Handling North American bison can pose risk to the handler and evoke stress in the animal. Moreover, this induced stress might affect qualities of semen collected by electroejaculation. The objective of this study was to investigate if a long acting neuroleptic tranquilizer (LAN) would reduce the stress of bison and thereby improve the quality of electroejaculated semen. Eight experimental replicates were conducted between May and November. In each replicate, the same six bison bulls were randomly assigned into LAN-treated (n = 3) and non-treated control (n = 3) groups. Pipothiazine palmitate (Piportil L4) was administered intramuscularly as a single dose of 100 mg in replicates 1–4 or 200 mg in replicates 5–8. Within each replicate, semen was collected by electroejaculation at 4, 6, 11 and 13 days post treatment. Behavioral parameters, sperm morphology and motility parameters were analyzed. A blood sample was collected before each electroejaculation and serum concentrations of testosterone, cortisol and corticosterone were determined. Treatment bulls with 100 mg of Piportil L4 reduced the restraint time and the struggling of bison bulls during handling compared to the control group (P < 0.05). Semen motility parameters and serum concentrations of testosterone, cortisol and corticosterone were not significantly affected when 100 mg of the LAN was administered (P > 0.05). However, giving 200 mg of Piportil L4 reduced the restraint time of bison bulls and the duration of semen collection (P < 0.05). Also, this treatment improved total and progressive sperm motilities when compared to the respective controls (P < 0.05). Interestingly, serum concentration of corticosterone, as an endocrine stress indicator, was decreased after administration of 200 mg of Pipothiazine palmitate, while testosterone concentrations were increased compared to those values in untreated control bulls (corticosterone: 0.10 ± 0.01 compared with 0.15 ± 0.02 ng/mL; testosterone: 9.11 ± 1.68 compared with 5.33 ± 0.74 ng/mL; P < 0.05). In conclusion, this study demonstrated that a treatment dose of 200 mg of Piportil L4 can decrease the behavioral and endocrine stress responses in bison bulls, which indirectly increasing testosterone concentrations and improving semen quality.

Brucellosis and tuberculosis are endemic in Wood Buffalo National Park, the largest reserve of wood bison (Bison bison athabascae) in Canada. Our goal is to produce and preserve disease-free embryos for the purpose of wood bison repopulation. This study was designed to determine if embryo collection is feasible in wood bison during the anovulatory season (May–July) and to test if progesterone priming is required for superovulation. A 2-by-2 design was used to determine the effectiveness of LH (Lutropin) or hCG (Chorulon) for induction of ovulation with or without intravaginal progesterone releasing device (PRID) in 32 wood bison cows. Follicular wave emergence was synchronized among bison by transvaginal ultrasound-guided follicle ablation. Synchronized bison were assigned to 4 groups: PRID+LH (n = 12), PRID+hCG (n = 4), no-PRID+LH (n = 12) and no-PRID+hCG (n = 4). A PRID was inserted on the day of follicular ablation in the respective groups. A single SC dose of 400 mg FSH (Folltropin) in a slow-release formulation was given the day after follicular ablation (i.e. on the expected day of a new follicular wave emergence, Day 0). The PRID was removed on Day 4 and either 25 mg LH or 2000 IU hCG was given IM on Day 5. Artificial insemination was done at 24, 36 and 48 h after LH or hCG treatment. Embryos were collected nonsurgically on Day 13 using commercial bovine equipment. Transrectal ultrasonography was done on Days 0, 5, 6, 7, 8 and 13 to record follicular and ovulatory responses. Count data (mean ± SEM) were analysed by two-way ANOVA and proportions by chi square. The number of ovulatory-sized follicles (≥10 mm) on Day 5 did not differ among groups (P = 0.33; Table 1). Ovulation rate (number of ovulations/number of follicles ≥10 mm) was greater in bison treated with hCG (P < 0.05; Table 1). The number of corpora lutea (CL) on Day 13 was greater in bison treated with hCG without a PRID (P < 0.05; Table 1). No differences in number of ova/embryos and transferable embryos were found among groups (P = 0.36 and P = 0.52, respectively; Table 1). In conclusion, progesterone priming (PRID) had no effect on ovarian superstimulation in wood bison in the anovulatory season. The ovulatory response was satisfactory only in bison treated with hCG. Embryo collection is feasible in wood bison, but the reasons for a low embryo collection rate in all groups remain unclear. Table 1.Response to superovulation and embryo collection in wood bison Funded by Advancing Canadian Agriculture and Agri-Food and Agri-Food Innovation.

The wood bison (Bison bison athabascae) is a threatened species, and recovery is constrained by endemic tuberculosis and brucellosis in wild herds in and around Wood Buffalo National Park, Alberta, Canada. The Committee on the Status of Endangered Wildlife in Canada has recommended the eradication of affected herds and repopulation with healthy bison obtained through the use of reproductive technologies. As part of the conservation effort, the specific objective of this study was to develop an effective ovarian superstimulatory protocol in bison that will permit collection of a large number of oocytes for the ultimate purpose of in vitro embryo production. Ovarian function was synchronized among female wood bison during the ovulatory season (December) by giving a luteolytic dose of prostaglandin followed 8 days later by transvaginal ultrasound-guided follicular ablation. On the day after follicular ablation (expected day of follicular wave emergence, Day 0), bison were assigned randomly to 2 groups (n = 11/group) and given either a single intramuscular dose of 2500 IU of eCG or subcutaneous doses of 200 mg of FSH given on Days 0 and 2. A luteolytic dose of prostaglandin was given on Day 2, and 25 mg of LH was given intramuscularly on Day 4. Cumulus–oocyte complexes (COC) were collected 24 h after LH treatment by transvaginal ultrasound-guided follicle aspiration using an 18 G × 2’ aspiration needle attached to a flexible tubing and a regulated vacuum pump (flow rate, 22 mL min–1). The COC were classified morphologically from 1 (excellent) to 4 (poor) based on attributes of the ooplasm and the number of surrounding cumulus cells. The number of COC in grade 1 to 3 was combined (i.e. grades acceptable for IVF) for purposes of statistical interpretation. Ovarian response and oocyte quality were compared between groups by t-tests, and COC collection rate was compared by chi-square analysis. The number (mean ± SEM) of follicles ≥5 mm was greater (P < 0.05) in bison treated with FSH than in those treated with eCG (12.2 ± 1.7 v. 5.8 ± 0.5), resulting in a greater number of follicles aspirated (11.2 ± 1.8 v. 5.6 ± 0.5; P < 0.05), more COC collected (7.2 ± 1.4 v. 3.4 ± 0.6; P < 0.05), and more COC of acceptable quality (6.1 ± 1.4 v. 2.5 ± 0.5). The COC collection rate (i.e. number of COC collected per number of follicles aspirated), however, did not differ between FSH- and eCG-treated groups [79/123 (64%) v. 37/62 (60%)]. The results document the successful collection of viable oocytes from superstimulated wood bison via tranvaginal ultrasound-guided follicle aspiration. The number of large follicles, follicles aspirated, and COC collected were more than twice as high in bison treated with FSH than in those treated with eCG. This work was supported by grants from Advancing Canadian Agriculture and Agri-Food Fund and Agri-Food Innovation Fund. The authors thank Bioniche Animal Health Canada for providing Folltropin, Lutropin, and Pregnecol.

Reclamation of Canada’s threatened wood bison (Bison bison athabascae) herd is complicated by endemic disease. As part of an overall goal to conserve bison genetics, the specific objective of this study was to develop a protocol to control follicular wave emergence in bison for the purposes of ovarian superstimulation and collection of disease-free oocytes. In an initial study, the synchronizing effect of follicular ablation and estradiol (E2) treatment was examined. Ablation was effective, but the effects of E2 (5 mg) were confounded by treatment-induced ovulation in some animals. Two experiments were done to determine the effect of a reduced dose of E2 or the addition of progesterone (P4) in comparison with follicular ablation. Bison cows (n = 19), ≥3 years old, were scanned for 14 days to determine the mean and variance in the interval to follicular wave emergence (control phase). In Experiment 1, bison were assigned randomly to 2 groups: follicular ablation (n = 9) or 2 mg of E2i.m. (n = 10). In Experiment 2, the same bison were randomly assigned to 2 groups: follicular ablation (n = 9) or 2 mg of E2+ 100 mg of P4 i.m. (n = 10). Ablation involved transvaginal ultrasound-guided aspiration of all follicles ≥5 mm. Wave emergence was determined retrospectively by identification of the follicle destined to become dominant at an initial diameter of 4 to 5 mm, with a concurrent increase in the number of follicles ≥4 mm. The interval and variation in the interval to emergence of a new follicular wave was compared among the control phase and treatment groups by ANOVA. In Experiment 1, the interval to new wave emergence (mean ± SEM) was 4.9 ± 0.66, 1.1 ± 0.11, and 2.4 ± 0.47 days from the start of the control phase, follicular ablation, and E2 treatment, respectively (P < 0.001). The degree of synchrony (residuals) was 2.4 ± 0.36, 0.2 ± 0.09, and 1.2 ± 0.24 days for the control phase, follicular ablation, and E2 treatment, respectively (P < 0.001). Ovulation was detected in 1 bison treated with E2. In Experiment 2, the interval to new wave emergence (mean ± SEM) was 4.9 ± 0.66, 1.2 ± 0.15, and 2.9 ± 0.31 days from the start of the control phase, follicular ablation, and E2 + P4 treatment, respectively (P < 0.001). The degree of synchrony was 2.4 ± 0.36, 0.2 ± 0.08, and 0.7 ± 0.20 days for the control phase, follicular ablation, and E2 + P4 treatment, respectively (P < 0.001), and no ovulations were detected. In conclusion, follicular ablation, E2, and E2 + P4 treatments all shortened and decreased the variability in the interval to new wave emergence in bison, but follicular ablation consistently produced a quicker and more synchronous response. Supported by grants from the Advancing Canadian Agriculture and Agri-Food Fund (ACAAF), the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.

As part of a project involving reproductive biotechnology as a method of preserving Canada’s threatened wood bison population, an experiment was designed to test the effectiveness of steroid-induced ovarian synchronization and fixed-timed AI. The experiment was performed in the early ovulatory season (September) with female wood bison (4 years old, n = 13) and plains bison (2 years old, n = 4, and 8 years old, n = 3). Based on results of a preceding study, progesterone was combined with estradiol as a synchronization treatment to mitigate against untimely ovulation. The bison were blocked by subspecies and assigned randomly to a control group (no treatment; n = 10) or given estradiol 17β (2.5 mg) + progesterone (50 mg) in canola oil i.m. and a progesterone-releasing intravaginal device (Cue-mate™, Bioniche, Belleville, Ontario, Canada) on Day 0 (n = 10). On Day 8, the Cue-mate™ device was removed and PGF (500 mg Estrumate®, Mallinckrodt Vet GmbH, Friesoythe, Germany) was given to induce luteolysis. On Day 10, all bison were given 5 mg of LH (Lutropin®-V, Bioniche) and artificially inseminated 12 h later with semen collected and frozen previously from wood bison of the same herd. The ovaries were examined daily by transrectal ultrasonography beginning 5 days before treatment and thereafter until the first post-treatment ovulation. Ultrasonographic pregnancy diagnosis was done 30 days post-insemination. No differences were detected between wood and plains bison for any end point, and data were combined for further statistical analyses. Ovarian follicular wave emergence occurred on Day 4.1 ± 0.8 (mean ± SEM) and 4.1 ± 0.3 in the control and treatment groups, respectively (P = 99.3). The interval to new wave emergence was less variable in bison treated with estradiol + progesterone than in untreated controls (residuals, 0.7 ± 0.2 and 1.9 ± 0.5 days, respectively; P < 0.05). The interval from LH administration to ovulation was 2.7 ± 0.6 and 5.4 ± 1.9 days for the treatment and control groups, respectively, and was less variable in the treatment group than in controls (residuals, 1.2 ± 0.4 and 5.3 ± 0.8 days, respectively; P < 0.05). The diameter of the preovulatory follicle was not different between groups and was, on average, 15.2 ± 1.1 mm. Pregnancy was diagnosed in 3 bison in the treatment group and 2 in the control group. In conclusion, treatment with estradiol and progesterone effectively synchronized the interval to wave emergence, and subsequent LH treatment resulted in a synchronous ovulatory response necessary for fixed-time AI in bison. Although the pregnancy rate was modest, perhaps because of issues with semen quality, timing of AI, or quality of the ovulated oocyte, this represents the first report of pregnancy in bison from fixed-time insemination. Supported by grants from the Advancing Canadian Agriculture and Agri-Food Fund, the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.

Endemic tuberculosis and brucellosis threaten Canada’s wood bison population. For the purposes of developing a procedure to harvest pathogen-free sperm from bison, a sperm separation and purification product, BoviPure, was tested to determine its effect on bison sperm viability before and after cryopreservation. BoviPure (NidaCon International AB, Mölndal, Sweden) is a density centrifugation gradient system that contains trypsin and is designed to separate motile from non-motile sperm and remove infectious pathogens. Spermatozoa were expelled from the caudal epididymis of bison testicles collected at slaughter and diluted in 3 to 5 mL of TCM-199 medium (Gibco, Burlington, Ontario, Canada). After 1 h of incubation at 37°C, 1 to 1.5 mL of diluted sample (approximately 300 × 106 sperm) was placed in (1) a BoviPure gradient containing trypsin (n = 18 samples), (2) a BoviPure gradient without trypsin (n = 10 samples), or 3) left untreated (control; n = 20). Gradients were centrifuged at 300g for 20 min. The sperm pellet was resuspended in 5 mL of TCM-199 medium and recentrifuged at 500 g for 10 min. Total and progressive motility were estimated using computer-assisted sperm analysis (CASA). Gradient-treated and untreated sperm samples were diluted in an egg yolk extender (Triladyl; Minitube Canada, Ingersoll, Ontario, Canada), placed in 0.5-mL straws (50-200 × 106 spermatozoa per straw), frozen, and stored in liquid nitrogen for a minimum of 24 h. Semen straws were thawed by plunging in a 37°C water bath for 30 s, and motility measurements were taken immediately by CASA. Total and progressive motility were compared among groups by analysis of variance. Before cryopreservation, total motility and progressive motility were not affected by gradient processing. Total motility was 85±5.1%, 80±4.8%, and 77 ± 3.7% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively (mean ± SEM), and progressive motility was 74 ± 2.7%, 77 ± 3.8%, and 68 ± 4.7%, respectively. After freezing and thawing, total motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (37 ± 3.6%, 33 ± 3.7%, and 29 ± 4.8% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). Similarly, progressive motility decreased compared to unfrozen samples (P < 0.0001) but was not different among groups (26 ± 3.6%, 21 ± 3.7%, and 14.5 ± 4.9% for untreated, BoviPure with trypsin, and BoviPure without trypsin, respectively). We conclude that the BoviPure gradient with or without trypsin does not influence total motility or progressive motility of bison sperm either before or after cryopreservation and has potential as a method of harvesting pathogen-free sperm from wild animals of unknown disease status. This study was supported by Canadian Adaptation and Rural Development in Saskatchewan.

Two methods for synchronizing ovarian follicular development in both wood bison (Bison bison athabascae) and plains bison (Bison bison bison) were tested as part of a project to conserve wood bison through the application of advanced reproductive technologies. A secondary objective was to test the effect of a long-acting neurolept tranquilizer, pipothiazine palmitate, on ovarian function in bison. Female wood bison (4 years old; n = 14) and plains bison (2-8 years old, n = 10), previously conditioned to daily examination in a chute, were divided randomly into 3 groups in which 1) ovarian follicles ≥5 mm were ablated by ultrasound-guided transvaginal follicle aspiration, 2) 5 mg of estradiol-17β in canola oil was given i.m. or 3) no treatment was given (control). The experiment was conducted in 3 replicates so that each animal rotated through each of the groups. Half of the bison, blocked by subspecies and treatment group, were given a single dose (150 mg) of pipothiazine palmitate i.m. The ovaries were examined daily by transrectal ultrasonography beginning 4 days before treatment and continuing until the dominant follicle of a new wave reached a diameter of 10 mm. No effect of treatment with pipothiazine palmitate was detected for any end point. In addition, no differences were detected between wood and plains bison for any end point. Consequently the pipothiazine palmitate and subspecies treatment groups were collapsed for further analyses. The interval and variation in the interval to new follicular wave emergence were compared by ANOVA. Wave emergence was detected on Day 1.0 ± 0.2 (mean ± SEM; Day 0 = day of treatment) in the follicle ablation group, and was earlier (P < 0.05) than in both the estradiol (Day 3.3 ± 0.3) and control (Day 4.0 ± 0.4) groups. The interval to follicle wave emergence was least variable in the follicle ablation group (P < 0.05) and tended to be less variable in the estradiol group (P = 0.09) than in the control group (residuals, 0.1 ± 0.04, 1.0 ± 0.2, and 1.6 ± 0.3 days, respectively). Ovulations occurred subsequent to estradiol administration in 10 of 23 (43%) bison. In conclusion, ovarian follicular wave emergence can be synchronized in bison during the anovulatory season. Follicular ablation consistently shortened and decreased the variability in the interval to new wave emergence. The synchronizing effect of estradiol was confounded by the induction of ovulation. Progesterone will be added in future studies to control the ovulatory effect of estradiol. Pipothiazine palmitate had no discernable effect on ovarian function and maybe useful in reducing the effects of handling stress on untrained animals. Supported by a grants from the Advancing Canadian Agriculture and Agri-Food Fund, the Agri-Food Innovation Fund, Parks Canada, the World Wildlife Fund, and the Northwest Territories.

Two experiments were done to test the effects of treatments designed to electively induce ovarian follicular wave emergence in wapiti for the purpose of group synchronization. In Experiment 1, hinds were assigned randomly to three groups and given saline im (controls; n = 5), 5 mg of estadiol-17ß im (n = 4), or 5 mg estradiol-17ß plus 100 mg progesterone im (n = 5). In Experiment 2, hinds were assigned randomly to two groups and given no treatment (controls; n = 6), or transvaginal ultrasound-guided follicle ablation (n = 7). In both experiments, ovarian follicular dynamics were monitored by daily transrectal ultrasonography from Day 0 (day of treatment) to Day 9. In Experiment 1, blood samples were collected at each examination for measurement of serum concentrations of progesterone and FSH. Both experiments were conducted during the late anestrous period (July and August). The mean (±S.E.M.) day of wave emergence did not differ between the control and estradiol alone groups, but tended to be later in the estradiol plus progesterone group Day 4.0 ± 0.7, Day 3.5 ± 0.3, and Day 5.2 ± 0.2, respectively; P = 0.06). The interval from treatment to wave emergence was less variable in the estradiol plus progesterone group (P < 0.05) and tended to be less variable in the estradiol-alone group (P = 0.07) than in the control group. The day of wave emergence was more variable (P < 0.05) and tended to be later (P = 0.10) in the control group compared to the ablation group (Day 2.5 ± 0.8 versus Day 1.4 ± 0.2). All three treatments were effective in synchronizing ovarian follicular wave emergence among a group of wapiti hinds. Follicle ablation may be an alternative method for synchronization of follicular waves in estrus synchronization and superstimulatory protocols.

The ovarian response to an empirically derived treatment protocol used commercially for fixed-time insemination in wapiti (Cervus elaphus) was evaluated by transrectal ultrasonography in hinds during transition into the ovulatory season. On September 29, hinds (n=7) were given an intravaginal progesterone-releasing device (CIDR-B, 1.9g of progesterone) or left untreated (controls, n=9). Fourteen days later, hinds in the treated group were given 200IU eCG and the CIDR was removed. Hinds in the control group ovulated randomly over a 15 day period. In the treated group, five hinds ovulated 3 days after eCG treatment, one ovulated 7 days after treatment, and one failed to ovulate by November 1. All extant dominant follicles ceased growth and/or began to regress within 2 days of CIDR placement. Two waves of follicular development were detected between CIDR insertion and removal; the first emerged 5.1±0.5 days after CIDR insertion and the second at 11.0±0.7 days. Serum progesterone concentration was 0.6±0.5ng/mL (range 1.0–0.3ng/mL) before CIDR placement, remained above 6ng/mL during CIDR placement, and fell to 0.8±0.9ng/mL after CIDR removal. In the control group, maximal luteal-phase progesterone concentration was lower (1.1±0.1ng/mL; P<0.05) and emergence of the first follicular wave was more variable (P=0.05) than in the treated group. The protocol to synchronize ovulation was effective in 5/7 (71%) hinds, and 4/7 (57%) became pregnant and calved. The pregnancy rate (6/9) and calving rate (5/9) was similar in the control group. In conclusion, synchronization with CIDR-B was effective; however, the protocol may be improved by shortening the interval of CIDR placement to ≤7 days and by reducing the circulating concentrations of progesterone to physiologic concentrations (<4ng/mL).

The ovaries of 12 mature wapiti hinds were studied by transrectal ultrasonography during the anovulatory season to characterize follicular dynamics and to test the hypothesis that follicle development occurs in a wave-like fashion. The hinds were examined daily, standing without sedation. Follicle size and numbers were recorded, and individual follicles were identified serially. Follicle development was considered wave-like if periodic changes in follicle numbers could be associated temporally with the development of a dominant follicle. There were non-random changes (P<0.01) in the number of follicles ≥4 mm in diameter detected per day. Each peak in follicle numbers was associated with the development of a single dominant follicle. The dominant follicle of the cohort was larger (P<0.05) than the other follicles 1 day after its emergence. Intervals between successive peaks (6.8±0.4 day) and troughs (6.8±0.4 day) in follicle numbers, and emergence of sequential dominant follicles (7.1±0.5 day) were not different (P=0.86). Results confirmed the hypothesis that ovarian follicles develop in a wave-like fashion in wapiti during the anovulatory season.