Improvement of porcine genetic resource preservation by improving in vitro production, vitrification and transfer of embryos.

Abstract

One of the main causes of poor in vitro embryo development (IVEP) efficiency is incomplete cytoplasmic maturation of pig oocytes. In contrast to nuclear maturation, there are no efficient markers to detect the cytoplasmic maturation. The overall objective of this dissertation was to investigate the effect of sorting oocytes using mitochondrial (MT) and cortical granules (CGs) distribution as cytoplasmic maturation markers of selected mature oocytes on improving embryonic development, polyspermy, embryo vitrification and transfer them to recipients. We hypothesized that embryos derived from sorted oocytes would be better than non-sorted oocytes in embryo development, quality, and viability after vitrification. For this purpose, we used MitoTracker green (MTG) and Peanut agglutinin (PNA) as fluorescent dyes to sort the oocytes based on MT and CGs distribution, respectively. In chapter 2, we examined the viability rate of oocytes after using of some fluorescent dyes. We used Propidium Iodide (PI) to check the viability rates of oocytes after staining with MTG, PNA and Rhodamine-123 (RH-123). Also, we used Fluorescein diacetate (FDA) to examine MitoTracker orange (MTO) and wheat germ agglutinin (WGA) for oocyte viability. We evaluated the viability rate then the MT and CGs distribution for oocytes derived from pre-pubertal gilts and adult sow pigs using both confocal and wide-field microscopes to find if the wide-field microscope is an adequate replacement to confocal microscope. We found that the concentrations of 200 nM of MTG and 625 nM of PNA were optimal to use in the next series of experiments with maximum visibility and viability. The viability rates for these concentrations were 94.7% ± 5.10 (MTG 200 nM) and 90.9% ± 0.83 (PNA 625 nM). Also, we found that the distribution of MT and CGs can be recognized using wide-field microscope in the same efficiency of confocal microscope. There was strong agreement between the wide-field and confocal microscopy (>96%) using MTG and PNA for pre-pubertal oocytes. In addition, there was no significant differences in MT and CG distribution using oocytes derived from pre-pubertal or sow pigs using wide-field microscope. In chapter 3, we developed a technique to segregate mature oocytes from harvested oocytes using MTG and PNA staining using wide-field microscope. We investigated the in vitro development of embryos, quality, and polyspermy to embryos derived from sorted and non-sorted oocytes. There were no significant differences between the matured sorted (diffused MT or peripheral CGs) and non-sorted oocytes in cleavage, morula, and blastocyst stages as well as the polyspermy. On the other hand, the quality of blastocysts was better morphologically in embryos derived from sorted oocytes than non-sorted oocytes. In addition, the polyspermy was significantly (P≤0.05) less in matured oocytes and non-sorted oocytes groups than immature patterns or non-inseminated groups. In chapter 4, we evaluated many maturation media and their effects on MT and CGs distribution, IVEP, and polyspermy. The media evaluated in this chapter were Tissue Culture Medium-199 (TCM-199), modified Whitten’s Medium (mWM), North Carolina State University 23 (NCSU23), North Carolina State University 37 with glucose (NCSU37G), and North Carolina State University 37 with pyruvate and lactate (NCSU37PL). There were no significant differences among the media used in this chapter in MT and CGs distribution, cleavage, morula, and blastocyst rates. On the other hand, the NCSU37PL had less (P≤0.05) polyspermy than other media in both matured sorted oocytes and non-sorted oocytes groups. In chapter 5, we evaluated the effect of MT-sorted oocytes and CG-sorted oocytes (mature pattern) on the viability, quality, and development of embryos after the vitrification and warming procedures. Also, we tried to examine the embryo transfer (ET) process for both vitrified and non-vitrified (fresh) morulae. We found that the vitrification and warming procedures had a negative affect (P≤0.05) on embryos viability, quality, and development for both sorted and non-sorted groups. However, there was no difference between sorted and non-sorted oocytes according to the previous parameters. In addition, there was no difference between the vitrified embryos and fresh embryos on the pregnancy after ET (no pregnancy for both groups), but one of the gilts in vitrified group had numerous corpora lutea when they checked by necropsy. In conclusion, results from the present dissertation support the hypothesis partially. The sorting of oocytes can be done using wide-field microscopy with fluorescent dyes such as MTG and PNA, and it is a useful technique to obtain high quality embryos after IVEP with less polyspermy. There is no significant effect of maturation media on IVEP outcome. We suggest additional experiments to improve and develop the vitrification and warming procedures as well as ET with a larger number of animals.