Veterinary World (May 2014)
Effect of harvesting technique and presence or absence of corpus luteum on in vitro development after parthenogenetic activation of oocytes recovered from buffalo ovaries
Abstract
Aim: The present study was aimed at assessing the influence of three oocyte harvesting (aspiration, puncture and slicing) methods and presence or absence of corpus luteum (CL) on recovery efficiency and subsequent in vitro maturation and parthenogenetic development of immature oocytes recovered from buffalo ovaries. Materials and Methods: Buffalo ovaries were collected from local slaughter house and oocytes were collected by using aspiration, puncture and slicing methods. Collected oocytes were matured in vitro in TCM-199 medium supplemented with 10% fetal bovine serum (FBS), 0.22 mM sodium pyruvate, 10 µg/mL of follicle-stimulating hormone, 6 IU/mL luteinizing hormone, 1 µg/mL 17-β estradiol, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. In vitro matured oocytes were activated by ethanol (7%) followed by 6-DMAP (2mM) and cultured to assess the in vitro developmental capacity. Results: The average total number of oocytes recovered per ovary was significantly (P<0.05) higher in slicing (7.88±0.54) than the aspiration (2.50±0.11) and puncture (3.59±0.18) methods. The number of culture grade oocytes per ovary was significantly higher in slicing (5.40±0.29) than in puncture (2.45±0.12) and aspiration (1.94±0.11) methods. However, the percentage of culture grade oocytes was higher in the aspiration method (77.67%), compared to the puncture (68.24%) or slicing (68.51%) methods. The oocyte recovery was significantly lower (P<0.05) in CL containing ovaries than that of ovaries without CL (3.01vs.5.17). However, the presence of CL did not affect the oocytes ability to reach the MII stage (73.07%vs.75.77%). The results showed that the rates of Cumulus oocyte complexes (COCs) that reached the metaphase-II (M-II) stage were similar in aspiration, puncture and slicing techniques (77.29, 76.12 and 73.92% respectively). The oocytes obtained were matured and parthenogenetically activated in vitro using ethanol and 6-DMAP. The oocyte collection methods and presence or absence of CL did not influence the subsequent embryonic developmental competence. Conclusion: It can be concluded that slicing method can be used as an alternative to aspiration and puncture methods to recover oocytes from buffalo ovaries; as slicing method resulted higher oocyte yield and similar in vitro development rate when compared with other methods.
Keywords
