Pig oocyte activation and developmental competence of parthenogenetically activated oocytes: in vitro and in vivo studies

Abstract

In somatic cell nuclear transfer in mammals, to clone a piglet is still a big challenge. Although many factors could contribute to the low success rate, such as quality of donor and recipient cells, type of donor cell including sources of animal breeds and tissues, number of passages and culture conditions, timing of cell cycle, procedures of nuclear transfer, techniques and the number of survival cloned embryos, embryos transfer, one of these factors is believed to be poor oocyte activation, especially in pig nuclear transfer. Therefore studies presented in this thesis aimed at the establishment of an in vitro culture system for pig oocyte maturation and embryo culture, based on this system an electrical activation protocol for pig oocytes was optimized and also tested by monitoring in vivo development of activated pig oocytes. Finally, the protocol was used for activating pig embryos reconstructed by transfer of somatic cells into enucleated ovulated oocytes and for production of pig parthenotes to maintain pregnancies of cloned pig embryos, which resulted in the birth of a cloned male piglet.The thesis comprises a total of 6 chapters. In addition to the review of literature (Chapter 1), general materials and methods (Chapter 2) and general discussion (Chapter 6), in Chapter 3 and 4, the studies focused on optimizing electrical parameters on pig oocyte activation and investigating the effects of activation conditions including temperature, activation medium, and concentrations of Ca2+ and Mg2+ in activation medium and diploidization of activated oocytes. These experiments were carried out in vitro, whereas experiments in Chapter 5 were II conducted in vivo to assess the in vivo developmental competence of in vitro matured (IVM) pig oocytes activated by the improved activation protocol.In Chapter 3, pig oocytes were matured in NCSU 23 medium + 10% pig follicular fluid (pFF) (1); NCSU 23 medium + 10% pFF + amino acids (essential and non essential) (2); NCSU 23 medium + pFF + lOpg/ml epidermal growth factor (EGF) (3) and NCSU 23 medium + pFF + amino acids + EGF (4) for 44 h. The medium (2) yielded the highest blastocyst rate of activated oocytes, which was significantly higher than those in the medium (3) and (4) (p<0.05). Oocytes matured in medium (2) resulted in a maturation rate of about 75% at 36 h of maturation, afterwards the rate reached over 90%. Subsequently, four experiments were carried out to examine the interactions among age of oocytes, electrical field strength, pulse number and duration. These results demonstrate that the best stimuli are 3 x 80 psec consecutive pulses of 1.0 kV/cm DC using 44 h post matured oocytes, and suggest that using 3 consecutive pulses of low electrical field strength for shorter duration is beneficial for activating oocytes and developmental potential.In Chapter 4, based on the improved electrical activation protocol the effects of activation conditions were examined including activation temperature, activation media, and Ca2+ and Mg2+ concentrations in activation medium as well as diploidization of the activated oocytes. The results reveal that temperature; Ca2+ but not Mg2+ concentration in activation medium and diploidization are very important factors for successful oocyte activation. The blastocyst rate of activated pig oocytes was highly correlated with the changes of temperature (R = 0.97; p<0.01). Similarly, the percentages of diploid blastocysts were also correlated with either the proportions of diploid oocytes or the blastocyst rates (R=0.89; p<0.05 and R= 0.959; p<0.05). Moreover, the results suggest that the minimum required time of cytochalasin B treatment to the activated IVM pig oocytes is 3 hours for achieving a reasonable blastocyst rate with over 70% of activated oocytes being diploid.In Chapter 5, IVM and ovulated pig oocytes were electrically activated and transferred into synchronous recipients in order to assess their in vivo developmental potential. The pregnancies of the transferred IVM pig parthenotes were not detected by ultrasound examination between 40-50 days of gestation. Pig parthenotes were surgically collected from 11 pregnancies on day 21, 30 and 35 of gestation, respectively. These results showed that either ovulated or IVM pig oocytes following activation with the protocol were able to develop in vivo for 30 days, suggesting that they stopped development around day 31 of gestation. Although these parthenotes were smaller and lighter, majority of them were morphologically normal. In addition, transferring the reduced number of day 2 cleaved oocytes into recipient animals, compared with the overnight cultured oocytes, seemed not to affect the pregnancy rate (85%; 11/13 vs. 75%; 3/4).