
TY  - JOUR
A1  - Penfold, L. M. 
T1  - 87 OSMOTIC SENSITIVITY OF OKAPI SPERMATOZOA AND DEVELOPMENT OF CRYOPRESERVATION PROTOCOLS USING CRYOMICROSCOPY
JF  - Reproduction, Fertility and Development
JO  - Reprod. Fertil. Dev.
Y1  - 2007
VL  - 20
IS  - 1
SP  - 124
EP  - 124
KW  - 
UR  - https://doi.org/10.1071/RDV20N1AB87 
N2  - Okapi would benefit from artificial insemination with frozen-thawed sperm in cases where aggression prevents mating or where individuals are geographically disparate. Effective sperm cryopreservation is a prerequisite to this goal. Ejaculates (n = 20) were collected from 7 anesthetized adult male okapi housed individually, or with a female for breeding, throughout the year by electroejaculation, and semen and sperm parameters were assessed. Semen aliquots were centrifuged; resuspended in 500 &#181;L of PBS with the osmolarity adjusted to 35, 75, 150, 600, 1200, and 2400 mOsm; and incubated for 30 min before returning to isosmotic conditions. Semen was extended in TEST containing 1%, 2%, or 4% glycerol with or without 0.5% Equex (Minitube, Verona, WI, USA); 5-&#181;L aliquots were cooled in a Latin square design on a Linkam BCS 196 cryomicrostage (Linkam Scientific, Tadworth, Surrey, UK) at 20°C min&#8211;1 to &#8211;6° &#8211;12°C at which point ice crystal formation was induced (seeded), and cooled further to &#8211;70°C before warming at 50°C min&#8211;1 to 35°C (okapi body temperature). To investigate cooling effects only, raw ejaculate was cooled to &#8211;6°C without seeding and warmed to 35°C. Percent sperm motility and plasma membrane integrity (PMI) were recorded before and after treatments. Differences were examined using one-way repeated measures analysis of variance. No differences in motility, total sperm numbers, or percent normal morphology were observed throughout the year (P  > 0.05), although the power of the test was low so that negative findings should be interpreted cautiously. Mean semen volume was 1.3 ± 0.19 mL, sperm motility was 29 ± 3.2%, with a PMI of 39 ± 6.8%; 48 ± 2.8% were morphologically normal. High proportions of non-motile, plasma membrane-damaged cells were noted in every ejaculate, and whiplash motility, possibly indicating spontaneous capacitation, was observed in several ejaculates 1 h after collection. Motility was dramatically reduced on either side of isosmotic conditions and was more sensitive to osmotic pressure than was plasma membrane integrity. Cooling of raw ejaculate to sub-zero temperatures without freezing did not result in any loss of motility or PMI, indicating cold tolerence. Superior results were obtained when sperm were frozen-thawed in TEST containing 4% glycerol with 0.5% Equex. Findings indicate that okapi semen collected by electroejaculation routinely contain high numbers of non-motile and plasma membrane-damaged spermatozoa, apparently unrelated to season or the length of time since the male was housed with a breeding female. Okapi spermatozoa are remarkably intolerant of departures from isosmotic conditions, indicating a lack of ability to regulate or withstand volume excursions during osmotic stress events; however, cooling to sub-zero temperatures in the absence of cryoprotectant did not reduce percent sperm motility or PMI, indicating resistance to cold shock. Increasing and maintaining proportions of motile, membrane-intact spermatozoa prior to and during cryopreservation will be critical for development of freezing protocols for this species. 
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