Taş M, Bacinoglu S, Cirit U, Ozgümüş S, Kaşgöz H, Pabuccuoğlu S
Department of Obstetrics and Reproduction, Faculty of Veterinary Medicine, Dicle University, 21280, Diyarbakir, Turkey.
Theriogenology. 2007 Oct 15;68(7):981-7. doi: 10.1016/j.theriogenology.2007.07.011. Epub 2007 Sep 11.
In the present study, we aimed to develop a polyacrylamide gel that could be used instead of bovine cervical mucus in the cervical mucus penetration test (CMPT) to obtain coherent and replicable results in bulls. The frozen semen samples of six Holstein bulls, which were divided into two fertility groups as low and high according to their non-return rate (NRR), were used. In this study, the modified CMPT (mCMPT) was carried out within 0.25 mL transparent plastic straws with an inner diameter 1.7 mm. The penetration ability of spermatozoa to bovine cervical mucus and to polyacrylamide gels swollen with two different solutions [NaCl (G1) and PBS (G2)] was compared. For the penetration test, the straws filled with cervical mucus and both gels were dipped into thawed semen samples and incubated at 37 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, the frozen straws were cut at 1.5-1.75 cm (penetration distance range=PDR1), 3.25-3.5 cm (PDR2) and 5.0-5.25 cm (PDR3), beginning from open-end of the straws. The separated frozen parts were then immediately transferred onto special counting slides by pushing with a mandrel and left to thaw. Thawed samples were covered with cover glass and penetrated spermatozoa in these parts were counted. The relation between the results and fertility of bulls was determined. In the tests performed using mucus, the number of spermatozoa determined in the high fertility group was found to be higher at PDR3 (p<0.0001) compared to the low fertility group, while in G1 spermatozoa number was significantly higher at PDR1 and PDR3 (p<0.0001). However, in G2 medium, no significant difference was observed between either of the fertility groups with respect to spermatozoa number determined at all distance ranges. In the study, we have determined that the gel swollen with NaCl produces better results and this gel can be used instead of bovine cervical mucus for the CMPT. Therefore, we have concluded that the penetration test performed by polyacrylamide gel swollen with NaCl can be a suitable technique for estimation of the potential fertility of bull spermatozoa.
在本研究中，我们旨在研发一种聚丙烯酰胺凝胶，其可在宫颈黏液穿透试验（CMPT）中替代牛宫颈黏液，从而在公牛身上获得连贯且可重复的结果。我们使用了6头荷斯坦公牛的冷冻精液样本，根据其返情率（NRR）将它们分为低生育力组和高生育力组。在本研究中，改良的CMPT（mCMPT）在0.25 mL内径为1.7 mm的透明塑料吸管内进行。比较了精子对牛宫颈黏液以及对用两种不同溶液[NaCl（G1）和PBS（G2）]溶胀的聚丙烯酰胺凝胶的穿透能力。对于穿透试验，将装有宫颈黏液和两种凝胶的吸管浸入解冻的精液样本中，并在37℃下孵育15分钟。孵育后，将吸管在液氮蒸气中冷冻，并在-20℃下保存。在评估当天，从吸管的开口端开始，将冷冻的吸管在1.5 - 1.75 cm（穿透距离范围=PDR1）、3.25 - 3.5 cm（PDR2）和5.0 - 5.25 cm（PDR3）处切割。然后用芯棒将分离出的冷冻部分立即推到特殊的计数载玻片上，并使其解冻。解冻后的样本用盖玻片覆盖，并对这些部分中穿透的精子进行计数。确定了结果与公牛生育力之间的关系。在使用黏液进行的试验中，发现高生育力组在PDR3处确定的精子数量比低生育力组更高（p<0.0001），而在G1中，PDR1和PDR3处的精子数量显著更高（p<0.0001）。然而，在G2培养基中，在所有距离范围内确定的精子数量方面，两个生育力组之间均未观察到显著差异。在该研究中，我们确定用NaCl溶胀的凝胶产生了更好的结果，并且这种凝胶可替代牛宫颈黏液用于CMPT。因此，我们得出结论，用NaCl溶胀的聚丙烯酰胺凝胶进行的穿透试验可以是一种评估公牛精子潜在生育力的合适技术。
