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In vitro embryo production in wood bison (Bison bison athabascae)

In vitro embryo production in wood bison (Bison bison athabascae)
Miriam Pilar Cervantes Flores

2016

Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5A2, CANADA.

ABSTRACT

The studies presented in this thesis were designed to develop efficient protocols for collecting competent oocytes and to produce disease-free wood bison embryos by in vitro fertilization and culture. In Chapter 3, the in vivo and in vitro maturational characteristics of cumulus-oocyte complexes (COC) collected from live wood bison during the anovulatory and ovulatory seasons were compared. The stages of nuclear maturation: germinal vesicle (GV), germinal vesicle break down (GVBD), metaphase I (MI) and metaphase II (MII) were determined in wood bison oocytes using anti-Lamin AC/DAPI staining. Additionally, the optimal interval of time after human chorionic gonadotrophin (hCG) treatment required for in vivo oocyte maturation in wood bison was determined. Nuclear maturation occurred more rapidly during in vitro versus in vivo maturation, but was associated with less cumulus cell expansion than with in vivo maturation. In vitro oocyte maturation was maximal after 24 h of in vitro maturation. In vivo oocyte maturation was more complete at 30 than 24 h after hCG treatment. Season had no effect on the maturational capacity of wood bison oocytes. Competence of the in vitro (Chapter 4) or in vivo (Chapter 5) matured oocytes to develop to the blastocyst stage in culture was evaluated in subsequent studies. In Chapter 4, the hypothesis that the morphological characteristics of wood bison cumulus-oocyte complexes (COC) affect the ability of the immature oocyte to develop in vitro following in vitro fertilization was tested. The effect of extending from 48 h to 72 h the FSH starvation period after superstimulation (FSH diluted in 0.5% hyaluronan) on number and size of the follicles at the time of collection by transvaginal ultrasound-guided follicular aspiration, on COC morphological characteristic, and on blastocyst development rate, was also investigated. Compact COC classified as good (>3 layers of cumulus cells) resulted in the highest blastocyst rate following in vitro maturation, fertilization and culture. There was no effect of extending the FSH starvation period by 24 h on the number of follicles ≥ 5 mm at the time of collection, the morphology of the COC or blastocyst rate on Day 7 or 8 after fertilization. The morphological characteristics of immature wood bison COC affect oocyte in vitro developmental potential to the blastocyst stage. In Chapter 5, the effect of an additional 4 h of in vitro maturation of in vivo matured oocytes collected 30 h after hCG treatment on subsequent embryo development was evaluated. In addition, the effects of extending the interval between hCG treatment and COC collection from 30 to 34 h on in vitro embryo production was evaluated. Results confirmed that an additional short period of in vitro maturation, or an extended period of in vivo maturation increased in vitro embryo production rates in wood bison. In the final chapter (Chapter 6), the effectiveness of the IETS washing procedures with or without antibiotics for removing Brucella abortus from in vitro-produced embryos infected in vitro with the pathogen was determined. Brucella abortus was removed from 100% of in vitro-exposed embryos following 10 washes of 100 fold dilution, with or without antibiotics. Results validated the embryo washing procedures for producing Brucella-free in vitro-produced wood bison embryos.