive processes, such as ovum pickup, it would be a benefit. This experiment was performed to examine the effect of IVM medium composition to produce goat embryos in vitro using oocytes already found denuded at collection (DOC). In total, 411 DOC and 141 intact COC (control treatment) obtained by slaughterhouse ovaries were analysed in 4 replicates. The maturation medium consisted in TCM199 supplemented either with (1) 10 ng mL^–1 of EGF and 100 µM cysteamine (simplified); (2) 10 ng mL^–1 of EGF, 5 IU mL^–1 of hCG, 10 IU mL^–1 of eCG, 19 ng mL^–1 of IGF-1, 2.2 ng mL^–1 of FGF, 5 µg mL^–1 of ITS, 90 µg mL^–1 of l-cystein, 0.1 mM β-mercaptoethanol, 75 µg mL^–1 of vitamin C, 720 µg mL^–1 of glycine, 0.1 mg mL^–1 of glutamine, and 110 µg mL^–1 of pyruvate (semidefined); or (3) 10% fetal calf serum (FCS), 100 µM cysteamine, and 50 ng mL^–1 of oFSH (complex). Both DOC and COC were subjected to IVM, IVF, and IVD as previously described (Souza-Fabjan et al. 2014, Theriogenology 81, 1021–31). The COC were matured only in simplified medium. On Day 8, all expanded blastocysts were fixed and stained with Hoechst for cell counting. Statistical analysis was performed using all tests with a significant interval of 95%. All variables were compared among treatments using ANOVA and SNK test. The results are described as mean per replicate ± s.e.m. No significant differences were found in simplified, semidefined, or complex medium, respectively, in cleavage rate (52 ± 7.5, 60 ± 9.4, or 51 ± 15.0%), blastocyst from cleaved (36 ± 3.9, 39 ± 9.3, or 41 ± 4.8%), blastocyst from initial DOC (19 ± 5.0, 23 ± 8.1, or 21 ± 3.3%), hatching rate (55 ± 22.9, 55 ± 15.9, or 52 ± 14.8%), or total blastomeres number (184 ± 12.6, 179 ± 12.4, or 190 ± 13.8). The control COC showed no significant differences to any DOC treatment on cleavage (77 ± 3.4%) and blastocyst from cleaved (60 ± 2.2%). However, the blastocyst rate from initial COC was higher (46 ± 0.5%; P < 0.05) than all DOC treatments. Even though the blastocyst yield was lower than COC (~21 v. 46%), it is reasonable to affirm that it is possible to produce embryos from oocytes that would not be utilised, which may represent additional number of embryos. The blastocyst cell numbers in COC (192 ± 13.7) were similar (P > 0.05) to DOC, indicating that the goat embryos produced were of good quality. In conclusion, the inclusion of more complex substances in IVM media did not increase the development rate of DOC and, therefore, more simple IVM media could be used for this purpose. Finally, the goat embryos produced had satisfactorily number of blastomeres, demonstrating that the in vitro development step is able to generate good quality embryos from grade III oocytes. Therefore, some oocytes that in general would be discarded will develop to blastocysts and may represent benefits, especially after ovum pick-up from genetically valuable goats.