Fibroblast inflammatory priming determines regenerative versus fibrotic skin repair in reindeer.

Adult mammalian skin wounds heal by forming fibrotic scars. We report that full-thickness injuries of reindeer antler skin (velvet) regenerate, whereas back skin forms fibrotic scar. Single-cell multi-omics reveal that uninjured velvet fibroblasts resemble human fetal fibroblasts, whereas back skin fibroblasts express inflammatory mediators mimicking pro-fibrotic adult human and rodent fibroblasts. Consequently, injury elicits site-specific immune responses: back skin fibroblasts amplify myeloid infiltration and maturation during repair, whereas velvet fibroblasts adopt an immunosuppressive phenotype that restricts leukocyte recruitment and hastens immune resolution. Ectopic transplantation of velvet to scar-forming back skin is initially regenerative, but progressively transitions to a fibrotic phenotype akin to the scarless fetal-to-scar-forming transition reported in humans. Skin regeneration is diminished by intensifying, or enhanced by neutralizing, these pathologic fibroblast-immune interactions. Reindeer represent a powerful comparative model for interrogating divergent wound healing outcomes, and our results nominate decoupling of fibroblast-immune interactions as a promising approach to mitigate scar.

Recombinant prion protein vaccination of transgenic elk PrP mice and reindeer overcomes self-tolerance and protects mice against chronic wasting disease.

Chronic wasting disease (CWD) is a fatal neurodegenerative disease that affects cervids in North America and now Europe. No effective measures are available to control CWD. We hypothesized that active vaccination with homologous and aggregation-prone recombinant prion protein (PrP) could overcome self-tolerance and induce autoantibody production against the cellular isoform of PrP (PrPC), which would be protective against CWD infection from peripheral routes. Five groups of transgenic mice expressing elk PrP (TgElk) were vaccinated with either the adjuvant CpG alone or one of four recombinant PrP immunogens: deer dimer (Ddi); deer monomer (Dmo); mouse dimer (Mdi); and mouse monomer (Mmo). Mice were then challenged intraperitoneally with elk CWD prions. All vaccinated mice developed ELISA-detectable antibody titers against PrP. Importantly, all four vaccinated groups survived longer than the control group, with the Mmo-immunized group exhibiting 60% prolongation of mean survival time compared with the control group (183 versus 114 days post-inoculation). We tested for prion infection in brain and spleen of all clinically sick mice. Notably, the attack rate was 100% as revealed by positive CWD signals in all tested tissues when assessed with Western blotting, real-time quaking-induced conversion, and immunohistochemistry. Our pilot study in reindeer indicated appreciable humoral immune responses to Mdi and Ddi immunogens, and the post-immune sera from the Ddi-vaccinated reindeer mitigated CWD propagation in a cell culture model (CWD-RK13). Taken together, our study provides very promising vaccine candidates against CWD, but further studies in cervids are required to investigate vaccine efficacy in the natural CWD hosts.

Acute BVDV-2 infection in beef calves delays humoral responses to a non-infectious antigen challenge.

Immunosuppressive effects of an intranasal challenge with non-cytopathic bovine viral diarrhea virus (BVDV) 2a (strain 1373) were assessed through acquired and innate immune system responses to ovalbumin (OVA). Concurrent BVDV infection was hypothesized to delay and reduce the humoral response to ovalbumin (administered on days 3 and 15 post-inoculation). Infected animals followed the expected clinical course. BVDV titers, and anti-BVDV antibodies confirmed the course of infection and were not affected by the administration of OVA. Both the T-helper (CD4(+)) and B-cell (CD20(+)) compartments were significantly (P < 0.05) reduced in infected animals, while the gamma-delta T-cell population (Workshop cluster 1+, WC1(+)) decreased slightly in numbers. Infection with BVDV delayed the increase in OVA IgG by approximately 3 d from day 12 through day 21 post-inoculation. Between days 25 and 37 post-inoculation following BVDV infection the IgM concentration in the BVDV- group decreased while the OVA IgM titer still was rising in the BVDV+ animals. Thus, active BVDV infection delays IgM and IgG responses to a novel, non-infectious antigen.

Une infection aiguë par le BVDV-2 chez les veaux retarde les réponses humorales face à un test à l'aide d'un antigène non infectieux. Les effets immunosuppressifs d'une inoculation défin intranasale à l'aide du virus non cytopathogène de la diarrhée virale bovine (VBVD) 2a (souche 1373) ont été évalués par les réactions acquises et innées du système immunitaire à l'ovalbumine (OVA). On a émis l'hypothèse que l'infection concomitante par le VBVD retardait et réduisait la réaction humorale à l'ovalbumine (administrée aux jours 3 et 15 après l'inoculation). Les animaux infectés ont suivi le cheminement clinique prévu. Les titres de BVDV et les anticorps anti-BVDV ont confirmé le déroulement de l'infection et ils n'ont pas été affectés par l'administration d'OVA. Les compartiments des lymphocytes T auxiliaires (CD4+) et des cellules B (CD20+) étaient significativement réduits (P < 0,05) chez les animaux infectés, tandis que la numération de la population de cellules T gamma-delta (WC1+) a diminué légèrement. L'infection par le VBVD a retardé l'augmentation de l'OVA IgG d'environ 3 jours, à compter du jour 12 jusqu'au jour 21 après l'inoculation. Entre les jours 25 et 37 après l'inoculation suivant l'infection par le BVDV, la concentration d'IgM dans le groupe VBVD a diminué tandis que le titre d'OVA IgM augmentait toujours chez les animaux positifs pour le VBVD. Par conséquent, l'infection active par le VBVD retarde les réactions IgM et IgG face à un antigène non infectieux nouveau.(Traduit par Isabelle Vallières).

Ovarian synchronisation in wood bison (Bison bison athabascae) during the anovulatory season.

Two experiments were performed in wood bison during the anovulatory season to establish an effective protocol for ovarian synchronisation. In an untreated control phase, bison cows (n=19) were examined daily to establish the interval to new follicular wave emergence (4.9±0.7 days) for the purposes of comparison with the experimental treatments. In Experiment 1, bison were treated by transvaginal ultrasound-guided follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol (n=10). In Experiment 2, bison were treated by follicular ablation (n=9) or with 2mg, i.m., 17ß-oestradiol +100mg, i.m., progesterone (n=10). In Experiment 1, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol-treated groups was 4.9±0.7, 1.1±0.1 and 3.1±0.4 days, respectively (P<0.05). The degree of synchrony was 2.4±0.4, 0.2±0.1 and 0.8±0.2 days, respectively (P<0.05). In Experiment 2, the interval to new wave emergence for control, follicular ablation and 17ß-oestradiol + progesterone-treated groups was 4.9±0.7, 1.2±0.2 and 3.3±0.3 days, respectively (P<0.05), and the degree of synchrony was 2.4±0.4, 0.2±0.1, and 0.8±0.2 days, respectively (P<0.05). The degree of synchrony did not differ between ablation and hormone treatment groups in either experiment, but was greater in treatment groups than in the untreated control phase. Both follicular ablation and hormone treatment shortened and decreased the variability in the interval to follicular wave emergence in bison, but wave emergence occurred earlier after follicular ablation.

Limited efficacy of Fever Tag(®) temperature sensing ear tags in calves with naturally occurring bovine respiratory disease or induced bovine viral diarrhea virus infection.

Temperature sensing ear tags were tested in 1) auction-derived calves with 50% incidence of bovine respiratory disease, and 2) specific pathogen-free calves infected with bovine virus diarrhea virus. There were no false positives, but tag placement, probe displacement, and a high threshold for activation all contributed to failure to reliably detect sick calves.

Efficacité limitée des étiquettes d'oreille Fever TagMDpour mesurer la température chez les veaux atteints de maladies respiratoires d'origine naturelle ou d'une infection induite par le virus de la diarrhée virale des bovins. Les étiquettes d'oreille pour mesurer la température ont été testées chez 1) des veaux provenant d'encans ayant 50 % d'incidence de maladies respiratoires et 2) des veaux exempts d'agents pathogènes spécifiques infectés par le virus de la diarrhée virale bovine. Il n'y avait aucun faux positif, mais le placement des étiquettes, le déplacement de la sonde et un seuil d'activation élevé ont tous contribué à l'échec de la détection fiable des veaux malades.(Traduit par Isabelle Vallières).

Transport versus on-farm slaughter of bison: physiological stress, animal welfare, and avoidable trim losses.

Ranched bison are typically less acclimated to handling than are domesticated livestock, suggesting that they might be more vulnerable to handling and transportation stressors. Grain-finished bison were slaughtered on-farm (n = 11), or held for 48 h, transported to a research abattoir, held in lairage for 18 h, and then slaughtered (n = 11). An additional group (n = 10) was sampled at a conventional fixed location abattoir. Measures included serum cortisol and corticosterone concentrations during on-farm handling and exsanguination, serum glucose, creatinine phosphokinase (CPK), aspartate aminotransferase (AST), and trim losses. Transport was associated with an increase in trim loss. On-farm, glucose was elevated, CPK was positively associated with handling order over 12 h, and corticosterone concentration, although lower than cortisol concentration, showed a greater response to prolonged disturbance. With appropriate on-farm handling facilities, the use of on-farm slaughter and mobile abattoir could avoid muscle damage and trim losses, and mitigate injuries sustained during handling and transport of bison.

Transport ou abattage des bisons à la ferme : stress physiologique, bien-être animal et pertes de viande évitables. Les bisons élevés dans un ranch sont habituellement moins accoutumés à la manipulation que le bétail domestiqué, ce qui suggère qu'ils sont plus vulnérables aux facteurs de stress liés à la manipulation et au transport. Des bisons engraissés au grain ont été abattus à la ferme (n = 11), ou détenus pendant 48 h, transportés à un abattoir de recherche, mis dans des installations d'attente pendant 18 h, puis abattus (n = 11). Un groupe additionnel (n = 10) a été échantillonné à un abattoir fixe conventionnel. Les mesures incluaient les concentrations de cortisol sérique et de corticostérone durant la manipulation et l'exsanguination à la ferme, le glucose sérique, la créatinine phosphokinase (CPK), l'aspartate aminotransférase (AST) et les pertes de viande. Le transport était associé à une hausse de la perte de viande. À la ferme, le glucose était élevé, la CPK était positivement associée à l'ordre de manipulation pendant 12 heures et la concentration de corticostérone, quoique inférieure à la concentration de cortisol, a montré une réponse plus élevée lors d'une perturbation prolongée. Avec des installations de manipulation à la ferme appropriées, l'utilisation de l'abattage à la ferme et d'un abattoir mobile pourrait éviter les dommages musculaires et les pertes de viande et atténuer les blessures subies durant la manipulation et le transport des bisons.(Traduit par Isabelle Vallières).

Systemic murine cathelicidin CRAMP safely attenuated colonic neutrophil infiltration in pigs.

Post-weaning diarrheic colitis, often caused by enteropathogens, are severe and potentially lethal diseases in young pigs. Conventional treatment with antibiotics is problematic due to increasing prevalence of multi-drug resistant bacteria. Few alternative treatments exist, so development of antibiotic-free therapies is urgently needed for livestock. Cathelicidin peptides, produced by epithelial cells and neutrophils, are microbicidal compounds capable of modulating innate immune and inflammatory responses. However, the effects of exogenous cathelicidin on gut homeostasis is poorly understood in pigs. We administered the murine cathelicidin CRAMP systemically to healthy pigs, to establish the peptide's safety and assess its ability to modulate colonic mucosal defenses. A single intraperitoneal injection of CRAMP was well tolerated up to two weeks and pigs remained clinically healthy. CRAMP caused some alteration of mucus glycosylation patterns in the colon by increasing sialylated mucins (P < 0.05) and decreased neutrophil influx close to the epithelium (P < 0.001). This study supports further investigation of CRAMP as an immunomodulatory treatment for infectious colitis in pigs.

Induction of humoral immune response in piglets after perinatal or post-weaning immunization against porcine circovirus type-2 or keyhole limpet hemocyanin.

The objective of this study was to test the hypothesis that porcine circovirus type-2 (PCV2) vaccination is efficacious when administered in the first week of life. Three groups of pigs were vaccinated with Circumvent either early (at the end of week 1), late (at the end of week 4), or not at all. All 3 groups were later challenged intranasally with PCV2 (at the end of week 5). Two other groups were immunized with keyhole limpet hemocyanin (KLH) as a novel antigen at the end of either week 1 or week 4. Weight, PCV2 genome copy number in serum and saliva, anti-KLH antibody titer, and serum PCV2-neutralizing antibodies were measured weekly. Early PCV2 vaccination or KLH antigen exposure resulted in earlier humoral responses that were slower to develop than in older piglets, yet converged with the responses to later vaccination within 5 wk. Both groups of vaccinated piglets had periods of higher PCV2-neutralizing antibody titers and lower viral levels shortly after weaning and PCV2 challenge, thus supporting the recent labelling of 1 Canadian PCV2 vaccine for use in week 1 and suggesting that early PCV2 vaccination can reduce piglet handling without compromising vaccine efficacy.

L'objectif de la présente étude était de vérifier l'hypothèse que la vaccination contre le circovirus porcin de type 2 (CVP2) est efficace lorsqu'administrée durant la première semaine de vie. Trois groupes de porcs ont été vaccinés avec Circumvent soit hâtivement (à la fin de la semaine 1), tardivement (à la fin de la semaine 4), ou pas du tout. Les trois groupes ont plus tard été inoculés par voie intranasale avec CVP2 (à la fin de la semaine 5). Deux autres groupes ont été immunisés avec de l'hémocyanine de patelle (KLH) à titre de nouvel antigène à la fin de soit la semaine 1 ou la semaine 4. Le poids, le nombre de copies du génome de CVP2 dans le sérum et la salive, le titre d'anticorps anti-KLH, et le titre d'anticorps sériques neutralisants CVP2 ont été mesurés à chaque semaine. La vaccination tôt contre CVP2 ou l'exposition à l'antigène KLH a donné des réponses humorales plus hâtives qui étaient plus lentes à se développer que chez les porcs plus vieux, mais qui convergeaient vers les réponses de la vaccination tardive à l'intérieur d'un délai de 5 sem. Les deux groupes de porcelets vaccinés avaient des périodes de titres d'anticorps neutralisants contre CVP2 plus élevés et des charges virales plus basses peu de temps après le sevrage et le challenge avec CVP2, soutenant ainsi l'étiquetage récent d'un vaccin canadien contre CVP2 pour utilisation dans la semaine 1 et suggérant qu'une vaccination tôt contre CVP2 peut réduire la manipulation des porcelets sans compromettre l'efficacité du vaccin.(Traduit par Docteur Serge Messier).

Diverse and highly recombinant anelloviruses associated with Weddell seals in Antarctica.

The viruses circulating among Antarctic wildlife remain largely unknown. In an effort to identify viruses associated with Weddell seals (Leptonychotes weddellii) inhabiting the Ross Sea, vaginal and nasal swabs, and faecal samples were collected between November 2014 and February 2015. In addition, a Weddell seal kidney and South Polar skua (Stercorarius maccormicki) faeces were opportunistically sampled. Using high throughput sequencing, we identified and recovered 152 anellovirus genomes that share 63-70% genome-wide identities with other pinniped anelloviruses. Genome-wide pairwise comparisons coupled with phylogenetic analysis revealed two novel anellovirus species, tentatively named torque teno Leptonychotes weddellii virus (TTLwV) -1 and -2. TTLwV-1 (n = 133, genomes encompassing 40 genotypes) is highly recombinant, whereas TTLwV-2 (n = 19, genomes encompassing three genotypes) is relatively less recombinant. This study documents ubiquitous TTLwVs among Weddell seals in Antarctica with frequent co-infection by multiple genotypes, however, the role these anelloviruses play in seal health remains unknown.

Ovarian follicular and luteal dynamics in wapiti during the estrous cycle.

The reproductive tracts of 13 mature hinds were examined daily by transrectal ultrasonography and blood samples were taken daily from October to January to characterize follicular, luteal, and endocrine dynamics in wapiti during the estrous season. Follicle development occurred in waves characterized by regular, synchronous development of a group of follicles in temporal succession to a surge in serum FSH concentration. The mean interovulatory interval was 21.3 +/- 0.1 d, but was shorter in hinds exhibiting two follicular waves than in hinds exhibiting three and four waves (P < 0.05). The interwave interval was similar among waves in two-wave cycles and the first wave of three-wave cycles. All other interwave intervals in three- and four-wave cycles were shorter (P < 0.05). The maximum diameter of the dominant follicle of the first wave was similar among two-, three-, and four-wave cycles. For all other waves in three- and four-wave cycles, the maximum diameter was smaller (P < 0.05). Corpus luteum diameter and plasma progesterone concentrations were similar between two- and three-wave cycles, but the luteal phase was longer (P < 0.05) in four-wave cycles. The dominant follicle emerged at a diameter of 4 mm at 0.4 +/- 0.1 and 0.8 +/- 0.1 d before the largest and second largest subordinate follicles, respectively. The follicle destined to become dominant was larger (P < 0.05) than the largest subordinate follicle one day after emergence, which coincided with the first significant decrease in serum FSH concentration. We concluded that the estrous cycle in wapiti is characterized by two, three, or four waves of follicular development (each preceded by a surge in circulating FSH), that there is a positive relationship between the number of waves and the duration of the cycle, and an inverse relationship between the number of waves and the magnitude of follicular dominance (diameter and duration of the dominant follicle).

Superovulation in wood bison (Bison bison athabascae): Effects of progesterone, treatment protocol and gonadotropin preparations for the induction of ovulation.

Experiments were done to determine the ovarian response and embryo production following superstimulation of wood bison. In Experiment 1 (Anovulatory season), the efficacy of pLH vs. hCG for inducing ovulation was compared in wood bison superstimulated with a single dose of pFSH in 0.5% hyaluronan and the effect of exogenous progesterone (PRID) on superovulatory response and embryo quality was examined. In Experiment 2 (Ovulatory season), the efficacy of pLH vs. hCG for the induction of ovulation was compared in wood bison superstimulated with pFSH in a single intramuscular dose vs. a two-dose regimen 48 h apart (split dose) in 0.5% hyaluronan. In Experiment 1, the number of CL was greater (P < 0.05) in bison treated with hCG than pLH (6.6 ± 1.8 vs. 2.8 ± 0.8) and in those that were not given PRID (6.0 ± 1.5 vs. 2.7 ± 1.0). There was no effect of progesterone treatment on embryo quality. In Experiment 2, the number of CL was greater (P < 0.05) in bison treated with hCG than with pLH (6.3 ± 0.8 vs. 3.8 ± 1.2) and in bison superstimulated with split dose vs. single dose of FSH (7.1 ± 0.9 vs. 3.0 ± 0.8). The number of ova/embryos and freezable embryos did not differ among groups in either experiment. In conclusion, hCG induced a greater ovulatory response than pLH in both seasons. Two doses of FSH induced the greatest superovulatory response during the ovulatory season. Exogenous progesterone did not improve embryo quality during the anovulatory season.

Use of azaperone and zuclopenthixol acetate to facilitate translocation of white-tailed deer (Odocoileus virginianus).

Thirteen wild-caught white-tailed deer (Odocoileus virginianus) from two different holding sites were chemically immobilized to facilitate capture, processing, and translocation to a new facility. The deer were divided into two treatment groups on the basis of age and received i.m. injections of the immobilizing agents via remote drug delivery systems. Group 1 (<1 yr old; n = 6) animals were immobilized with a combination of xylazine 1 mg/kg i.m. and azaperone 0.3 mg/kg i.m. At the end of the procedure the deer received zuclopenthixol acetate 1 mg/kg i.m. and (to antagonize the xylazine) tolazoline 2 mg/kg i.m. Group 2 (>1 yr old; n = 7) deer were administered xylazine 1 mg/kg i.m.. tiletamine-zolazepam 1 mg/kg i.m., and ketamine 1 mg/kg i.m. The deer in this group received a combination of azaperone 0.3 mg/kg i.m. and zuclopenthixol acetate 1 mg/kg i.m. before reversal with tolazoline 2 mg/kg i.m. All deer were recovered in a trailer before being moved in small groups to the new facility and released into the new enclosures. Azaperone and zuclopenthixol acetate provided short- and long-term relief from anxiety and allowed the animals to gradually become familiar with their new surroundings without excitement, injuries, or mortalities. Two deer exhibited mild signs of extrapyramidal side effects, which suggests that they may have received a relative overdose of the tranquilizers.

Ovarian superstimulation and oocyte collection in wood bison (Bison bison athabascae) during the ovulatory season.

The objective of the study was to establish an effective ovarian superstimulatory protocol and subsequently obtain oocytes from bison by transvaginal ultrasound-guided follicular aspiration. Two experiments involving 22 wood bison were done during the breeding season (September to December). In experiment 1, the bison were given a luteolytic dose of prostaglandin (Day 0) and underwent follicular ablation (Day 8) to induce ovarian synchrony. Synchronized bison were then assigned randomly to two groups (n = 11 per group) and given either 200 mg FSH diluted in saline sc, or 200 mg FSH diluted in a proprietary slow-release formulation (SRF) im on Days 9 and 11. Prostaglandin was given to both groups on Day 11 followed by 25 mg LH on Day 13. Oocytes were collected by transvaginal ultrasound-guided aspiration of follicles ≥5 mm on Day 14. In experiment 2, bison were synchronized as in experiment 1 and assigned randomly to one of two groups (n = 11 per group) and given either a single dose of 2500 IU eCG im on Day 9, or 200 mg FSH sc on Days 9 and 11. Prostaglandin was given to both groups on Day 11, and LH (25 mg) was given on Day 13. Oocyte collection was done as described in experiment 1. Cumulus-oocyte-complexes (COC) were classified according to morphologic characteristics. In experiment 1, more follicles ≥5 mm were detected on Day 14 in bison treated with FSH versus eCG (12.2 ± 1.73 vs. 5.8 ± 0.52; P < 0.05), and more COC were collected from FSH-treated animals (7.2 ± 1.41 vs. 3.4 ± 0.62; P < 0.05). In experiment 2, the FSH-saline and FSH-SRF groups had a similar number (mean value ± standard error of the mean) of follicles ≥5 mm on Day 14 (12.4 ± 1.49 vs. 13.8 ± 1.24, respectively) and a similar number of COC were collected (6.5 ± 1.13 vs. 6.3 ± 0.96, respectively). The proportion of COC collected per follicle aspirated and the percentage of compact, expanded, and denuded oocytes did not differ between groups in either experiment 1 or 2. In summary, a two-dose regimen of FSH diluted in saline and given sc or in a SRF and given im induced a similar ovarian response in wood bison, whereas a single dose of eCG resulted in a significantly lower ovarian response. Overall, COC were collected from 55% of follicles after transvaginal, ultrasound-guided needle aspiration in wood bison.

Superovulation in waptiti (Cervus elaphus) during the anovulatory season.

The objective was to evaluate the efficacy of three previously unreported ovarian superovulatory treatment protocols in wapiti. Protocols were initiated specifically at the time of ovarian follicular wave emergence, and intended to enable determination of the effects of frequency of treatment (i.e., animal handling) and LH supplementation on ovarian response. Thirteen parous wapiti hinds, 2 to 4 y of age, were used late in the anovulatory season (July). The ovaries were examined daily by transrectal ultrasonography. Hinds were given 5 mg estradiol 17-ß im (day of treatment designated as Day 0) to induce a new wave of ovarian follicular development. On the expected day of wave emergence (Day 3), hinds were assigned randomly to three treatment groups and given: (1) 100 mg FSH im once a day for 4 days (N = 5); (2) 200 mg FSH sc on Day 3 and Day 5 (N = 4); or (3) 200 mg FSH plus 2.5 mg LH sc on Day 3 and Day 5 (N = 4). All hinds were given 10 mg LH im on Day 6 to induce ovulation. The mean (± SEM) number of ovulations per animal in the respective groups was 6.2 ± 2.0, 15.5 ± 5.9, and 14.8 ± 2.7. In conclusion, the technique of inducing follicular wave emergence to initiate superovulatory treatment at the time of wave emergence was effective in wapiti during the anovulatory season. The most efficient and effective method of ovarian superovulation in this study involved administration of estradiol 17-ß on Day 0, followed by 200 mg FSH sc on Days 3 and 5, and induction of ovulation (10 mg of LH) on the evening of Day 6. Compared with conventional methods that require 14 days and handling the hinds six times, the protocol used herein reduced the treatment period to 8 days and the number of animal handlings to four.

Serial ovarian ultrasonography in wild-caught wood bison (Bison bison athabascae).

The objectives of this study were to determine the feasibility of daily examination of wild-caught wood bison and to characterize the ovarian function using serial transrectal ultrasonography and blood hormone analysis. Ten 2-year-old wood bison heifers obtained from Elk Island National Park were placed in a corral adjacent to a handling system designed for restraining bison. The handling system was left open to the corral allowing the bison to explore it freely for 2 months. Active acclimation followed for a 2-week period, during which the bison were herded daily through the handling system and rewarded with whole oats. Finally, the bison were restrained in the handling system and rewarded with whole oats upon release. Once conditioned, daily transrectal examination of the ovaries was completed in 100% of attempts for 30 days (January-February) using a B-mode scanner with a 5 to 10-MHz linear array. Follicle size and numbers were recorded, and individual follicles were identified serially. Blood samples were collected daily and the serum was analyzed for FSH concentrations. Nonrandom changes were detected in the number of follicles ≥ 4 mm in diameter per day (P < 0.05). Each peak in follicle numbers was associated with the development of a single dominant follicle. The interval between the emergence of successive dominant follicles was 6.8 ± 0.6 days (mean ± SEM). The maximum diameter of the dominant follicle was 9.9 ± 0.4 mm. In conclusion, wild-caught wood bison were amenable to daily examination and blood sampling, and ovarian dynamics were characterized by wave-like development of anovulatory antral follicles. The demonstrated success of this approach to the study of ovarian function will be useful for characterizing the annual reproductive pattern in wood bison, which is necessary for the development of bison-specific protocols for controlling ovarian function for species conservation.

Superstimulatory response and oocyte collection in North American bison during the non-breeding season.

A 2×2 design was used to compare the ovarian response and oocyte collection characteristics in bison given a superstimulatory dose of eCG or FSH, with or without a follow-up dose of LH. Follicular wave emergence was synchronized by follicle ablation (Day -1) and bison were assigned randomly to two superstimulatory treatment groups (n=10 per group): (i) a single intramuscular dose of 2500IU of eCG given on Day 0, or (ii) two subcutaneous doses of 200mg of FSH given on Days 0 and 2. On Day 4, 200mg of LH was given intramuscularly in 5 bison in each superstimulatory treatment group. The study was done in two replicates (n=20 per replicate) involving a crossover design so that each animal was given the opposite superstimulatory treatment (eCG or FSH) during successive replicates. Cumulus-oocyte complexes (COC) were collected by transvaginal ultrasound-guided follicle aspiration, and were classified according to morphologic attributes as compact, expanded, or denuded. Superstimulatory treatment with FSH (vs. eCG) resulted in the development of more follicles ≥5mm (14.2±1.41 vs. 8.2±0.67; P<0.05; mean±SEM), and more follicles aspirated (12.4±1.3 vs. 6.3±0.6; P<0.04). Follow-up treatment with LH (vs. no LH) resulted in a greater proportion of expanded COC (37% vs. 15%; P<0.05), and a tendency for a higher COC collection rate (61% vs. 54%; P=0.08). In summary, superstimulation with FSH (vs. eCG) resulted in twice as many follicles available for aspiration and nearly twice as many COC collected in bison during the anovulatory season, and follow-up treatment with LH increased the proportion of expanded COC collected.

Superovulation and embryo transfer in wood bison (Bison bison athabascae).

Two experiments were done to develop an effective superovulatory treatment protocol in wood bison for the purpose of embryo collection and transfer. In experiment 1, donor bison were assigned randomly to four treatment groups (N = 5 per group) to examine the effects of method of synchronization (follicular ablation vs. estradiol-progesterone treatment) and ovarian follicular superstimulation (single slow-release vs. split dose of FSH). Recipient bison were synchronized with donor bison by either follicular ablation (N = 8) or estradiol-progesterone treatment (N = 9). In experiment 2, bison were assigned randomly to four treatment groups (N = 5 per group) to examine the ovarian response to two versus four doses of FSH, and the effect of progesterone (ovarian superstimulation with or without an intravaginal progesterone-releasing device). Donor bison were inseminated with fresh chilled wood bison semen 12 and 24 hours after treatment with GnRH (experiment 1) or LH (experiment 2). The ovarian response was assessed using ultrasonography. In experiment 1, the number of large follicles (≥ 7 mm) increased in response to both FSH treatments, but the diameter of the largest follicle detected 4 and 5 days after the start of ovarian superstimulation was greater in bison treated with a single dose of FSH than in those treated with two doses (P < 0.05). A total of 10 ova and/or embryos were collected. One blastocyst was transferred to each of five recipient bison resulting in the birth of two live wood bison calves. In experiment 2, two doses of FSH resulted in a greater number of large follicles (≥ 9 mm) on Days 4, 5, and 6 (P < 0.05) after beginning of superstimulation (Day 0), and more ovulations than four doses of FSH (11.2 ± 2.4 vs. 6.4 ± 0.8; P < 0.05). Embryo collection was performed on only five donors, and a total of 19 ova and/or embryos were recovered. In summary, fewer FSH treatments were as good or better than multiple treatments, consistent with the notion that minimizing handling stress improves the superovulatory response in bison. Follicular ablation and estradiol plus progesterone treatment were effective for inducing ovarian synchronization in embryo donor and recipient bison, and an intravaginal progesterone-releasing device during superstimulatory treatment did not influence the superovulatory response or embryo collection. Delaying ovulation-inducing treatment (GnRH or LH) to 5 days after superstimulatory treatment resulted in a greater number of ovulations and improved embryo collection efficiency (experiment 2). Embryo collection and transfer resulted in live offspring from wild wood bison.