Effects of cooling, freezing and glycerol on penetration of oocytes by spermatozoa in dogs.
A homologous zona penetration assay was used to evaluate dog spermatozoa after selected treatments and associated gamete interaction with sperm characteristics. Canine semen, diluted in egg-yolk extender, was treated to compare samples quickly cooled to 0 degree C (after 0.5 and 3 h), slowly cooled to 0 degree C after 3 h or held for 3 h at room temperature. In a second study, slowly cooled spermatozoa with or without the addition of 4% glycerol were studied. After each treatment, spermatozoa were incubated with canine oocytes for 18 h, stained with Hoechst and examined for bound sperm heads. After 3 h, cells that had been cooled quickly demonstrated lower numbers of spermatozoa per ovum than fresh or slowly cooled treatments (2.4 +/- 0.6 versus 5.2 +/- 1.1 and 3.6 +/- 0.9 sperm per oocyte, respectively) and coincided with a reduction in acrosomal integrity and motility (58.6% fast cool 3 h versus 83.1% fresh). Addition of glycerol effected no change in acrosomal status or motility, but resulted in a decline in spermatozoa bound per oocyte to 1.5 +/- 0.3. While freezing caused a further decline in motility, acrosomal status and oocyte penetration, there was no difference between freezing with and without seeding (0.3 +/- 0.08 versus 0.2 +/- 0.04 spermatozoa per ovum). Evaluation of sperm penetration of homologous oocytes provides new insights into the effects of cooling, seeding and glycerol on canine spermatozoa.
Hay
M A
MA
Metro Toronto Zoo, Scarborough, Ontario, Canada.
King
W A
WA
Gartley
C J
CJ
Leibo
S P
SP
Goodrowe
K L
KL
D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004285:Dogs; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005990:Glycerol / Q000494:pharmacology*; D008297:Male; D009865:Oocytes / Q000502:physiology*; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*
D003451:Cryoprotective Agents; D005990:Glycerol