ABSTRACT
Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1M sucrose in HSOF+6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P<0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P<0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P<0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC.
Subject(s)
Cats , Cryopreservation/veterinary , Oocytes , Animals , Animals, Wild , Cell Differentiation , Cell Survival , Cryopreservation/instrumentation , Cryopreservation/methods , Cryoprotective Agents , Cumulus Cells/cytology , Endangered Species , Felidae , Female , Fertilization in Vitro/veterinary , In Vitro Techniques , Male , Oocytes/cytology , Reproductive Techniques, Assisted/veterinaryABSTRACT
Cryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen-thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen-thawed sperm sample was tested by determining the motility (54.7 +/- 11.3% and 32 +/- 13.1% respectively for cat spermatozoa; 38.3 +/- 18.7% and 21.5 +/- 16.8% respectively for tiger spermatozoa), viability (74.3 +/- 8.6% and 45.2 +/- 9.4% respectively for cat spermatozoa; 42.4 +/- 14.5% and 33.5 +/- 12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species.
