Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 1. Effect of epithelial oviductal monolayers and heparin on in vitro sperm motility and penetration of in vitro matured oocytes.
In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P<0.001). Penetration rates were similar for 4-12h pre-incubation of sperm on COEM (P>0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.
Berg
D K
DK
Reproductive Technologies Group, AgResearch Ruakura, Private Bag 3123, Hamilton, New Zealand. debra.berg@agresearch.co.nz
Thompson
J G
JG
Asher
G W
GW
D000818:Animals; D017077:Culture Media, Conditioned; D046508:Culture Techniques; D003670:Deer / Q000196:embryology*; D005314:Embryonic and Fetal Development; D004848:Epithelium; D005187:Fallopian Tubes; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006493:Heparin / Q000494:pharmacology*; D008297:Male; D009865:Oocytes / Q000502:physiology; D013075:Sperm Capacitation / Q000187:drug effects; D013081:Sperm Motility / Q000187:drug effects*; D013084:Sperm-Ovum Interactions / Q000187:drug effects*
D017077:Culture Media, Conditioned; D006493:Heparin