In Vitro Production And Development Of Porcine Embryos Derived From Intracytoplasmic Sperm Injection

Protein supply has been one of the greatest challenges in Malawi and Africa as a whole. Malawi is one of the small countries whose pig industry is still at its infant stage but with great potential. Local pig population accounts for over3,000,000distributed countrywide. Pigs play a vital role in both rural and urban areas. Pork consumption, live sales and organic manures are some of the importances justifying the presence of pigs in the communities. However, Malawi Government emphasizes on pig production like any other livestock. Good housing, good feeding, proper breeding, disease and parasite control in pigs are among the areas of intensification to improve pig performance. Cross breeding with the exotic breeds has been practiced aiming at improving carcass quality, litter sizes and quantity of pork. Government is strict on control and prevention of pig diseases like African swine fever. Policy stresses to produce more pork and disease free to safeguard human health which should be consumed locally and the surplus to be exported. In Malawi, little study has been done on reproductive performance of local pigs than in other domestic species. The population of local pigs dwindles due periodic out breaks of major diseases like African swine fever which sweeps pigs in many areas. Another challenge is the occurrence of both internal and external parasites. The presence of diseases and parasites yields away the productive and reproduction performance pigs at all ages leading to economic losses at smallholder and national level. Pigs have high fertility rates leading to multiple litter numbers per year. Information on animal genetic resources conservation is scanty. In situ conservation is done in Malawi as opposed to ex situ. Research recommends ex situ which is performed through cryopreservation of boar semen, oocytes from sows or gilts and embryos. Cryopreservation is the preservation of gametes or embryos in liquid nitrogen. This is a practical means of saving breeds from extinction in the event of disasters in their original location.The main purpose of this study was to explore one of the ex situ ways in form of in vitro production and development of pig embryos derived from intracytoplasmic sperm injection (ICSI) as way of genetic resources conservation of local Malawian pigs. ICSI is one of useful assisted reproductive technologies, commonly used to produce embryos in mammals. In this study, oocytes were collected from a nearby slaughterhouse in order to produce ICSI, IVF and SCNT embryos. The non-atretic follicles2-5mm in diameter were picked and frozen thawed boar semen was used. The experiment was divided into two parts:The first part was carried to culture oocytes and embryos by using different media solutions. In first place oocytes were cultured to observe the effect of oocyte quality on the maturation. The oocyte maturation medium used in the experiment was porcine zygote maturation medium (PZM-3) supplemented with0.57mM cysteine,1mM dibutyryl cAMP,5μg/mL insulin,50μM B-mercaptoethanol,10IU/mL PMSG,10IU/mL hCG and10%(v/v) porcine follicular fluid. It showed that the maturation rate of porcine oocyte in group A and B was higher than that of C (73.3%and63.6%vs36.1%).This was followed by the culture of oocytes on effect of oocytes quality on the development of porcine ICSI embryos to reach cleavage and blastocyst formation. PZM-3with0.4%BSA,75μg/mL potassium penicillin G, and50μg/mL streptomycin sulfate was used. Oocytes in group A showed high developmental rate73.3%at cleavage while Group B had64.1%lower than group A. Finally group C managed to reach51.6%. It has been observed that oocyte quality has a significant bearing on subsequent development to either cleavage or blastocyst stages (P<0.05). Poor quality led to no development to blastocyst stage for embryos in group C. The embryos exposed to various electric field strengths in order to observe the effect on cleavage and blastocyst development rates of embryos. At the electric field strength of1.4kv/cm, the cleavage rate was77.5%for group B which was followed by group A embryos (76.9%) and group C (73.7%) activated at1.5kv/cm. In terms of blastocyst rate of group A, B and C managed to develop as7.7%,15.0%and13.2%, respectively. There was no difference (P>0.05) among groups for cleavage rate but the blastocyst rate of group B and C was significantly higher than that of group A (P<0.05). To evaluate the in vitro production of porcine embryos using ICSI method. NCSU-23and PZM-3were used. PZM-3was supplemented with0.57mM cysteine,1mM dibutyryl cAMP,5μg/mL insulin,50μM β-mercaptoethanol,10IU/mL PMSG,10IU/mL hCG and10%(v/v) porcine follicular fluid. PZM-3had high effect71.1%on the maturation of embryos while NCSU-23had slightly lower cleavage rate62.5%. PZM-3had significantly high blastocyst rate10.9%than NCSU-23which had2.9%. This indicated that PZM-3suitable than NCSU-23. Effect of PZM-3and50nM TSA on developmental competence of porcine ICSI embryo was evaluated. PZM-3showed high influence on the embryo developmental competence cleavage rate77.8%vs25.9%blastocyst rate. The developmental process in vitro of ICSI, IVF, PA and SCNT embryos were compared and IVF group had high cleavage rate77.8%and good blastocyst rate19.4%which were followed by SCNT group which reached72.94%at cleavage and16.47%blastocyst rate. ICSI embryos followed and had cleavage rate72.7%and16.9%blastocyst rate. PA group had cleavage rate66.0%vs17.02%blastocyst rate. In experiment it was indicated that that all groups of embryos had different developmental competence on cleavage and same developmental pattern on blastocyst formation.The second part in the experiment was to evaluate effects of50nM TSA on developmental competence of porcine ICSI embryos. Using PZM-3medium only or supplemented with50nM TSA had the same cleavage rate (55%vs55%). The blastocyst rate derived from PZM-3group was slightly higher but no statistical difference as compared with PZM-3plus TSA group (13.3%vs8.6%). The effect of25nM TSA was conducted on developmental competence of porcine ICSI embryos. PZM-3showed high influence on the embryo developmental competence77.8%at cleavage and25.9%blastocyst rate while PZM-3with TSA yielded70.4%at cleavage and18.5%at blastocyst (P>0.05). Finally the effect of50nM TSA on developmental competence of porcine SCNT embryos was conducted. PZM-3with or without50nM TSA resulted in69.39%vs72.94%of cleavage rate and25.51%vs16.47%of blastocyst rate. The relative expression of Aurora-A in both IVF and ICSI embryos at different stages was compared. Expression in IVF embryos was evidently higher than that of ICSI embryos. At12&16h in IVF group the expression remained high but it was low at16h in ICSI embryos.Results in our experiment indicate that quality has more positive influence on developmental competence of both porcine oocytes and embryos. ICSI and IVF methods combining with PZM-3medium may be the suitable options for producing pig embryos.