Jesus Manuel Palomino DVM, MSc University of Saskatchewan 41st Annual Conference of the IETS, Versailles, France January 10-13, 2015 Superovulation: A tool to produce disease free embryos in wood bison (Bison bison athabascae) Jesus Manuel Palomino DVM, MSc PhD Candidate University of Saskatchewan Recipient of: Peter W. Farin Trainee Travel Scholarship Award

The Problem Reproductive Germ plasm Disease-free Hartop et al., 2009 Genetic diversity Brucellosis and tuberculosis Disease-free areas Population decline Loss of genetic diversity Reproductive Germ plasm Disease-free Technologies bio-bank offspring

Reproductive technologies to produce disease-free wood bison embryos In vitro embryo production Superstimulation and oocyte collection In-vivo-derived embryos Superovulation and embryo collection

Progression of thesis research  Study 1. Inducing ovulation in Wood Bison Study 2. Effect of LH vs hCG and progesterone on superovulation Study 3. Does eCG improve the superovulatory response and embryo quality in wood bison? Study 4. … Study 5. …  

What did we learn from Studies 1 to 3? Ovarian superstimulation: - 8 vs 2 vs 1 administrations of FSH Induction of ovulation: - hCG more effective than LH Adjunct treatments eCG: Did not improve superovulation or embryo quality Exogenous progesterone not needed: anovulatory season Problem Number of embryos collected: 4 embryos/cow Number of Grade 1 & 2 (Freezable) embryos: 2 embryos/cow Can we improve these numbers?

Study 4 Effect of lengthening of FSH treatment on superovulation and embryo quality in wood bison Rationale: In cattle, lengthening the period of FSH treatment induced a greater number of ovulations and CL compared with conventional treatment (Garcia Guerra et al., 2012) Lengthening FSH treatment will rescue more subordinate follicles in the wave and they will grow to a sufficient stage of maturity to respond to LH and ovulate

Hypothesis Lengthening FSH treatment will improve the ovulation rate and embryo quality in superovulated wood bison. Objective: Determine the effect of lengthening of FSH treatment on superovulatory response and embryo quality in wood bison during the ovulatory and anovulatory season

Experiment 1: Anovulatory season Experimental design:

Experiment 2: Ovulatory season Experimental design:

Animals and facilities Alternative Livestock Center, University of Saskatchewan 50 adult female and 4 male wood bison Hydraulic chute to handle bison

Synchronization: Follicular ablation Day -1 Day 0

FSH 400 mg (Folltropin, Bioniche) Superovulation FSH 400 mg (Folltropin, Bioniche) Hyaluronic acid (Map 5, Bioniche) hCG 3000 IU (Chorulon, Merck)

Artificial Insemination: Chilled semen Semen collection: electroejaculation, 3 bulls, pooled Extender: Tryladil (MOFA) + 20% egg yolk Chilled semen 5º C (1-3 days)

Embryo collection: Non-surgical

Embryo classification (IETS, 2010) cvzd

Table 1: Anovulatory season FSH Short FSH Long Number of bison 16 Number of follicles ≥9 mm on Day 5 10.8 ± 2.0 10.8 ± 1.1 Ovulation rate (# ovulations / # follicles ≥9 mm) 52% (67/130) 54% (70/130) Number of CL 4.5 ± 0.9 5.7 ± 1.0 Total ova-embryos 2.6 ± 0.5 3.2 ± 0.7 Grade 1 & 2 0.5 ± 0.3 0.3 ± 0.2 Grade 3 0.1 ± 0.1 Grade 4 1.2 ± 0.4 1.8 ± 0.6 Recovery rate (# ova-embryos/# CL) 48 % (26/54) 51% (35/68) Results are presented as Mean±SEM, otherwise stated.

Table 2: Ovulatory season FSH Short FSH Long P - value Number of bison 14 Number of follicles ≥9 mm on Day 5 8.1 ± 1.1 9.8 ± 1.4 0.40 Ovulation rate (# ovulations / # follicles ≥9 mm) 60% (67/130) 74% (73/98) 0.07 Number of CL 4.6 ± 0.6 6.5 ± 0.9 0.13 Total ova-embryos 2.2 ± 0.5 4.4 ± 0.7 0.04 Grade 1 & 2 1.1 ± 0.4 2.3 ± 0.6 0.09 Grade 3 0.2 ± 0.1 0.8 ± 0.4 0.03 Grade 4 0.8 ± 0.2 1.7 ± 0.6 0.05 Recovery rate (# ova-embryos/# CL) 48 % (22/46) 68% (44/65) 0.06 Results are presented as Mean±SEM, otherwise stated. Garcia Guerra et al., 2012 Dias et al., 2013

Conclusions Extended superstimulatory protocol improved embryo production during the ovulatory season Embryo quality was not affected by lengthening the superstimulatory protocol Repeat experiment with greater number of bison

Production of disease-free embryos from superovulated wood bison Study 5: Production of disease-free embryos from superovulated wood bison Objective: Determine the effectiveness of washing procedures for removing Brucella abortus from bison embryos exposed in vitro Abstract 140

Level 3 laboratory – Intervac/VIDO

Acknowledgments Department of Veterinary Biomedical Sciences Miriam Dr. Adams Dr. Mapletoft Dr. Woodbury Dr. McCorkell Dr. Muir/ Dr. Machin Dr. Anzar Dr. Mastromonaco Miriam Rodrigo, Hayder, Alisha Nick, Brad Kosala, Lyle Department of Veterinary Biomedical Sciences