RESUMEN
BACKGROUND: Vitrification by Rapid-I method could be essential for felid rescue programs to protect wild felid in the future. OBJECTIVE: This study was aimed at adapting the Rapid I method and evaluating the viability of serval and Pallas cat oocytes compared to oocytes of the domestic cat. MATERIALS AND METHODS: Oocytes after collection and in vitro maturation were vitrified using Cryotech medium (Cryotech, Japan) and a Rapid-I device (Vitrolife, Sweden). To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. RESULTS: Survival rate in the control group (domestic cat) was 75 %. In the experimental group, 70% (serval) and 60% (pallas cat) viable oocytes were found. CONCLUSION: The Rapid-I method can be applied successfully for the vitrification of wild felid oocytes.
Asunto(s)
Supervivencia Celular , Criopreservación/veterinaria , Felidae , Felis , Oocitos/citología , Animales , Criopreservación/métodos , Crioprotectores , Femenino , VitrificaciónRESUMEN
BACKGROUND: Vitrification is commonly used for cryopreservation of gametes. OBJECTIVE: The study aimed at evaluating viability and developmental competence of bovine oocytes vitrified by Rapid-I method. MATERIALS AND METHODS: Oocytes after collection (group 1) and IVM (group 2) were vitrified using medium containing 18% Ficoll, 40% ethylene glycol, 0.3 M sucrose. To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 0.5 µM ionomycin in TCM 199 combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum. RESULTS: Survival rate in group 1 was 58%, and 88% in the control. In group 2, 63% viable oocytes were found, compared to 82% in the control group. After parthenogenetic activation 27.2% morulas were observed. This percentage was lower than in the non-vitrified group (31%). CONCLUSION: Maturity stage of bovine oocytes has no effect on their survival after vitrification.
Asunto(s)
Criopreservación/veterinaria , Oocitos , Vitrificación , Animales , Bovinos , Supervivencia Celular , Glicol de Etileno , Femenino , Fertilización In VitroRESUMEN
The effect of filly age on morphology of the ovaries, collected oocytes and their capacity for in vitro maturation (IVM) was examined. The ovaries of slaughtered fillies were classified into three groups, according to filly age: (I) <10 month old (<10MF); (II) approximately 1 year old (1YF); and (III) approximately 1.5 year old (1.5YF). The ovaries of mares were used as a control group. Ovarian morphology and collected oocytes were evaluated. Only oocytes with expanded (Ex) and compact (Cm) cumuli were used for IVM. In <10MF, 1YF, 1.5YF and mare groups, corpora lutea were found in the ovaries of 9.3%, 36.7%, 59.6% and 80.9% females, respectively (P<0.001). Based on this observation, we found that about 37% of fillies reach puberty at approximately 12 months of age. No relationship was found between filly age and morphology of the oocytes obtained. In comparison to mares, fewer (P<0.05) Cm oocytes were collected from filly ovaries. Among Cm groups, fewer filly (28.4-35.5%) than mare oocytes (50.0%) reached metaphase II stage, but the difference was only significant when compared to oocytes of the <10MF group (P<0.05). In the Ex groups, a similar proportion of oocytes of fillies (40.8-51.1%) and mares (48.4%) attained the metaphase II stage. In conclusion, in the culture conditions applied, Cm oocytes of fillies younger than 10 months showed lower meiotic competence than mare oocytes. Oocytes of older fillies showed meiotic competence similar (P>0.05) to mare oocytes. Both filly and mare oocytes with expanded cumuli displayed the same capacity for IVM.
Asunto(s)
Envejecimiento , Caballos/fisiología , Oocitos/citología , Ovario/anatomía & histología , Ovario/fisiología , Animales , Femenino , Meiosis , Oocitos/fisiología , Tamaño de los Órganos , Maduración SexualRESUMEN
The binding of a spermatozoon to the zona pellucida is the first step in fertilization. The number of spermatozoa bound to a zona pellucida may reflect the functional status of both the oocyte and spermatozoa. The aim of the present study was to determine whether the stage of maturation of the equine oocyte affects the capacity of the zona pellucida to bind with spermatozoa. Cumulus-oocyte complexes (COCs) were collected from the ovaries of mares from abattoirs or were obtained in vivo by ultrasound-guided follicular aspiration. Oocytes were inseminated directly after collection or after 30 h of in vitro maturation (IVM) with semen from the same ejaculate. The results indicate that the immature equine oocytes collected from abattoirs and inseminated directly after collection bound fewer spermatozoa than did mature (metaphase I or II) oocytes inseminated after 30 h of IVM. A similar correlation was observed with the oocytes collected in vivo. The mean number of spermatozoa bound by mature oocytes collected from preovulatory follicles and inseminated directly after collection was 324 +/- 29, whereas the mean number of spermatozoa bound by immature oocytes from nonpreovulatory follicles inseminated without maturation was 222 +/- 26 (P < 0.05). This difference was not observed in the oocytes inseminated after maturation. It is postulated that immature equine oocytes bind fewer spermatozoa than do oocytes matured either in vivo or in vitro.
Asunto(s)
Caballos/fisiología , Oocitos/fisiología , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiología , Zona Pelúcida/fisiología , Animales , Femenino , MasculinoRESUMEN
The effect of epidermal growth factor (EGF) on the in vitro maturation rate of equine oocytes was examined. Oocytes were collected from an abattoir (Expt 1) or using ultrasound-guided follicular puncture in vivo (Expt 2). All oocytes with a compact or expanded cumulus at recovery were cultured for 30 h in: medium 1 (TCM199 + fetal calf serum (FCS) + crude equine gonadotrophin (CEG) + oestradiol + antibiotics); medium 2 (TCM199 + EGF); medium 3 (medium 1 without FCS + EGF); or medium 4 (medium 1 without CEG + EGF). In Expt 1, 84% (37/44) and 87% (40/46) cumulus expansion (P > 0.05), and 39% (22/57) and 9% (5/57) (P < 0.01) nuclear maturation, were observed in medium 1 and 2, respectively. In Expt 2, cumulus expansion was observed after culture in medium 1, 3 and 4 (30/30, 31/31 and 29/29, respectively). The nuclear maturation rate was significantly lower in medium 3 (6%, 2/36) than in medium 1 (43%, 16/37) (P < 0.01) and was higher in medium 4 (64%, 25/39) than in medium 1, although the effect was not significant (P = 0.07). In conclusion, 50 ng EGF ml(-1) alone was an effective substitute for crude equine gonadotrophin and the presence of EGF improves the nuclear maturation rate of equine oocytes.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Caballos/fisiología , Oocitos/fisiología , Animales , Núcleo Celular , Medios de Cultivo , Citoplasma , Femenino , Gonadotropinas Equinas/farmacología , Meiosis/efectos de los fármacosRESUMEN
From the ovaries of 256 slaughtered mares a total of 1713 follicles were isolated from which 1641 (95.8%) oocytes were recovered (6.4/mare). A total of 564 follicles and oocytes were evaluated for the degree of vascularisation of the follicle wall, the appearance of the follicular fluid and the location and morphology of the cumulus-oocyte-complex. Follicles with a diameter of >10 mm displayed more numerous, well branched and more pronounced blood vessels than the smaller ones (4-10 mm diameter) and most of them contained clear, yellowish fluid with few granulosa cells. The percentage of oocytes with compact cumuli increased significantly with an increasing diameter of the follicle, being 233%, 43.9%, 55.6% and 64.2% (P<0.01) for the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. The percentage of oocytes attached to the follicle wall also increased with increasing follicle size, being 48.0%, 59.6%, 81.5% and 90.1% (P<0.01), respectively. On the contrary, the percentage of oocytes floating in the follicular fluid decreased with increasing follicle diameter, from 52.0% in the smallest follicles to 9.9% in the biggest ones. A significantly greater percentage of oocytes found on the follicular wall than in the follicular fluid had a compact cumulus (56.6 versus 21.3%; P<0.01). For in vitro culture were accepted 30.4%, 54.3%, 60.7% and 77.8% (P<0.01) of oocytes from the follicles with diameters of 4-10, 11-15, 16-20 and 21-35 mm, respectively. After culture for 28-40 h in TCM 199 medium, 90 of a total of 165 (54.5%) oocytes reached the metaphase II stage of maturation.