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a Conservation & Research Center, National Zoological Park, Smithsonian Institution, Front Royal, Virginia 22630
Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25°C (no cooling; control), 2) cooled to 5°C in a commercial refrigerator for 30 min (rapid cooling; ~4°C/min), 3) placed in an ice slush at 0°C for 10 min (ultrarapid cooling; ~14°C/min), or 4) cooled to 0°C at 0.5°C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4°C. All samples then were warmed to 25°C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25°C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling (~4°C/min) to 5°C (N, 65.6%; T, 27.5%) or ultrarapid cooling (~14°C/min) to 0°C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5°C/min) to 5°C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0°C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.
2 Correspondence: Budhan Pukazhenthi, Reproductive Physiology Program, National Zoological Park and Conservation & Research Center, Smithsonian Institution, 3001 Connecticut Ave. NW, Washington, DC 20008. FAX: 202 673 4733; bpukazhenthi{at}nzp.si.edu
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