Effect of different cryo-devices on in vitro maturation and development of vitrified-warmed immature buffalo oocytes.

The aim of the study was to identify a cryo-device that would be best suited for the vitrification of buffalo immature cumulus-oocyte complexes (COCs) as judged by viability and meiotic competence of the vitrified-warmed oocytes and their development ability following in vitro fertilization (IVF). The expression of oocyte secreting factors and their receptors (GDF9, BMP15, BMPR2, TGFBR1) and apoptosis related genes (BCL2, BAX, P53, C-MYC) were compared in vitrified-warmed oocytes after in vitro maturation. COCs from the ovaries of slaughtered buffaloes were vitrified in a combination of dimethyl sulfoxide, ethylene glycol, and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). The fresh COCs were exposed to vitrification and warming solutions as in other vitrification methods without plunging in to liquid nitrogen (EC). The viability of vitrified-warmed COCs, 2 h post warming in HS and CT was similar to fresh and EC groups but significantly higher than CS and OPS methods. The proportions of oocytes with first polar body after 24 h in vitro maturation were significantly higher in HS and CT methods than in CS, OPS and CL methods. The development ability of these vitrified-warmed oocytes to blastocyst stage following IVF in all vitrified groups was significantly lower than control and EC groups. Among the vitrified groups, the blastocyst rate in HS, CT and CL groups was significantly higher than in OPS and CS groups. It was also observed that the expression levels of GDF9, BMP15, BMPR2, TGFBR1, BCL2, BAX, P53 and C-MYC genes in vitrified-warmed COCs in CT, HS and CL groups were similar to control. The results indicated that HS, CT and CL are more suitable cryo-devices for vitrification of buffalo immature oocytes.

Meiotic maturation of oocytes recovered from the ovaries of Indian big cats at postmortem.

In order to increase the available sources of genetic material for endangered members of the great cat family, this study was designed to assess the meiotic competence of oocytes recovered from postmortem ovaries of the Indian leopard, tiger and lion. The average number of oocytes that were recovered per ovary was 11.0 ± 5.0, 11.0 ± 3.5 and 21.3 ± 8.8 for tiger, lion and leopard, respectively. The proportion of culture grade oocytes for tiger, lion and leopard were 72.7, 78.8 and 71.9%, respectively. The culture grade oocytes were matured in tissue culture medium 199 modified with sodium bicarbonate supplemented with 0.3% BSA (fatty acid-free) (w/v), 10 µg/ml FSH, 6 IU/ml LH, 1 µg/ml 17ß-estradiol, 0.36 mM pyruvate, 2.2 mM calcium lactate, 2.0 mM L-glutamine, 100 IU/ml penicillin and 0.1 mg/ml streptomycin in an incubator with 5% CO2 under humidified air at 38.5°C for 36 h. After in vitro maturation, 56.3, 53.8 and 58.7% of the tiger, lion and leopard oocytes, respectively, were matured. The proportion of oocytes that extruded first polar body was significantly higher when the oocytes were collected from the animals of less than 15 yr of age compared to above 15 yr. These findings suggest that the oocytes recovered from ovaries of tiger, lion and leopard immediately postmortem can be successfully matured to MII stage.

Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes.

The aim of the study was to evaluate meiotic maturation, and expression of genes coding for oocyte secreted factors (GDF9, BMP15, TGFBR1, and BPR2) and apoptosis (BCL2, BAX and P53) after vitrification of immature goat cumulus oocyte complexes (COCs) and in vitro maturation. COCs were vitrified in a solution containing ethylene glycol, dimethyl sulfoxide and sucrose using either a conventional straw (CS), open pulled straw (OPS), cryoloop (CL), hemistraw (HS) or cryotop (CT). Freshly collected COCs (Control), COCs exposed to vitrification and dilution solutions without cryopreservation (EC) and vitrified-warmed COCs were matured in vitro for 27h. The viability of vitrified-warmed COCs 2 h post warming and in vitro maturation was similar for CL, HS and CT. The proportion of oocytes that extruded a 1st polar body and reached TI/MII was significantly higher with CT and HS followed by CL, OPS and CS. Gene expression of GDF9, BMP15, BMPR2, BAX and P53 were comparable to control levels for OPS, CL, HS and CT. The gene expression pattern in CS vitrified COCs was by contrast changed in that GDF9, BMP15, TGFBR1 and BAX were up regulated and BMPR2, BCL2 and P53 down regulated. In conclusion immature goat COCs vitrified using CT and HS showed that viability, maturation rates and expression of genes coding for oocyte secreted factors and apoptosis were similar to non-vitrified controls.

Meiotic maturation of vitrified immature chousingha (Tetracerus quadricornis) oocytes recovered postmortem.

The ability to recover and cryopreserve oocytes from postmortem ovaries of endangered or wildlife species holds tremendous potential for conservation using assisted reproductive technologies. The objective of this study was to assess the in vitro meiotic maturation of chousingha (four-horned antelope) oocytes following vitrification using open pulled straw (OPS) method. The average number of oocytes recovered per ovary was 65.6. The proportion of oocytes that matured was significantly lower in vitrified oocytes (29.4%) when compared with fresh oocytes (69.3%). The study provides evidence that it is possible to cryopreserve immature oocytes by vitrification collected from the ovaries of chousingha at postmortem and also demonstrates that these cryopreserved oocytes retain their potential to undergo in vitro meiotic maturation.