Study On Wapiti (Cervus Elaphus) Sperm Capacitation In Vitro And Its Ultrastructural Variation

Sperm capacitation is an essential mature process before mammalian fertilization. Making sperm capacitated in vitro is an important prerequisite to complete fertilization in vitro. Wapiti (Cervus elaphus) sperm capacitation in vitro and its ultrastructural change were studied in order to choose a set of perfect and feasible protocol of wapiti sperm capacitation in vitro, and to provide a basis data on the research of wapiti capatitation in vitro and the mechanism of fertilization.To make the wapiti sperm capacitated in vitro, we selected and used the method of sperm-up. By making the rate of acrosome reaction as a main index, and the sperm energy, the survial time and the fertilizated rate of wapiti sperm and hamster mature oocytes as auxiliary indexes, it were carried out to compare the effects of wapiti sperm capacitation in vitro under different temperature, different density of pyruvate sodium, caffeine and heparin and different culture medium. These results showed that:1. It can succeed in making wapiti sperm capacitation in vitro by use of the method sperm-up. The suitable proportion of sperm to sperm-washing medium was 1:3~5, and the suitable rate and time of centrifugal washing were 2200rpm × 10min and 2000rpm × 5min.2. By use of the modified method of triple-stain technique could evaluate the level of wapiti acrosome reaction effectively.3. Cultivated in vitro with 5% carbon dioxide and saturated humidity, wapiti sperm had a greatly significant higher acrosome reaction rate under 38.5℃ than under 37℃ (P<0.01).4. When the intensity of pyruvate sodium in BO medium was 0.5mM, the rate of wapiti sperm acrosome reaction in vitro was significant higher than 1.25mM(P<0.05).5. Cultivated with TALP basic medium, the rate of wapiti sperm acrosome reaction in vitro was significant higher than the two medium BO ( Ⅰ ) and BO (Ⅲ) (P<0.05); but these two BO medium had an insignificant difference(P>0.05).6. Using TALP with 6mg/ml BSA and 10μg/ml heparin, the wapiti sperm acrosomereaction rate in vitro with 5Mm caffeine was obviously higher than with OmM >. 2.5mM and lOmM caffeine (P<0.05); Increasing the density of heparin to 2Qag/ml and 3Qug/ml, the effects of wapiti sperm acrosome reaction rate in vitro hadn't had a significant difference under 2.5mM and 5mM caffeine (P>0.05).7. Using TALP with 6mg/ml BSA and 2.5mM caffeine, the wapiti sperm acrosome reaction rate in vitro was obviously higher with 3Qug/ml heparin than with lQug/ml and 20/ig/ml heparin (P<0.05); when the density of caffeine increased to 5mM, the wapiti sperm acrosome reaction rate was significant higher with lQMg/ml heparin than with 3Qwg/ml (P<0.05).8. mKRB and HIS could all made wapiti sperm capacitated in vitro, but the rate of acrosome reaction were very lower, comparing with the other experiments.9. In the all 15 experiments we found that the suitable program of wapiti sperm capacitation in vitro were experiment 2, 9,10,11.All kinds of the wapiti sperm samples during the capacitation in vitro had been dealt with a series of procedure to get into slices, which need to be dyed, the sperm ultrastructure were observed and the results showed that:1. Wapiti sperm was consisted of three parts the head(58.75 ± 2. 35^m long), the neck(8.93 ±0. 24/jm long) and the tail(48.18+l. 18/jm long). The shape of the head was like a stick and most part of it was occupied by concentrated sperm nucleus with high-electronic density. The acrosome which was almost 2/3 of the head and had an expanded fore part was like a cap upon the sperm nucleus. Between the head and the tail there was the neck, and the length of the part was not long. The tail consisted of the middle, the principal and the end pieces.2. The axoneme structural type of wapiti sperm tail was "9+2"; the microtubule structural type of the end piece of the tail was "9+9+2".3. Before wapiti capacitation the plasma membrane and the acrosome of the sperm head were intact. After capacitation, partial vesiculation of some acrosome plasma membrane occurred, and some even broke and total lost. Some of the acrosome swelled and the outer membrane brgan to sink and formrd vesiculation. There was no difference of the ultrastructure among all the cultivated mediums.