Abstract

A split-ejaculate comparison was made between a traditional method of cryopreservation using a standard bovine freezing curve (IMV Technologies) and a novel method of cryopreservation using a directional cooling technique.¹ The objective was to test the hypothesis that directional cooling controls the rate of ice crystal propagation and ice crystal morphology more efficiently, thus reducing mechanical and osmotic stress exerted on spermatozoa by random seeding and slow cooling. Additionally, the effects of centrifugation and long versus short equilibration of spermatozoa at 4ºC on post-thaw sperm viability was evaluated. In conclusion, neither of the treatments applied produced acceptable post-thaw sperm viability; therefore, successful freezing of Bennett’s wallaby semen remains an enigma. Follow-up work on ultrastructure of both fresh and frozen sperm membranes is being undertaken.

Introduction

Macropod sperm viability has historically been extremely poor post-freeze-thaw using conventional methods of cryopreservation.^3,7 Increasing sperm viability over time will help maximise success of future artificial breeding in macropod species that are not widely held and/or are endangered. Ways of increasing viability while minimising the amounts of toxic cryoprotectants used to protect the sperm from rupture due to the freezing process need to be investigated. A directional freeze technique has recently been perfected by harmony cryocare (www.harmonycryocare.com [VIN editor: link was not accessible as of 2-5-2021.]) with apparent improvements in sperm viability in bulls and stallions. We aim to test this technique against standard cryopreservation practices in the Bennett’s Wallaby.

Methods

Epididymides were collected from seven Bennett’s Wallabies. The samples were processed based on previously published methods for marsupials.^2-4 HEPES-buffered MEM (minimum essential medium) was warmed in a water bath to 35°C. Each epididymis was separated from the testicle and placed in the MEM. The head of each epididymis was removed. Surface connective tissue, including blood vessels, was gently peeled back from the head using dissection scissors and forceps, while the epididymis was held in an MEM soaked gauze swab. The head was then removed from the rest of the epididymis and placed into 100 mm diameter Petri dishes, lined with dental wax, and holding 40 ml MEM. Each head was then scored in a cross hatch pattern with a scalpel blade to free the spermatozoa and dishes placed in an incubator set to 35°C to allow swim-up for 45 minutes. The samples were then split, half being centrifuged, and the resultant pellet re-extended with TRIS-citrate buffer to be chilled at 4°C for two hours, the other half not centrifuged and extended in a 1:1 mix of MEM:Tris buffer. Half the non-centrifuged samples were held at 4°C for two hours, as per the centrifuged samples. The other half were cooled to 4°C and frozen immediately. Glycerol (at concentrations of 0%, 5%, 15%, and 20% ) was added with the buffer at 4°C. Samples were frozen routinely for each freezing process and thawed and analysed two weeks post-freeze.

Results and Discussion

Our results thus far are preliminary, but we have found that centrifugation of wallaby sperm prefreezing (a common method of concentrating in domestic livestock) dramatically reduces sperm motility (from 75% to 5%). Six percent of sperm were alive after centrifugation, compared to 78% (5% glycerol) and 40% (20% glycerol) in samples that weren’t centrifuged.

Conversely, a short equilibration of sperm at 4°C of 10–15 minutes, did not appear to affect motility when compared to keeping them chilled at 4°C for over one hour. There was minimal motility of all samples post-thaw, and close to 100% membrane rupture was confirmed using 1 mg/ml propidium iodine and 5 mg/kg Hoechst 33342 (bisbenzimide stain).

The spermatotoxic effects of glycerol have been well-documented; however, some cryopreserver is required to protect the sperm membranes from cryoinjury. Results from Cooper, et al. (1995) indicate that the effect of 2% glycerol on the percentage of sperm showing forward motility is minimal when compared to motility without glycerol.² However, viability of sperm post-freeze with glycerol any lower than 15% has been unsuccessful (Holt, personal communication). Prefreeze results from the present experiment indicate that a glycerol percentage of 2.5% to 5% yielded best prefreeze motility.

Freezing and recovery of electro ejaculated sperm in Tammar wallabies was conducted by Molina and Roger (1996).⁷ The spermatozoa were screened for toxicity in diluents containing a range of cryo-protectants. The most promising was 7.5% glycerol + 10% DMSO. Pellets were thawed to 35°C and spermatozoa were washed by centrifugation (200 g for five minutes) and resuspended in diluent to minimize cryoprotectant toxicity. Progressive motility was high (three on a five scale) but only 10% of motile sperm were recovered (the best post-thaw motility found for any macropod species). This was considered by the researchers as possibly adequate for in vitro fertilization and AI. This has yet to be tested.

Diluent selection is critical for successful preservation of sperm in any species. Johnston, et al. (2000) found that TRIS-citrate buffer was superior to PBS as a preservation diluent at all temperatures for koala spermatozoa.⁶ This indicates that methods developed for the preservation of eutherian spermatozoa (in this case, the widespread use of PBS) may not necessarily be suitable for marsupial semen. The present experiment indicates that TRIS-Citrate and MEM combination maintains a high motility percentage in wallaby sperm.

Antibiotic use in extended semen, to control the growth of bacterial contaminants is standard practice for domestic species but was not included in the current experiment. When koala spermatozoa were extended in diluents containing 4000 IU/ml penicillin and 400 µg/ml of gentamicin, the rate of sperm motility declined significantly after four hours storage.⁵ Also, in the current experiment, the sperm were harvested directly from the epididymis, thus eliminating contamination of preputial sperm, but contamination while in storage is a potential issue.

Marsupial spermatozoa can survive for about one week at 4°C if left in the reproductive tract.¹⁰ Koala spermatozoa remained motile even after 42 days storage at 5°C in TRIS-citrate buffer diluent.⁶ This has implications for artificial insemination in macropod species not widely held (for example the tree kangaroos, allowing selective outbreeding of an inbred population), and needs to be investigated further. Processing for non-frozen sperm from recently deceased individuals can be conducted as outlined in the present experiment.

The largest issue for post-freeze viability in marsupials appears to be damage to lipid membranes sustained on thawing. Electron microscopy work is currently being undertaken to examine the ultrastructure of fresh sperm and those undergoing the freezing process to investigate specific freezing injury. Until this issue can be resolved, successful cryopreservation of macropod sperm will remain problematic.

Acknowledgments

Thanks to staff and Management of Whipsnade Wild Animal Park for their assistance in conducting this experiment, and Professor Michael McGowan for technical assistance.

Literature Cited

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