Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season
Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season
复制标题
DOI：
10.1016/s0378-4320(00)00214-1
复制
发表时间：
2001-01-31
影响因子：
2.2
通讯作者：
Torner, H
中科院分区：
文献类型：
--
作者：
Blottner, S;Warnke, C;Torner, H
这项研究比较了繁殖季节和非繁殖季节种马精液的质量和冷冻性能。于5月(n=24)和12月(n=24)每周两次采集4匹种马的精液。精液与脱脂牛奶稀释剂混合，离心后再悬浮在新鲜稀释液中。将这种精子悬液的等量从稀释液中分离出来，并在TALP培养液中或用冷冻稀释剂(“Gent”型或Triladyl(R)和脱脂牛奶的组合)稀释以进行精子评估。0.5毫升的样品用程序冷冻机在吸管中超低温保存。在冷冻/解冻前后对精子质量参数进行评估。这些指标包括活动精子和形态完整精子的百分比。扫描电子显微镜(S.E.M.)显示典型损伤。顶体状态用荧光素标记的花生凝集素显示，顶体反应由钙离子载体A 23187诱导。染色质稳定性用吖啶橙试验评价，冬季活动率和形态正常精子的平均百分率分别为67%和74.3%。分别高于5月份繁殖期(59%和65.9%：P<；0.05)。冷冻/解冻后，活精子和完整精子的比例显着降低。活动精子的数量在5月和12月分别下降到15%和18%(范围为5%-40%)，形态完整的精子数量在两个季节都下降到51%。扫描电子显微镜检查显示，精子顶体区有典型的膜破裂，部分精子颈部异常。冬季出现自发性顶体反应的冷冻精子比例较高(86.5%比77.0%)。表明膜的反应性较高。精子染色质变性的比例很低，在新鲜状态和冷冻状态、种马和季节之间显示出极小的差异。用木瓜酶和DTE进行的另一项解冻处理显示，冷冻保存后，带有可变性DNA的精子数量略有增加，尤其是在12月份(5.4+/-1.3%)。冷冻延长剂对大多数精子参数的影响并不显著，但公马之间有很高的变异性。综上所述，各因素对精子质量的影响大小顺序为：冷冻>；种马>；季节。不同的细胞结构似乎对物理化学压力有不同的敏感性。12月份冷冻保存精子的结果与繁殖季节测得的存活率相似，对于成功保存精子来说，更重要的是选择合适的精液捐赠者。(C)2001 Elsevier Science B.V.保留所有权利。
The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl(R) and skim milk). Samples of 0.5 mi were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were evaluated before and after freezing/thawing. These included percentages of motile spermatozoa and of morphological intact sperm. Typical injuries were demonstrated by scanning electron microscopy (S.E.M.). The acrosomal status was visualised using FITC-conjugated peanut agglutinin, and the acrosome reaction was induced by calcium ionophore A 23187. The chromatin stability was estimated by acridine orange test.In winter, the average percentages of motile and morphologically normal sperm (67 and 74.3%. respectively) were higher than during the breeding season in May (59 and 65.9%: P < 0.05). After freezing/thawing the proportions of vital and intact sperm decreased significantly. The number of motile sperm declined to 15 and 18% in May and December (range 5-40%), and of morphologically intact sperm to 51% in both seasons. Results of S.E.M. showed typical membrane ruptures in the acrosomal region and some sperm with abnormal necks. The proportion of frozen sperm with spontaneous acrosome reaction was higher during winter (86.5 versus 77.0%). suggesting a higher degree of membrane reactivity. Percentages of spermatozoa with denaturated chromatin were minimal and showed minimal differences between fresh and frozen state, stallions or seasons. An additional decondensation treatment with papain and DTE revealed a slightly enhanced number of spermatozoa with denaturable DNA after cryopreservation, especially in December (5.4 +/- 1.3%). The influence of cryoextenders was not significant for most sperm parameters, but there was a high variability between the stallions. Altogether, the influence of factors on the quality of spermatozoa has the following rank order: cryopreservation > stallion > season. Different cellular structures seem to have different susceptibilities to physicochemical stress. The cryopreservation of sperm during December results in survival rates similar to those measured during the breeding season, even more important for successful preservation is the selection of suitable semen donors. (C) 2001 Elsevier Science B.V. All rights reserved.