Hermansson Ulrika, Ponglowhapan Suppawiwat, Forsberg Catharina Linde, Holst Bodil Ström
Department of Clinical Sciences, Division of Comparative Reproduction, Obstetrics and Udder Health, Swedish University of Agricultural Sciences, Box 7054, SE-750 07 Uppsala, Sweden.
Theriogenology. 2006 Sep 1;66(4):717-25. doi: 10.1016/j.theriogenology.2006.01.043. Epub 2006 Feb 23.
The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Ström Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.
精液样本的受精能力可通过体外试验评估精子来预测。透明带结合试验（ZBA）考虑了多个参数，并解释精子与卵母细胞之间的相互作用。本研究分为两个部分。第一个实验的目的是评估不同卵母细胞库中卵母细胞的精子结合能力是否存在差异。每次透明带结合试验均使用来自不同母犬的卵母细胞，并使用来自同两只公犬的混合冻融精液。精子 - 卵母细胞复合体孵育1小时。与透明带（ZP）结合的精子数量在六个重复试验之间存在显著差异，这表明不同卵母细胞库中ZP的精子结合能力不同。第二个实验的目的是评估精子的五种不同处理对ZBA的影响，以及评估精子 - 卵母细胞复合体的两种不同孵育时间。ZBA试验使用以下精液进行：新鲜精液；在进行ZBA试验前冷藏1天或2天的精液；以及添加或未添加Equex冷冻的精液。卵母细胞和精子孵育1小时或4小时。对于新鲜精液和未添加Equex冷冻的精液，孵育1小时导致每个卵母细胞结合的精子数量高于孵育4小时（P<0.0001）。当评估不同精子处理对与ZP结合的精子数量的影响时，发现共同孵育1小时和4小时后，新鲜精子结合的精子数量均高于冷藏或冻融精子（P<0.0001）。精子 - 卵母细胞复合体孵育1小时后，冷藏1天的精子比冷藏2天的精子表现出更好的透明带结合能力，未添加Equex冷冻的精子比添加Equex冷冻的精子具有更好的透明带结合能力。新鲜精液中的精子活力和精子质膜完整性高于冷藏和冻融精液。所有处理组精液的顶体完整性均较高。总之，精子 - 卵母细胞复合体孵育1小时似乎足以用于新鲜和冷藏精液。当评估冻融精液时，需要进一步研究以确定精子 - 卵母细胞复合体的最佳孵育时间，因为添加Equex冷冻的精液和未添加Equex冷冻的精液之间的比较结果因孵育时间是1小时还是4小时（本研究中）或6小时[斯特罗姆·霍尔斯特B，拉尔松B，林德 - 福斯伯格C，罗德里格斯 - 马丁内斯H。使用透明带结合试验评估冷藏和冻融犬精子。
