46 Morphometric evaluation of cattle immature and matured oocytes pre- and post-cryopreservation

Successfully cryopreserving both immature and mature cattle oocytes would facilitate the management of the timing of IVM, fertilization, and culture. To date, oocyte cryopreservation is the most promising and cost-effective option for the storage of female germplasm. However, cryopreservation can alter oocyte morphology, potentially affecting viability and developmental competence. The aim of this study was to evaluate the morphometric parameters and morphology of immature and mature cryopreserved and fresh (non-cryopreserved) oocytes. The heterogeneous cattle ovaries of unknown reproductive status were collected at the local abattoir. The aspiration method for oocytes retrieval from ovaries was carried out using 10-mL disposable syringes and an 18 G sterile hypodermic needle. The collected oocytes were washed in modified Dulbecco’s PBS and modified M199 and then matured in 500 µL of TCM-199 medium supplemented with 10% fetal bovine serum, FSH, LH, and estradiol hormone before being covered with 250 µL of mineral oil. The oocyte maturation rate was determined by expansion of cumulus–oocytes complexes (COCs) after 22 h of IVM pre-cryopreservation. The retrieved oocytes were either non-cryopreserved (fresh oocytes) or cryopreserved (immature and mature oocytes) using the conventional straw vitrification method, in which they were exposed to equilibration and vitrification solutions. The vitrified oocytes straws were loaded on the aluminum cryocane, then stored inside the liquid nitrogen tank (−196°C) until thawing. The vitrified oocytes straws were removed from the liquid nitrogen tank and exposed inside warm (37°C) water for 1 min during thawing. The oocytes straws were cut at both ends and emptied into thawing solutions to remove the intracellular cryoprotectants. Following thawing, oocytes were fixed in formaldehyde with 4% PBS for morphometric analysis. The diameters (µm) of the oocyte ooplasm and oocyte zona pellucida (ZP), zona pellucida width (ZPW), granulosa cell width (GRSW), and zona pellucida granulosa cell width (ZP GRSW) were measured with the aid of a microscope connected to computer-assisted sperm analysis system (Sperm class Analyzer®, Microptic S.L.) at ×10 magnification for both vitrified and fresh oocytes. The data were analyzed using ANOVA statistical program of GenStat® program (VSNi). A significance level of P < 0.05 was used. Treatment means were separated using Fisher’s protected t-test. The data are presented as mean ± standard deviation. In the current study, the cytoskeletal structure of the oocytes was observed pre- and post-cryopreservation. The results showed no significant differences in oocyte ZP and ZPW between vitrified (immature and mature) and fresh oocytes. However, a significant difference was observed in oocyte GRSW (vitrified mature oocytes = 67.56 ± 32.61 µm, vitrified immature oocytes = 78.46 ± 33.51 µm, and fresh oocytes = 104.23 ± 27.21 µm) and ZP GRSW (vitrified mature oocytes = 232.39 ± 50.34 µm, vitrified immature oocytes 247.25 ± 66.92 µm, and fresh oocytes = 289.30.80 ± 42.19 µm). In conclusion, the cryopreservation process did not affect the structural integrity of ZP in fresh and vitrified oocytes. However, it affected the granulosa cells in both vitrified immature and mature oocytes, with the most significant reduction seen in mature oocytes, which could potentially affect the developmental competence and viability of the oocytes post-thawing.