In Vitro Fertilization of Vitrified Immature or Mature Oocytes as Affected by Mechanical or Natural Denudation

Abstract

he current study aimed to evaluate the role of cumulus-corona radiate complex of immature or in vitro matured bovine oocytes on their vitrification, in vitro fertilization and embryo development. Bovine ovaries were collected from an abattoir and follicular oocytes (3-8 mm in diameter) were aspirated. Oocytes were examined and classified into cumulus oocytes-complexes (COCs), natural denuded oocytes (NDOs) and mechanically denuded oocytes (MDOs). Natural COCs were mechanically denuded by repeated pipetting in phosphate buffer solution (PBS). All types of oocytes were cryopreserved by vitrification as immature or in vitro matured for 24 h prior to vitrification by open-pulled straw cryodevice. After at least 2 weeks of vitrification, all types of vitrified oocytes were evaluated for survival and viability (Normal and abnormal oocytes). Morphologically normal immature oocytes were matured and fertilized in vitro, while morphologically normal mature oocytes were directly fertilized in vitro for determination of cleavage rate (CR). After co-culture of cleaved oocytes for 7 days, rate of morula (MPR) and/or blastocysts (BPR) production was recorded. Results show that survival (SR) and normality (NR) rates were the highest for CCOs (84.47 and 81.73%), followed by MDOs (73.28 and 62.0%), and the lowest for NDOs (60.71 and 54.39%), respectively (P<0.05). SR was higher (P<0.05) for mature than immature oocytes (78.03 vs. 67.60%), while NR was nearly similar for both. CR, MPR and BPR were higher (P<0.05) for CCOs (45.28, 18.05 and 15.28%) than those of MDOs (29.72, 6.06 and 3.03%) and NDOs (25.24, 7.69 and 7.69%), respectively. CR was higher (P<0.05) in mature than in immature oocytes (41.93 vs. 29.36%), while MPR and BPR were nearly similar in both. Based on the obtained results, cumulus cell surrounding cumulus oocytes-complexes play a vital role for survival of bovine oocytes during vitrification and successful in vitro maturation and fertilization as well as embryonic development to morula and blastocyst stages.