Comparative analysis of two different techniques to obtain porcine cumulus-oocyte complexes”

LORENZO, MS.; TELLO, MF.; MARURI, A.; GAMBAROTTA, MC.; FISCHMAN, ML.; LOMBARDO, DM.

Buenos Aires

Jornada; III Jornadas Internacionales del Instituto de Investigación y Tecnología en Reproducción Animal – INITRA; 2012

Instituto de Investigación y Tecnología en Reproducción Animal - INITRA. Facultad de Ciencias Veterinarias. UBA

The oocyte quality is one of the factors that determine the success of in vitro embryo production; the methodology used to obtain cumulus oocytes complexes (COCs) affects this quality. In others species it has been reported that the amount of oocytes collected per ovary with slicing is higher than with others techniques such as follicles aspiration and dissection; in addition it has been demonstrated that the recovery of oocytes by slicing technique decreases the apoptosis rates in immature COCs. Apoptosis can be used as a quality indicator because most of the follicles degenerate during their developmental by a process, follicular atresia, in which apoptosis is involved. The aim of this study was to compare two methods to obtain porcine COCs from slaughter ovaries. In aspiration technique, follicles from 3 to 6 mm were puncture using a 18G needle an a 10mL syringe, whereas in slicing COCs were obtained by slice ovary surface. They were classified under stereomicroscope basing on their morphology and those who had at least a complete layer of cumulus cells surrounding the oocyte were selected to in vitro maturation (IVM). In oocytes and cumulus cells from immature and in vitro maturated COCs Annexin V essay was made to evidence early apoptosis, while late apoptosis was assessed by TUNEL technique. After 44-48 hs of IVM the percentage of nuclear maturation was measured by staining denuded oocytes whit Hoechst 33342 (observing the metaphase II and the first polar body extrusion). The amount of COCS recovered per ovary (average) was higher using slicing (11, 79 versus 26, 66). The nuclear maturation rates differed between techniques, obtaining 62% by aspiration and only 34% by slicing. Both early and late apoptosis were found in cumulus cells but only early apoptosis was found in oocytes from immature COCs whereas in in vitro maturated COCs early and late apoptosis was observed both in cumulus cells and oocytes. Results were contradictory when comparing apoptosis between techniques. Although slicing method allowed us to draw on pre pubertal ovaries, those smaller follicles contain lower quality oocytes, affecting their ability to mature in vitro. There was an increase in apoptosis during in vitro maturation, supporting the theory that biochemical changes occurring during atresia are similar to those that happen in oocyte maturation.