Artificial Insemination in the Scimitar-Horned Oryx as a Conservation Management Tool
Despite the implementation of effective small population management strategies, insufficient funding and enclosure space can preclude breeding genetically valuable individuals, and this can adversely impact gene diversity. For species like the scimitar-horned oryx (Oryx dammah), which thrives in large numbers in captivity, artificial insemination (AI) has potential for eliminating the risks of animal transport, and for optimizing the use of limited enclosure space, while simultaneously preserving extant gene diversity. Maximizing the full potential of AI, however, requires both effective sperm cryopreservation techniques and the ability to reliably produce offspring using frozen-thawed sperm. Substantial progress has been made in the scimitar-horned oryx towards understanding the female’s reproductive cycle, manipulating estrus/ovulation, developing safe and reliable approaches for collecting and storing viable spermatozoa, and optimizing methods for proper deposition of sperm at the appropriate time and site within the reproductive tract. Recent results15 demonstrate that non-surgical AI in scimitar-horned oryx using frozen-thawed sperm results in pregnancy rates approaching 40% after a single fixed-time insemination. Summary data from 72 inseminations across three separate AI trials conducted at the Conservation & Research Center and the Wilds are presented here to illustrate how AI, combined with genome resource banking, can augment captive breeding management of the scimitar-horned oryx.
Less than a century ago, hundreds of thousands of scimitar-horned oryx inhabited the Sahel, a semi-arid transition zone south of the Sahara from Senegal to Sudan, and the northern edge of the Sahara from Morocco to Egypt. Although well adapted to harsh, arid environments, poaching, habitat destruction, recurrent drought and civil unrest led to the extinction of scimitar-horned oryx across their historic range.
The managed global population of captive scimitar-horned oryx exceeds 1,500 individuals, including small populations that have been re-introduced into fenced habitats in Tunisia and Morocco. Amazingly, scimitar-horned oryx now occupy >7% of the 16,000 spaces set aside for hoofstock species within zoological institutions worldwide. Metapopulation management is problematic because all captive oryx (descended from 40–50 wild founders) exist in fragmented populations widely dispersed at dozens of locations worldwide. This scenario has mandated that global ex situ management tactics focus both upon moderately reducing captive numbers of scimitar-horned oryx while still maintaining adequate genetic diversity to avoid inbreeding depression.
Compared to most exotic ungulates, the reproductive database for scimitar-horned oryx is extensive. Previous studies have investigated the ovarian cycle10,13,14 and ovulation induction2,4,7,13-16,20,21, as well as semen collection and cryopreservation5,6,7,11,17-19. And while methods for synchronizing estrus and ovulation have been reported for the scimitar-horned oryx2,7,12,13,15,16,20,21, there are few published reports describing fertility after estrus synchronization7,15. Numerous factors affect AI success rates including sire selection (which impacts pre-freeze semen quality), efficacy of estrus synchronization, semen freezing methods (which impacts post-thaw sperm quality), sperm concentration and total number of sperm inseminated, and timing of insemination. Since our intention was to integrate our research with a semen cryobanking program and strictly managed breedings, there was little that could be done to affect sire selection. Of the remaining factors, the total number of motile sperm inseminated could be regulated if post-thaw sperm motility and concentration were known. Thus, our AI studies were designed to investigate (1) two ovulation synchronization protocols, (2) chilled vs. frozen-thawed sperm, and (3) timing of AI relative to synchronization of ovulation.
The present paper reviews results of three AI trials (28, 20, and 24 animals, respectively) conducted consecutively over 3 yr (1996–1999). Study animals for each trial were divided equally between the Conservation & Research Center (CRC, Front Royal, VA) and the Wilds (Cumberland, OH). Strategies for future development of semen banking to augment captive genetic management and re-introduction of scimitar-horned oryx also are discussed.
Trial 1: Comparing Ovulation Synchronization Protocols for AI15
Fourteen females per group (n=28 total) were inseminated with frozen-thawed sperm after receiving either two i.m. injections of prostaglandin-F2α (PGF2α-only, 500 µg) or the same treatment combined with a modified progesterone-containing intravaginal CIDR-B device (11 day insertion interval; CIDR+PGF2α) to synchronize ovulation. Females were transcervically inseminated 56.0±1.1 hr (x±SEM) after CIDR withdrawal and/or the second PGF2α injection using 27.9±1.6x106 motile, thawed sperm, divided equally between both uterine horns. Semen for all AI trials was diluted in EQ extender (20% egg yolk, 5.5% lactose, 1.5 % glucose and 0.25% triethanolamine lauryl sulfate; 5% final glycerol concentration) and frozen in 0.5 ml straws directly on dry ice for 10 min before plunging and storage in liquid nitrogen.17 Post-thaw sperm motility averaged 46.4±1.6% (forward progressive motility, 3.0±0.1, scale 0–5), and there were no differences (p>0.05) between pregnant (23.7±1.2x106) and non-pregnant (29.1±1.8x106) females in the number of motile sperm inseminated.
AI was conducted non-surgically in females that were anesthetized using a combination of medetomidine (0.03–0.09 mg/kg) and ketamine (2.5–3.0 mg/kg); atipamezole (0.15–0.20 mg/kg) was used as antagonist. A detailed description of the efficacy to this anesthetic combination will be published in a separate manuscript. At both the CRC and the Wilds, animals were anesthetized in, or near their home pens, transferred to a nearby location for AI, and then returned to their home pen to recover (see exception described in trial 2). To ensure consistency between locations, animals were matched for parity and age among treatment groups, similar animal handling and restraint facilities were used, identical research and anesthesia protocols were followed, the same semen donors were used, and the same technical staff conducted veterinary and insemination procedures.
The increase in fecal progestogen excretion (indicative of corpus luteum [CL] development) was delayed (p<0.05) in 5/10 of the CIDR+PGF2α females (16.8±2.5 days) compared to the remaining CIDR+PGF2α individuals (7.6±0.7 days) and all PGF2α-only (8.6±0.8 days) females. The induced luteal cycle lengths (nadir to nadir fecal p) for non-conceptive PGF2α-only females was 25.3±0.3 days, whereas CIDR+PGF2α females exhibited either normal (n=5, 26.0±0.7 days) or delayed (p<0.05) luteal cycles (n=5; 35.5±3.8 days). The lack of between treatment differences (p>0.05) in fecal estrogen excretion after CIDR withdrawal/PGF2α administration suggested that follicle development was not adversely affected by short-term progesterone treatment. However, the 9–23 day delay in the onset of the post-synchronization luteal phase (i.e., the approximate time required for a new ovulatory follicle to be recruited, ovulated, and luteinized) in several of the CIDR+PGF2α females suggested that estrogenic follicles either failed to ovulate, or that ovulated follicles failed to fully luteinize. In summary, more pregnancies (p<0.05) resulted in PGF2α-only treated females (35.7%; 5/14 diagnosed pregnant; 4 live births) compared to CIDR+PGF2α counterparts (0/14).
Trial 2: Chilled vs. Frozen-Thawed Sperm for Producing Offspring After Transcervical AI
We hypothesized that chilled (vs. frozen-thawed) scimitar-horned oryx semen would provide superior conception rates since results from a preliminary trial showed that sperm longevity was prolonged compared to thawed sperm (i.e., fresh sperm diluted in EQ without glycerol at 4°C was motile for at least 88 hr). Thus, a second AI trial was designed to compare the efficacy of freshly collected, chilled semen to frozen-thawed sperm for producing offspring after fixed-time intrauterine AI. Nineteen females (one CRC female died during anesthetic induction before AI was conducted) synchronized with PGF2α-only were transcervically inseminated 54.2±0.3 hr after the second PGF2α injection using ∼40x106 motile, thawed (n=12) or chilled sperm (n=12) into each uterine horn. One ejaculate from a single male at each location was used for all inseminations. Half the pooled semen batch (chilled slowly to 4°C) was frozen,17 and the remainder was stored (4°C, <24 hr) until used for inseminations. Pre-insemination motility of chilled sperm was >70%, and progressive status was >3.0. Acrosome integrity of chilled sperm (82.3±4.9% intact acrosomes) was superior (p<0.05) to frozen-thawed sperm (69.3±2.1% intact acrosomes). Semen extenders, semen freezing methods, anesthesia protocols and AI techniques were identical to trial 1, but endocrine monitoring was not conducted.
Despite using a proven ovulation synchronization protocol and inseminating with greater numbers (trial 2, 40x106/horn vs. trial 1, 14x106/horn) of high-quality sperm (trial 2, ∼70% motility @ 3.0 status vs. trial 1, ∼45% @ 3.0 status), only a single female became pregnant (the Wilds, frozen- thawed sperm).
There were three notable protocol differences in trial 2 vs. trial 1. First, the mean time to AI after the second PGF2α injection was shortened by 2 hr (trial 2, 54.2±0.3 hr vs. trial 1, 56.1±1.1 hr; p<0.05); this unplanned result derived from the increased speed and experience of the I team. Second, CRC females were taken by trailer to the veterinary hospital 2–3 days before AI procedures to ensure proximity to medical instrumentation and to achieve consistent ambient temperatures during the AI procedures (research protocols at the Wilds were unchanged from trial 1). Third, half the research subjects were inseminated with chilled semen.
Previous research12 showed that onset (range, 29–44 hr) and duration (range, 3–41 hr) of behavioral estrus (in the presence of a vasectomized bull) in scimitar-horned oryx was highly variable following PGF2α administration, both among females, and between repeated experiments. Despite such high variability, pregnancy rates following AI at 54 hr (after a second PGF2α injection) were excellent in trial 1.15 We speculated that the stress associated with moving animals to a new location before AI may have blocked or delayed ovulation in females during trial 2.22 The mismatch between ovulation and insemination may have been further exacerbated by the advancement in the mean time to AI. Although fecal corticoid metabolites were not documented in the present study, recent results (C. Morrow, unpublished data) suggested that scimitar-horned oryx taken by trailer to the veterinary hospital for medical treatments excreted increased fecal corticoids over a prolonged interval.
Although subjective measures of sperm motility and progressive status, and objective measures of acrosomal integrity, suggested that chilling and storage for up to 24 hr had little impact on sperm viability, we could not rule out the possibility that prolonged storage at 4°C inhibited sperm capacitation.
Trial 3: Impact of AI Timing on Fertility
Trial 3 was designed to examine the impact of the timing of AI relative to ovulation synchronization on fertility. We tested the hypothesis that no fertility differences existed among scimitar-horned oryx cows inseminated 56, 64 or 72 hr after the second PGF2α injection. Twenty-four PGF2α-only synchronized females were transcervically inseminated 56±0.3, 64.3±0.3 or 72.0±0.3 hr after the second PGF2α injection with ∼50x106 motile, thawed sperm into each uterine horn. Post-thaw sperm motility ranged from 70–80%, and progressive status ranged from 3.0–3.5. Semen extenders, semen freezing methods, anesthesia protocols and AI techniques were identical to trials 1 and 2.
Nine pregnancies—three in each treatment group—were achieved in trial 3. Although numbers of study animals were relatively small, the null hypothesis that no fertility differences existed across an 18-hr interval (56–72 hr) after the second PGF2α injection was accepted. Six of 12 animals (50%) became pregnant at CRC, whereas 3/12 (25%) conceived at the Wilds. While treatment groups were balanced within location for age (p>0.05) and parity, females at the Wilds (10.4±0.5 yr) were older (p<0.05) than CRC females (8.2±0.7 yr). There were no among-treatment differences in pregnancy outcome, but increased cervical dilation and reduced cervical tone in the 56- and 64-hr treatment groups facilitated passage of insemination pipettes for intrauterine insemination.
Births resulting from AI have been reported in only four antelope species, including one Speke’s gazelle1 (Gazelli spekei), one addax3 (Addax nasomaculatus), six blackbuck9 (Antilope cervicapra) and six scimitar-horned oryx7,15. Despite these successes, semen cryopreservation and AI still has not been used for managing captive antelope populations.8,23 Inclusive of the three AI trials described in the present paper, we have produced 15 pregnancies in scimitar-horned oryx, and have achieved conception rates with frozen-thawed semen that range from 25–50% after a single insemination. These results provide strong incentive for the establishment of a genome resource bank for oryx semen.
While it was not surprising that environmental and/or management factors may have influenced pregnancy outcome, our results demonstrated that these AI techniques can be readily adapted to accommodate the diversity of management and husbandry schemes likely to be encountered within zoological institutions worldwide. This adaptability will be particularly important when regional sub-Saharan African re-introduction programs for scimitar-horned oryx are initiated. It is clear that integrated genetic management efforts will continue to be vital for protecting and managing animal populations within native habitats, as well as for captive breeding programs. We are confident that semen banking and routine offspring production after AI will become a useful tool for bridging ex situ and in situ conservation management programs for scimitar-horned oryx.
Special thanks go to the animal management, veterinary support and keeper staff at the Conservation & Research Center and the Wilds. Research was funded by grants from the Scholarly Studies Program of the Smithsonian Institution, the Friends of the National Zoo and the Morris Animal Foundation.
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