| Summary: | Wood bison (Bison bison athabascae) are a threatened subspecies native to North America. Endemic disease in the largest and most genetically diverse wood bison population in Wood Buffalo National Park has resulted in strict monitoring of populations and restricted the transfer of genetics to outside the park. To safeguard the future of wood bison, we are establishing a germplasm biobank through the use of advanced reproductive techniques. Recovery of embryos and semen collected from at-risk populations would reunite bison herds separated by time and space to produce healthy offspring and restore the genetic diversity of wood bison. The recovery of cumulus oocyte complexes (COC) from wild, free-roaming bison herds requires the use of minimal handling COC collection protocols. The following studies (Chapters 2 – 6) investigated novel approaches to improve in vitro embryo production (IVP) through a field-friendly approach. Chapter 2 investigated the effects of in vitro maturation, conventional morphologic COC grading and minimal handling ovarian superstimulation (sustained release follicle stimulating hormone [FSH]) on nuclear and cytoplasmic characteristics of bison COC and IVP through a 3D computer-assisted quantitative assessment. A greater proportion of active mitochondria was located in the central region of the ooplasm and greater mitochondrial clustering was present in low quality oocytes than high quality oocytes. However, additional analysis is required to characterize mitochondria distributions without quantitative software analysis. The bison oocytes had a high incidence of large lipid droplets similar to extended FSH starvation groups in cattle. Unfortunately, the minimal handling ovarian superstimulation treatments implemented in the present study did not increase the number of COC collected or improve embryo production rates. Chapter 3 investigated the effects of ovarian synchronization and a single dose superstimulation protocol using equine chorionic gonadotrophin (eCG) for COC collection and IVP in wood bison. A single dose of 5,000 IU eCG increased the number and size of follicles available for COC collection, more than doubled the number of COC collected for IVP and resulted in the production of more embryos than other groups (approximately 2 embryos per bison). Nonsuperstimulated COC collections done after follicle wave synchronization resulted in greater embryo production than collections done at random stages of the follicular wave. Repeated COC collections after successive wave synchronization resulted in similar follicular counts and embryo production rates within individuals. The greatest number of follicles aspirated, COC collected, and embryos produced occurred in the anovulatory season. Chapter 4 investigated the effects of restraint (lateral recumbency after chemical immobilization vs standing position in a hydraulic chute) and follicular wave status (random vs synchronized) on COC collection efficiency in non-superstimulated bison. It examined the effects of superstimulation treatment (single dose of eCG vs multiple doses of FSH plus human chorionic gonadotrophin [hCG]) and method of administration (field dart vs manual injection) on COC collection efficiency and IVP. The COC collections were done as effectively on sedated recumbent bison as those restrained in a standing position in a hydraulic chute. Ovarian superstimulation using the single-dose eCG protocol was as effective as a multiple-dose FSH protocol and field darting was an effective method of administering superstimulation treatments. The use of ovarian superstimulation resulted in greater IVP efficiencies than random, non-stimulated collections (3.1 vs 1.1 embryos per bison). Chapter 5 investigated the effects of reproductive status (non-pregnant, pregnant, and prepubertal), advancing gestation (pregnant bison, 90- vs 120- days) and follicular wave status (random vs synchronized) on ovarian follicles, COC collection and IVP. Oocytes were collected and embryos were produced from bison at both 90- and 120- days of gestation, but the position of the ovaries at the more advanced stage reduced COC collection efficiency. Yearling pre-pubertal bison provided the most promise as COC donors in the field. They had the highest number of follicles available for aspiration and produced a similar number of embryos (approximately 1 freezable embryo per collection) compared to non-pregnant mature bison. Yearling pre pubertal bison are available for collection throughout the winter season without the limitations imposed by advanced stages of pregnancy or unknown pregnancy status. Chapter 6 investigated the effect of in vitro wood bison embryo characteristics (stage of development, grade, and freeze day) on the post-thaw viability of pre-implantation IVP embryos characterized by microscopic morphology and establishment of pregnancy after embryo transfer. Morphologic grading was positively related to quantitative viability characteristics, with higher quality embryos having less cryo-damage and higher cell counts than low quality embryos. Morulae survived cryopreservation with less damage than blastocysts, and subsequent transfer resulted in a higher pregnancy rate at 30-days and lower pregnancy loss between 30 to 60 days than blastocysts. The vitrification method of cryopreserving embryos resulted in a pregnancy rate similar to the transfer of fresh embryos. The study produced nine pregnancies at 30 days postovulation, of which 6 made it to term and the birth of five calves. In conclusion, COC collection and IVP methods and procedures investigated throughout this thesis have made COC collection from wild, free-roaming herds a feasible task. Bison stakeholders may now implement these techniques to access inaccessible bison genetics in and around Wood Buffalo National Park.
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