Introduction

Artificial insemination (AI) has been recognized as a valuable conservation tool for ex situ management of endangered species.¹¹ Techniques for AI in non-domestic antelope have met with modest success.^1,3,4,6 For species such as the fringe-eared oryx (Oryx gazella callotis), successful AI has the potential to enhance one of the most challenging aspects of captive non-domestic species propagation and management: the promotion of genetic diversity. Reliable AI techniques could allow for safe, controlled, and efficient reproduction without risk of disease transmission. Furthermore, a genetically diverse captive population could be maintained through the use of cryogenically preserved semen. Procedures developed for scimitar-horned oryx (Oryx dammah) after years of systematic study have been among the most successful in antelope species^6,7 and it has been proposed that these techniques may be effective across a broad range of related antelope species.

The purpose of this study was to determine if proven semen cryopreservation and AI protocols developed in the scimitar-horned oryx^7,8 could be applied to the closely related fringe-eared oryx with similar success. However, due to the fractious nature of the fringe-eared species, and the necessity of working animals through a hydraulic restraint device on a regular basis for treatment and sample collection, the use of a neuroleptic was introduced into the study design in an effort to alleviate stress.

Experimental Design

Semen was collected from two adult male fringe-eared oryx by electroejaculation and was cryopreserved using standardized protocols developed for scimitar-horned oryx.⁸ Nine adult female fringe-eared oryx housed at the Wilds were utilized as a single insemination treatment group. For sample collection and injections, individual animals were physically restrained in a hydraulic restraint chute (Tamer^®, Fauna Products, Red Hook, NY, USA) weekly throughout the study period (~50 days). To minimize stress, all individuals were administered perphenazine enanthate (Trilafon^®, Schering-Plough, Kenilworth, NJ, USA) (100 mg IM Q 7 days), a long-acting neuroleptic tranquilizer. Estrus synchronization consisted of two injections of prostaglandin F2α (PGF2α), in the form of cloprostenol sodium (Estrumate^®, Bayer Corp., Shawnee, KS, USA) (500 µg IM Q 11 days), as previously described for scimitar-horned oryx.⁷ Timed artificial insemination was performed approximately 48 hours after the second PGF2α injection. For insemination procedures, animals were immobilized by IM injection with a combination of carfentanil citrate (Wildlife Laboratories, Inc., Fort Collins, CO, USA) (0.0175–0.0185 mg/kg IM), xylazine HCl (Butler Co., Columbus, OH, USA) (0.065–0.07 mg/kg IM) and ketamine HCl (Ketaset^®, Fort Dodge Animal Health, Fort Dodge, IA, USA) (0.65–0.7 mg/kg IM) and were supplemented with ketamine IV as needed. Reproductive ultrasound examinations were conducted using a 7.5 MHz linear array transducer (Aloka 500). All animals were examined via ultrasonography and palpation of the uterus per rectum prior to insemination to determine ovarian structures present. Following post-thaw sperm assessment, both uterine horns were inseminated (due to the duplex uterine anatomy) with a 0.5 cc semen straw containing 5.5×10⁶ to 16.3×10⁶ motile sperm from the same male. Antagonism of the immobilizing agents was achieved with IV administration of naltrexone HCl (Trexonil™, Wildlife Pharmaceuticals, Fort Collins, CO, USA) and yohimbine HCl (Antagonil™, Wildlife Pharmaceuticals, Fort Collins, CO, USA). Blood samples were collected by jugular venipuncture on a weekly basis from the start of estrus synchronization to 6 weeks post-insemination. Progesterone and cortisol serum concentrations were measured by a colorimetric enzyme linked immunosorbent assay (ELISA) generated against a standard curve.

Significant Findings

Semen Collection and Storage

Qualitative and quantitative comparisons between scimitar-horned oryx and fringe-eared oryx semen were made. Semen volume, concentration, and sperm motility prior to cryopreservation were lower in the fringe-eared oryx donors compared to those of scimitar-horned oryx donors. However, the post-thaw motility average of 41% (n=18) and the measurements of acrosome integrity and forward progressive status of the fringe-eared oryx sperm closely approximated those for scimitar-horned oryx.^5,7 The result of these characteristics was a lower total number of motile sperm upon insemination in the fringe-eared oryx.

Based on these parameters, the protocol for collection and cryopreservation developed for scimitar-horned oryx was considered applicable to fringe-eared oryx.

Serum Progesterone and Cortisol Analysis

Two of the female oryx in this study were apparently acyclic, as demonstrated by static serum progesterone levels. The reason for this is undetermined. Cyclicity in the other females appeared normal and serum progesterone reached basal levels on the day of insemination, indicating successful synchronization. Compared to the scimitar-horned oryx, progesterone rises following estrus lagged by 1–2 days, possibly reflecting a delayed ovulation. At 50 days post-insemination, no animals were found to be pregnant as determined by declining progesterone levels, and no other signs of pregnancy were detected beyond this point. Serum cortisol levels are currently undergoing analysis.

1.  Pregnancy was not achieved in these study animals.

2.  The findings demonstrate effective estrus synchronization with PGF2α in fringe-eared oryx, when treated as previously described for scimitar-horned oryx.⁷

3.  Estrus synchronization was achieved while the animals were on a concurrent therapy with the neuroleptic drug perphenazine enanthate, although the neuroleptic treatment may have contributed to a delayed ovulation.

Morphology of the Reproductive Tract

Rectal palpation and ultrasound examination allowed detailed evaluation of the reproductive tract. Bifurcation of the uterine horns (duplex uterus) was typically noted at the level of the cervix, while one animal had two separate cervices that shared an external os.

These anatomical findings are similar to those described for scimitar-horned oryx.⁷

Variation in estrus-related morphologic characteristics were recorded as follows: fluid was noted in the uterus of five animals; uterine tone varied from flaccid to firm and contracted; development of the primary follicle ranged from 5–17 mm in size; four animals exhibited a corpus luteum on one ovary, varying from 5.5–12.5 mm in size. Ovarian structures were not evaluated by Morrow et al., 2000 in the scimitar-horned oryx study.

Despite apparent success in estrus synchronization based on serum progesterone measurements, there were inconsistencies detected in estrus-related uterine and ovarian characteristics among individuals.

Conclusion

No pregnancies were detected in this study; however, the possibility of early embryonic death cannot be ruled out. The reasons for failure of conception or pregnancy in the study are unknown. Potential factors include the following:

1.  Lack sperm penetration and capacitation. This possibility could be attributed to the lower number of motile sperm inserted during AI in the fringe-eared oryx compared to the number used in the scimitar-horned oryx study, although the degree of acrosome integrity and forward progressive status of the sperm were considered very good.

2.  Ovarian disturbance caused by transrectal ultrasound examination. It has been suggested that ovarian manipulation at the time of AI may result in ectopic ova through displacement of the fimbriae.

3.  Animal stress. Although the animals in this project underwent fewer hands-on procedures than did the scimitar-horned oryx in the reference study, the variable effects of stress in non-domestic hoofstock are still largely unknown and therefore cannot be ruled out as factors in these study results.

4.  Unknown side effects of perphenazine administration. Little information is available on reproductive side effects of perphenazine and related compounds.

Report of previous trials on concurrent estrus synchronization and neuroleptic drug treatment could not be found after an exhaustive literature search. Studies with various species have demonstrated such effects as reproductive hormone inhibition, delayed ovulation, estrus inhibition,^2,10 and delayed nidation¹⁰. Given these previous findings, it is possible that perphenazine accounted for the apparently delayed ovulation in this study. Further investigation into the each of these factors as potential causes of failure to develop pregnancy during assisted reproduction is warranted. Despite the lack of reproductive success in this study, key aspects of assisted reproduction in fringe-eared oryx were elucidated and these results should facilitate the advancement of future studies.

Literature Cited

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2.  Boothe, N.H. 1990. Psychotropic agents. In: Veterinary Pharmacology and Therapeutics, 6th ed. Iowa State Press, Ames, Iowa. 363–395.

3.  Densmore, M.A., M.J. Bowen, S.J. Magyar, M.S. Amoss, R.M. Robinson, P.G. Harms, D.C. Kraemer. 1987. Artificial insemination with frozen, thawed semen and pregnancy diagnosis in addax (Addax nasomaculatus). Zoo Biol. 6:21–29.

4.  Holt, W.V., H.M. Moore, R.D. North, T.D. Hartman, J.K. Hodges. 1988. Hormonal and behavioral detection of estrus in blackbuck, Antilope cervicapra, and successful artificial insemination with fresh and frozen semen. J Reprod Fertil. 82:717–725.

5.  Kouba, A.J., A.D. Rowson, M.W. Atkinson, A.R. Gandolf, T.L. Roth. 2001. Species-specific sperm-egg interaction affects the utility of a heterologous bovine in vitro fertilization system for evaluation antelope sperm. Biol Reprod. 65:1246–1251.

6.  Monfort, S.L., B.A. Wolfe, E.S Blumer, M.W. Atkinson, R.E. Spindler, B.S. Pukazhenthid, M. Bush. D.E. Wildt, T.L. Roth, C.J. Morrow. 1999. Artificial insemination in the scimitar-horned oryx as a conservation management tool. In: Proc AAZV. 77–82.

7.  Morrow, C.J., B.A. Wolfe, T.L. Roth, D.E. Wildt, M. Bush, E.S Blumer, M.W. Atkinson, S.L. Monfort. 2000. Comparing ovulation synchronization protocols for artificial insemination in the scimitar-horned oryx (Oryx dammah). An Repro Sci. 59:71–86.

8.  Roth, T.L., R.B. Weiss, J.L. Buff, D.E. Wildt, M. Bush. 1998. Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah). Biol Reprod. 58:475–482.

9.  Roth, T.L., L.M. Bush, D.E. Wildt, R.B. Weiss. 1999. Scimitar-horned oryx (Oryx dammah) spermatozoa are functionally competent in a heterologous bovine in vitro fertilization system after cryopreservation on dry ice, in a dry shipper, or over liquid nitrogen vapor. Biol Reprod. 60:493–498.

10.  Shani, J., M. Anit, Y. Givant. 1979. Effect of the timing of perphenazine administration on pregnancy in the rat. J Endrocinol. 80:409–411.

11.  Wildt, D.E. 1992. Genetic resource banking for conserving wildlife species: justification, examples and becoming organized on a global basis. Anim Reprod Sci. 28:247–257.