RESUMEN
We aimed to establish efficient donor cells to produce piglets by somatic cell nuclear transfer (SCNT) of the endangered Vietnamese I pig. In Experiment 1, we assessed the effects of cell passages on the in vitro development of SCNT embryos. Cells with five and six passages showed significantly cleaved and blastocyst formation rates (86.72 and 86.64; 35.68 and 35.51, respectively, P < 0.05). The highest average total cell number per blastocyst was observed in groups of cells with five and six passages (50.45 and 50.18, respectively). Experiment 2 was performed to assess the sex of donor cells on the subsequent development of SCNT embryos. There was no significant difference in the cleaved and blastocyst formation rates, and the average total cell between female and male groups (86.51 % vs 86.94 % and 35.31 % vs 35.08 %, 50.29 % vs 50.67 %, respectively, P > 0.05). Experiment 3 was performed to assess the effect of cell lines on the development of SCNT embryos. Our results showed no significant difference in the success rate of fibroblast nuclear transfer into recipient oocytes, the cleaved and blastocyst formation rates, and the average total cell number per blastocyst among the cell lines 6004, 9154, 9155, 9156 and 9157 (P > 0.05). Experiment 4 was performed to assess the ability of SCNT embryos to induce pregnancy and to develop term. SCNT embryos were produced from I fibroblast cells established based on the results of Experiments 1, 2 and 3. Transfer of blastocyst stage embryos into 19 recipients (100-120 embryos in each) resulted in 14 pregnancies, in which 8 pregnant females terminated on Day 22-42 and 6 others produced 20 cloned piglets from donor cells of a female pig but 5 piglets died before birth and 15 healthy cloned piglets. However, 3 out of 15 healthy piglets died of unknown causes within 24h of birth and 3 out of 15 healthy piglets died at 3-5 days of age due to diarrhoea, 9 out of 15 healthy piglets are now 3 months of age. Finally, we established a protocol for the donor cell production which enabled the production of the endangered I pig embryos by SCNT and maximized blastocyst production rate by more than 35 % and pregnant rate after the transfer of cloned I pig embryos to recipients at 73.68 % for the first time in Vietnam.
RESUMEN
We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus-oocyte complexes were collected from antral follicles of 7-8 mo old female cyclic Ban pigs and vitrified in micro-drops. Oocyte morphology, lipid content, post-warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post-warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo-tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.
Asunto(s)
Criopreservación/métodos , Oocitos , Porcinos Enanos , Conservación de Tejido/métodos , Animales , Blastocisto , Supervivencia Celular , Células Cultivadas , Desarrollo Embrionario , Femenino , Fertilización In Vitro , Técnicas de Maduración In Vitro de los Oocitos , Metabolismo de los Lípidos , Oocitos/citología , Oocitos/metabolismo , Oocitos/fisiología , Manejo de Especímenes/métodos , PorcinosRESUMEN
We compared the efficacy of the microdrop and minimum volume cooling (MVC) methods for the vitrification of in vitro-produced porcine zygotes and blastocysts after equilibration in low concentrations of cryoprotectant agents. Zygotes and blastocysts were equilibrated in 2% (v/v) ethylene glycol and 2% (v/v) propylene glycol for 13-15 min. Then, they were vitrified in a medium comprised of 17.5% ethylene glycol, 17.5% propylene glycol, 0.3 M sucrose, and 50 mg/ml polyvinylpyrrolidone either by either dropping them directly into liquid nitrogen (microdrop method) or placing them on Cryotop sheets in a minimum volume of medium and plunging into liquid nitrogen (MVC method). Both zygotes and blastocysts were successfully vitrified. For the vitrification of zygotes, the MVC and microdrop methods were equally effective; however, for blastocyst vitrification, MVC was superior. For both methods, the vitrification of zygotes produced higher-quality embryos than the vitrification of blastocysts.