Preliminary results the cytogenetic evaluation of in vitro oocyte maturation in dogs

SEBASTIÁN DEMYDA PEYRÁS; JESUS DORADO; MANUEL HIDALGO; DANIEL ACHA VALLS; KARINA CAÑON ; MIGUEL MORENO MILLÁN

Krakow

Congreso; 19th Colloquium of Animal Cytogenetics and Gene Mapping; 2010

National research institute of animal production department of animal immuno and cytogenetics, Poland

Cytogenetic study of in vitro maturation of canine oocytes has no development at present time. The lack of validated protocols in this species requires a preliminary work to adjust existing ones in other species to the special requirements of canine oocytes. The aim of this study was to evaluate two different protocols for cytogenetic analysis previously used for bovine oocytes in canine in vitro matured oocytes. Moreover, these two protocols were evaluated at two different maturation times: 48 and 72 h. Eight bitch ovaries were obtained by surgical castration. Oocytes were obtained by dissecting and washing ovaries with TCM-199 media under stereomicroscope. Those who had more than three granulose cell layers and a diameter greater than 120 μm were set to mature in TCM-199 culture medium plus 20% FCS and FSH, LH and 17B estradiol at standard concentrations at 38.5°C in a 5% CO2 humid atmosphere. At 48 and 72 h were processed following two different protocols. In the protocol A, oocytes were treated with hyaluronidase to be denuded. After that hypotonic shock and fixation with a 2:1 Carnoy's fixative solution were made. Protocol B was similar to the previous but the oocytes were incubated with 0.75 μg/ml of colchicine for 3 h in the same culture media prior to processing. All the preparations were observed in direct microscopy at ×1,000 determining how many were suitable for cytogenetic analysis. Statistically significant differences were found indicating that the best preparations suitable for cytogenetic analysis are obtained in 48 h with protocol B. The rate of in vitro oocytes maturation in the dog which is slow and inefficient means that the techniques previously used in other species must bevalidated in this species.