几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。

Effects of some extenders and monoamines on sperm cryopreservation in tree shrews (Tupaia belangeri)

The tree shrew may be an important experimental animal for disease models in humans. The effects of some extenders and momamines on sperm cryopreservation will provide helpful data for experimentation of strains and conservation of genetic resources in tree shrews. Epididymal sperm were surgically harvested from male tree shrews captured around Kunming, China and sperm motility, acrosome integrity and fertility were assessed during cryopreservation. In Experiment 1 eight extenders (TTE, TCG, TCF, TTG, BWW, BTS, DM, and SR) supplemented with 0.4 mol/L DMSO were used to dilute the sperm: only TTE, DM and SR showed no differences in motility and acrosome integrity compared to fresh controls after equilibration. After freezing and thawing, sperm in any extender showed lower motility than fresh control and sperm in DM showed higher motility than other groups. However, BWW produced the lowest motility. For acrosome intergrity, TTE and DM showed higher than BWW, BTS and SR after equilibration. The parameter in DM was higher than other groups (except TTE) after thawing. In Experiment 2 four penetrating cryoprotectant agents (CPA) dimethyl-formamide (DF), formamide (F), dimethylacetamide (DA), and acetamide (A)] at 0.2 mol/L, 0.4 mol/L, 0.8 mol/L, and 1.2 mol/L, respectively were added to the DM extender. Motility showed no difference among CPA groups and non-CPA group (control) after equilibration, but all thawed sperm showed lower values in motility and acrosome intergrity than pre-freezing groups. However, sperm in 0.8 mol/L DF and 0.4 mol/L DMSO showed higher values in both parameters than that in other CPA groups (P>0.05). In Experiment 3 the fertilization rate of oocytes inseminated with 0.4mol/L DMSO (50%) were higher than that with 0.8mol/L DF (16%). In conclusion, non-ion extenders supplemented with egg yolk may be better for sperm cryopreservation in tree shrews and cryoprotectant effects of monoamines agents should be further studied in this species.