Barbara A. Wolfe

Comparing ovulation synchronization protocols for artificial insemination in the scimitar-horned oryx (Oryx dammah)

Ovarian response and pregnancy success in scimitar-horned oryx (n=28) were compared, following treatment with two synchronization protocols and fixed-time artificial insemination (AI) with frozen-thawed semen. Each oryx received two injections of 500 microg of prostaglandin-F(2alpha) analogue (PGF(2alpha)-only) 11 days apart, and half received PGF(2alpha) in combination with an intravaginal progesterone-releasing device (CIDR11+PGF(2alpha)). Semen was collected by electroejaculation from anaesthetised adult oryx and cryopreserved. Anaesthetised females were transcervically inseminated 56.0+/-1.1 h (+/-S.E.M.) after PGF(2alpha) injection and/or device withdrawal using 28.0+/-1.5x10(6) motile thawed sperm. Ovarian endocrine response was monitored in 20 females by analysing faecal oestrogen and progesterone metabolites. Periovulatory oestrogen peaks were detected in 19/20 (95%) females after synchronization. There were no between-treatment differences in oestrogen concentrations or peak characteristics (P0.05). Luteal development after synchronization was delayed in half the progesterone treated (CIDR11+PGF(2alpha)) females, and faecal progestin excretion profiles indicated that the ovulatory follicle associated with synchronization either failed to ovulate or to fully lutenise. Pregnancy was diagnosed by ultrasonography and/or rectal palpation and was monitored by faecal progestin excretion. More (P=0. 013) pregnancies resulted from the PGF(2alpha)-only treatment (37.5%, 5/14) than from the CIDR11+PGF(2alpha) treatment (0/14), and four healthy scimitar-horned oryx calves were born, three after gestation intervals of 247 days and one after 249 days.

Reproductive physiology and artificial insemination studies in wild and captive gerenuk ( Litocranius walleri walleri ).

Gerenuk antelope in North American zoos are descended from 28 founders imported from Kenya approximately 20 years ago. Intensive management is required to prevent inbreeding depression. Artificial insemination has potential for augmenting genetic management, but successful application requires a thorough understanding of species reproductive norms. Semen collected from captive (n = 10) and wild (n = 6) gerenuk contained low numbers of morphologically normal spermatozoa (approximately 40%). Age, but not season, influenced (P < 0.05) the proportion of morphologically normal spermatozoa (mean +/- s.e.m., 12-17 months of age, 10.3 +/- 1.9%; 18-26 months of age, 34.4 +/- 6.2%; 3-6 years of age, 40.0 +/- 4.7%). Seasonality was investigated by analysing faecal testosterone and progesterone in males and females, respectively, by radioimmunoassays. Females cycled all year (ovarian cycle length, 18.7 +/- 0.9 days). Testosterone in males did not vary (P > 0.05) with time of year. Three females (3/9, 33%) became pregnant by insemination with 9.75-54.0 x 0(6) motile fresh or frozen sperm after oestrus synchronisation with two prostaglandin F(2alpha) injections, 12 days apart. One female inseminated with frozen-thawed sperm delivered a full-term stillborn calf after 213 days gestation. These results characterise gerenuk reproductive norms and indicate that artificial insemination may be a useful tool in the genetic management of gerenuk.

Fundamental Studies of the Reproductive Biology of the Endangered Persian Onager (Equus hemionus onager) Result in First Wild Equid Offspring from Artificial Insemination

ABSTRACT We studied the Persian onager (Equus hemionus onager), an endangered equid subspecies. The objective was to characterize endocrine patterns and ovarian follicular dynamics of females as well as seminal traits and sperm sensitivity to cryopreservation in males as a prerequisite to testing the feasibility of artificial insemination (AI). Urinary progesterone and estrogen metabolite profiles were determined by enzyme immunoassay in 11 females. Serial ultrasonography of ovarian activity was performed for 2 mo in a subset of four females. Females were seasonally polyestrous (June–November). Ovarian morphometry via ultrasonography and urinary progesterone profiles were more reflective of reproductive events than urinary estrogen patterns, and preovulatory follicle size was smaller than reported for other equid species. There was evidence for lactational suppression of estrus for up to 1.5 yr in nursing dams. Electroejaculation allowed recovery of highly motile sperm from 7, anesthetized males on 57% of occasions. Spermatozoa, including motility and acrosomal integrity, were resilient to freeze-thawing. Artificial insemination was successful in 2 of 3 females following detection of a dominant follicle and deslorelin administration, resulting in births of a healthy female and male foal by using fresh/chilled and frozen/thawed sperm, respectively.

Abnormal Reproductive Patterns in Przewalski's Mares Are Associated with a Loss in Gene Diversity

ABSTRACT The ex situ population of the Przewalskis horse (Equus ferus przewalskii) is not self-sustaining (20% foaling rate), and the demography is skewed toward aging individuals with low gene diversity. We designed the present study to gain a better understanding of the reproductive biology of the Przewalskis mare and to determine whether age and gene diversity influenced reproductive function. Urine samples were collected 3–7 days/wk from 19 mares from May to September, and ultrasound examinations of follicular structures were performed 3 days/wk for 5 wk from May through July in nine individuals. A high proportion of mares exhibited abnormal (endocrine, 5 [26.3%] of 19; follicular, 2 [22.2%] of 9) or acyclic (endocrine, 4 [21.1%] of 19; follicular, 3 [33.3%] of 9) reproductive patterns. In four cyclic mares, estrous cycle length was 25.1 ± 1.2 days, with 12.2 ± 0.9 days of diestrus. Follicles in cyclic mares grew 1.2 ± 0.6 mm per day and ovulated after reaching 40.4 ± 8.9 mm. Mares with a high coefficient of inbreeding excreted reduced levels of mean urinary estrogens (r2 = 0.476, P < 0.05), but age had no significant impact on reproductive patterns in this population. Overall, these data suggest that long-term genetic management of this population is necessary to maintain reproductive fitness.

Analysis and cytologic characterization of hemocytes from freshwater mussels (Quadrula sp.).

BACKGROUNDnFreshwater mussels are among the most endangered taxa in North America and minimally invasive techniques to evaluate their health are needed.nnnOBJECTIVEnThe objective of this study was to develop a standardized approach for identifying and enumerating the cellular components of freshwater mussel hemolymph.nnnMETHODSnHemocyte clumping, total hemocyte count, and hemocyte morphology were compared in untreated hemolymph or hemolymph treated with formalin, sodium citrate, sodium heparin, EDTA, water, or l-cysteine. Morphology was then used to categorize hemocytes and perform a 100-cell differential.nnnRESULTSnTreatment with formalin or >25 mg/mL l-cysteine reduced hemocyte clumping, although only formalin significantly increased the total hemocyte count. However, formalin also induced crenation that impaired hemocyte identification. Both EDTA and sodium citrate-induced hemocyte degranulation while sodium citrate and >40 mg/mL l-cysteine-induced cell lysis. Hemocytes could be categorized into 2 groups of granulocytes (eosinophilic or basophilic) and 2 groups of agranulocytes (large or small) for performing a cytologic differential. The differential was not significantly altered by anticoagulant treatments providing cell morphology was adequate for obtaining a differential. Eosinophilic granulocytes predominated (59%) with fewer large agranulocytes (27%) and basophilic granulocytes (13%). Small agranulocytes comprised 2% of the total population.nnnCONCLUSIONSnNo single treatment provided an optimal method to evaluate freshwater mussel hemolymph. Maximal hemocyte counts were obtained following formalin treatment. l-cysteine reduced clumping and maintained hemocyte morphology for performing a cytologic differential. These techniques provide a standardized approach for the hematologic evaluation of freshwater mussels.

Nontargeted metabolomics reveals biochemical pathways altered in response to captivity and food limitation in the freshwater mussel Amblema plicata

Effective conservation of freshwater mussels (Mollusca: Bivalvia: Unionidae), one of the most endangered groups of animals in North America, is compromised by limited knowledge of their health. We address this gap in knowledge by characterizing the metabolic profile of Amblema plicata in the wild and in response to captivity and food limitation. Eight mussels brought into captivity from the wild were isolated for 18 days without a food source. Hemolymph samples were taken prior to, and 9 and 18 days after the start of the experiment; these samples were analyzed by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. We detected and identified 71 biochemicals in the hemolymph of freshwater mussels; of these, 49 showed significant changes during captivity and/or food limitation (p<0.05). Fasting resulted in severe metabolite depletion. Captive (but fed) mussels experienced changes similar to (albeit less severe than) fasting mussels, suggesting that mussels may experience nutritional deficiency under common captive conditions. A. plicata responded to food limitation stress by preferentially using energy reserves for maintenance rather than growth. Carbohydrate and energy metabolism exhibited down-regulation in captive, food-limited, and wild mussels. Lipid metabolism was up-regulated in captive/food-limited mussels and unchanged in wild mussels. Amino acid metabolism was up-regulated in wild mussels and down-regulated in captive/food-limited mussels. Nucleotide metabolism was up-regulated in the wild mussels, down-regulated in food-limited mussels, and unchanged in captive mussels. The different responses between treatment groups suggest potential for nucleotide metabolism as a biomarker of health status for freshwater mussels.

Glucocorticoid response to changes in enclosure size and human proximity in the Persian onager (Equus hemionus onager)

This study investigated glucocorticoid (GC) responses to season and changes in enclosure size and human proximity in the Persian onager (Equus hemionus onager). Enzyme immunoassays were validated to measure GC metabolites in urine and feces (fGCM). Fecal samples were collected from 10 female onagers while in a large pasture, after transport to smaller yards (in greater proximity to people), and 2 months thereafter. Urine samples were collected for 1 year while females were in smaller yards to examine seasonal GC activity. Approximately, 2-fold increases (P < 0.05) were observed in fGCM levels after transport from pasture to yards with increased human exposure, followed by a rapid decline (within ∼17 days) to baseline (pasture) values. However, responses varied among onagers during the 30 days after translocation, with one female failing to acclimate. Mean fGCM concentrations in smaller yards 2 months after transport were comparable to those in pasture. Seasonal GC concentrations were lowest (P < 0.05) during winter, indicating modest seasonal variability. Results demonstrate an acute increase in GC secretion in Persian onagers that moved from large to small enclosures coincident with increased human activities. Most animals acclimated within 3 weeks, suggesting that this rare equid has retained mechanisms to acclimate to marked alterations in an ex situ environment.

COMPARISON OF BUTORPHANOL-DETOMIDINE VERSUS BUTORPHANOL-AZAPERONE FOR THE STANDING SEDATION OF CAPTIVE GREATER ONE-HORNED RHINOCEROSES (RHINOCEROS UNICORNIS)

Abstract:u2003 Three adult and two subadult greater one-horned rhinoceroses (Rhinoceros unicornis) were sedated a total of nine times using two different intramuscular sedative combinations in order to compare the effectiveness of these combinations in inducing consistent standing sedation in this species. The sedation protocols compared were butorphanol tartrate (50–60 mg) and detomidine hydrochloride (20–30 mg; BD) versus butorphanol tartrate (80–120 mg) and azaperone (80–120 mg; BA). Specific doses were adjusted according to age and sex class, and based on previous experience. Parameters compared included time to achieve defined levels of sedation, time to recovery following antagonism, physiological parameters including heart rate, respiratory rate, indirect arterial blood pressure, and venous blood gas values. A hydraulic restraint chute was utilized to mechanically restrain animals during the procedures, and blood collection and ophthalmic examinations were conducted on all animals. Both protocols resulted in standing sedation for ≥22.3 ± 2.9 min or until antagonists were administered. The BD protocol resulted in deeper and more consistent sedation, compared to the BA protocol. Naltrexone hydrochloride (250–300 mg) and tolazoline hydrochloride (1,500–2,000 mg) were administered intramuscularly to antagonize protocol BD, whereas naltrexone alone (200–500 mg) was used to antagonize BA. Time to full antagonism, defined as normal mentation and ambulation following administration of antagonists, was prolonged in the BD protocol (132.3 ± 17.2 min) compared with the BA protocol (7.5 ± 2.5 min). Venous blood gas analysis did not reveal any significant blood gas deviations during sedation when compared with either conscious equine or white rhinoceros (Ceratotherium simum) venous reference ranges. In summary, both combinations resulted in adequate standing sedation for minimally invasive procedures, although BD resulted in more profound and consistent sedation.

Oral and injectable synthetic progestagens effectively manipulate the estrous cycle in the Przewalski's horse (Equus ferus przewalskii)

To date, there has been limited research on manipulation of the estrous cycle in endangered equids. The objectives of this study were to assess the efficacy of using combinations of: (a) oral altrenogest and PGF2α, and (b) injectable altrenogest and PGF2α for manipulation of ovarian activity in Przewalskis mares. Reproductive cycles were monitored by assessing follicular changes with rectal ultrasound and changes in urinary steroid hormones. In Study 1, five cycling mares were treated with oral altrenogest (n=11 cycles) for 14 days. In Study 2, cycling mares were treated with oral altrenogest for 12 days (n=5 cycles; n=5 mares) or a single injection of biorelease altrenogest (n=10 cycles; n=6 mares). In all study groups, PGF2α was given 2 days before cessation of progestagen treatment. In Study 1, mares responded in six of 11 cycles (54%) where treatment occurred with normal ovarian follicular development post hormone therapy. In Study 2, mares responded in four of five (80%, oral altrenogest) and eight of 10 (80%, injectable altrenogest) cycles with the development of an ovulatory follicle. With the use of injectable altrenogest, there was an obvious suppression of urinary estrogens and progetsagens. These results indicate that manipulation of the estrous cycle of Przewalskis mares can be achieved by administering oral (12 days) or injectable form of altrenogest in conjunction with PGF2α. Findings in the present study may have long term application for the development of timed artificial insemination as a genetic management tool for this critically endangered equid.

Clinical Parelaphostrongylus tenuis infection in two captive American bison (Bison bison).

CASE DESCRIPTIONn2 juvenile (17 and 19 months of age) male American bison (Bison bison) were examined because of acute bilateral hind limb weakness and ataxia; 1 animal also had urinary incontinence.nnnTREATMENT AND OUTCOMEnGiven the clinical signs and rapid deterioration in the condition of these 2 animals, obtaining a definitive diagnosis was considered essential to minimizing the risk of disease in the remaining bison herd and among other animals at the facility. Therefore, both affected animals were euthanized. At necropsy, no gross abnormalities were seen. Histologic examination of sections of the brains from both animals revealed mild to moderate multifocal aggregates of eosinophils and mononuclear cells in perivascular regions of the meninges and gray matter of the cerebrum, cerebellum, and brainstem. Systematic examination of multiple sections of brain and spinal cord revealed evidence of nematode sections and aberrant parasite migration.nnnCLINICAL RELEVANCEnFindings suggested that CNS migration of Parelaphostrongylus tenuis in American bison may cause clinical signs. These findings have implications for the management of captive bison and free-ranging bison sharing ranges with white-tailed deer (Odocoileus virginianus), the definitive host, and elk (Cervus elaphus canadensis).