Faculty of Agricultural Sciences, Department of Agricultural Production, University of Caldas, Manizales, Colombia.
Biotechnology of Animal and Human Reproduction (TechnoSperm), Unit of Cell Biology, Department of Biology, Institute of Food and Agricultural Technology, Faculty of Sciences, University of Girona, Girona, Spain.
Theriogenology. 2020 Mar 15;145:181-189. doi: 10.1016/j.theriogenology.2019.10.023. Epub 2019 Oct 31.
Variation between and within boar ejaculates in terms of their ability to withstand freeze-thawing is a limitation for sperm cryopreservation. Consequently, searching for freezability markers not only in sperm but also in seminal plasma (SP) is imperative. The present study aimed to evaluate the relationship between cholesterol content, relative levels of NPC2 and AQN-1 at two different holding times (0 h: HT0 and 24 h: HT24) at 17 °C, and boar sperm freezability. Forty-five ejaculates were cryopreserved and subsequently classified as of good (GFE) or poor (PFE) freezability according to their post-thaw sperm viability and total motility. Prior to cryopreservation, relative abundances of two SP proteins (NPC2 and AQN-1) and cholesterol content in sperm and SP were determined through immunoblotting and colorimetric methods, respectively. These determinations were made after ejaculation (HT0) and after 24 h of storage at 17 °C (HT24). Two bands for NPC2 protein (16 kDa and 19 kDa) were identified. Relative amounts of the 16 kDa-band were significantly (P < 0.05) higher in poor (PFE) than in good (GFE) freezability ejaculates both at HT0 and HT24, whereas those of the 19 kDa-band were significantly (P < 0.05) higher in PFE than in GFE at HT24 only. In the case of AQN-1, no significant differences between GFE and PFE were observed. In addition, no variations in the cholesterol content of sperm and SP were observed either between HT0 and HT24 or between GFE and PFE. We can conclude that the content of two NPC2 isoforms in SP, but not of that of spermadhesin AQN-1, may be involved in the sperm resilience to withstand freeze-thawing procedures and may predict ejaculate freezability. While a possible mechanism through which NPC2 during HT could affect boar sperm cryotolerance is suggested to be related to its ability to bind the plasma membrane cholesterol, further research is warranted.
公猪精液在抗冻能力方面存在个体间和个体内的差异，这是精子冷冻保存的一个限制因素。因此，不仅要在精子中寻找可冷冻性标记物，还要在精浆（SP）中寻找。本研究旨在评估胆固醇含量、NPC2 相对水平和 AQN-1 在 17°C 下两个不同的孵育时间（0 h：HT0 和 24 h：HT24）与公猪精子可冷冻性之间的关系。45 份精液被冷冻保存，然后根据解冻后精子活力和总运动性将其分为良好（GFE）和差（PFE）可冷冻性。在冷冻保存之前，通过免疫印迹和比色法分别测定精子和 SP 中两种 SP 蛋白（NPC2 和 AQN-1）和胆固醇含量的相对丰度。这些测定是在射精后（HT0）和在 17°C 下储存 24 小时后（HT24）进行的。鉴定出 NPC2 蛋白的两条带（16 kDa 和 19 kDa）。在 HT0 和 HT24 时，16 kDa 带的相对含量在差（PFE）冷冻可育性精液中明显（P < 0.05）高于好（GFE）冷冻可育性精液，而 19 kDa 带的相对含量在仅 HT24 时 PFE 中明显（P < 0.05）高于 GFE。在 AQN-1 的情况下，GFE 和 PFE 之间没有观察到显著差异。此外，无论是在 HT0 和 HT24 之间，还是在 GFE 和 PFE 之间，精子和 SP 中的胆固醇含量都没有变化。我们可以得出结论，SP 中两种 NPC2 同工型的含量，而不是精子结合素 AQN-1 的含量，可能与精子对冷冻-解冻过程的抵抗力有关，并可预测精液的可冷冻性。虽然提出 NPC2 在 HT 期间可能通过与其结合质膜胆固醇的能力影响公猪精子抗冻性的可能机制，但还需要进一步的研究。
