Wapiti (Cervus Elaphus) Sperm Capacitation And Evaluation System And Its Fertilization In Vitro

Sperm capacitation is an essential physiological procedure in normal fertilization. Studies on sperm capacitation and related issues are certainly necessary and important in reproductive biology field. Wapiti (Cervus elaphus) was as a test object in this study. The effect of some kinds of factors on sperm in vitro capacitation and acrosome reaction were analyzed, and it was thereby selected that practical operational programme of wapiti sperm in vitro capacitation culture and exactly quickly evaluating wapiti sperm capacitation level. On the base of above, it was studied that wapiti in vitro fertilization and physiological and morphological changes during wapiti sperm in vitro capacitation and acrosome reaction. The purpose of this study was to offer new thinkings to deeply research on deer in vitro fertilization and to provide data and material for basical theorical research on reproductive biology and related subjects.The main results obtained from studies on wapiti sperm in vitro capacitation culture and evaluation are as follows:1. Temperature made significant effect on wapiti sperm in vitro capacitation. Temperature 37℃and 37.5℃lower to wapiti average body temperature were unfavorable to wapiti in vitro capacitation (P＜0.05). The suitable temperature range was 38～39.5℃, and the best one was 38.5℃.2. The three kinds of methods swim-up, incubation and Percoll, could all be used in wapiti sperm in vitro capacitation. But swim-up was best if making a general survey of AR rate, motility and curvicaudate rate.3. Fresh semen and frozen-thawed semen could all be induced to capacitate in vitro. The rates of SPA, AR, motility and curvicaudate of frozen-thawed semen were all lower than the fresh, but the difference was not significant (p＞0.05).4. Ca²⁺, Mg²⁺, K⁺, glucose and Na-pyruvate in the culture medium could all affect wapiti in vitro capacitation. Using modified BO could improve wapiti sperm capacitation level (p＜0.05), but using HIS couldn't (p＜0.01).5. Different kinds and concentration of serum and BSA could all affect on wapiti sperm capacitation. The results were better that using 10%stag serum, 15%FBS, 6mg/mlBSA or 9mg/mlBSA, and the AR rates were all beyond 60%. Using too high concentration of BSA or FBS in the culture received worse results on the contrary.6. Concentration and affecting time of heparin directly influenced wapiti sperm in vitro capaeitation level. Among the heparin concentration range of 0～200μg/ml, the suitable range to wapiti was 20～30μtg/ml, and the best one was 20μg/ml. Using 20μg/ml heparin inducing wapiti sperm capacitation in vitro, affecting time 15min was best (p＜0.05) of 0～120min, and extending time hadn't improve capacitation level.7. Progesterone (P₄),γ-aminobutyricacid (GABA), calcium ionophore A23187 (A23187) and valinomycin(Val) could all induce wapiti capacitated sperm acrosome react in vitro. The most suitable concentration and affecting time of P₄, GABA, A23187 and Val were 10μmol/L×10min, 0.5mmol/L×10min, 0. lμmol/L×5min and 0.5μmol/L×5min separately. The AR rates of the above four units were all beyond 70% and the highest one was P4 (78.4±4.3%).8. Making sperm penetration of zona-free hamster egg assay (SPA) rate as test standard, it was compared that the three effects of technique for electron microscopy (TEM), triple-stain technique (TST), coomassie brilliant blue staining (CBB) separately on wapiti capacitated sperm evaluation. The results were that only TST showed no difference to SPA, and the other methods showed difference to SPA, but no difference to each other. SPA needed higher skills and couldn't be completed by one person in practice, however TST was easy to operate and could save time relatively.9. The selected wapiti sperm in vitro capaeitation culture system was that using modified BO culture medium supplemented with 6mg/mlBSA, the method of swim-up, heparin (20μg/ml×l5min) induced wapiti sperm capacitate in vitro at 38.5℃under a humidified atmosphere of 5%CO₂ in air. After comparison it was found that this system could obviously improve SPA rate and AR rate (p＜0.01).10. The selected evaluation system of wapiti sperm in vitro capacitation level was that using P₄(10μmol/L×10min) to induce wapiti sperm acrosome react, TST to evaluate acrosome morphology, assisting with sperm motility and eurvicaudate rate, and necessarily with SPA.Studied on wapiti in vitro fertilization using the above wapiti sperm in vitro capacitation culture system. Results are as follows:1. 10%FBS, 20%FBS, or 10%hind serum supplemented to M199 mature medium had no difference on successuful wapiti oovytes in vitro maturation and fertilization (p＞0.05). But supplementing with 20%FBS made higher 2-cell rate (p＜0.05) in early embryo in vitro culture.2.Making use of oviduct epithelial cell monolayers (COEM) into wapiti oocytes in vitro fertilization and early embryo in vitro culture, it was found that the fertilized rate was 60%, 66.7% embryo cleavaged, and some embryos eleavaged to 4-cell stage. In the control group embryos had only cleavaged to 2-cell stage and no one to 4-cell stage.Measuring the concentration of Ca²⁺, Mg²⁺, Na⁺, K⁺, CI^- and P in the culture medium of different stage of wapiti sperm in vitro capacitation and acrosome reaction by inductive coupled plasma (ICP) and ion chromatograph, it was found that all the ions concentrations had changed at different stage and there was being ions transmembrane transport between the intrcellular and exracellular of wapiti sperm.Results of wapiti morphological changes before and after sperm in vitro capacitation and freezing are as follows:1. The ultrastructure of wapiti sperm was similar to other mammals. There were three parts the head, the neck and the tail consisting wapiti sperm under TEM. The head was oval and mainly composed of sperm nucleus. Acrosome was map-alike on the head, ahead of which there was elephant-nose-alike drop. The neck was fragile and the tail was easy to fallout from it. The tail was consisted of middle piece, main piece and end piece. The axial filament structura type was "9+2".2. Under the TEM, the plasma membrane and the acrosome of the sperm head were intact before wapiti sperm capacitation. But after capacitation, dilation even and fragmentation or losing occurred in the plasma membrane, and outer acrosomal membrane vesiculated by different ways leading to inner acrosomal membrane naked.3. Under the TEM, 31.7±5.2% parts of the plasma membrane of wapiti sperm had dilated after being frozen-thawed. The damage of acrosome was a little. Only few acrosome reaction similar to the capacitation group occurred, and the rates of it was 4.6±3.8%. The ultrastructure changes had no different between wapiti fresh semen and frozen-thawed semen after in vitro capacitation.