The role of different extenders in pre-centrifugation for optimizing equine semen cryopreservation

IF 1.3 3区 农林科学 Q2 VETERINARY SCIENCES
B.M. de Zutter , C.P. Freitas-Dell'Aqua , J.A. Dell'Aqua Jr , C.M. Trinque , M.S. Frasson , L.E.F. Canuto , T. Troncarelli , G.A. Monteiro , M.A. Alvarenga , F.O. Papa
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引用次数: 0

Abstract

Cryopreservation can cause injuries to sperm, thus the inclusion of extenders is crucial in maintaining viability outside the reproductive tract. This study evaluated the effectiveness of 4 extenders: Skim Milk (G1), Skim Milk + Pentoxifylline (G2), Skim Milk + Cholesterol (G3), and Cholesterol + Casein (G4) in maintaining the kinetics and integrity of the cytoplasmic membrane of equine spermatozoa. Semen was collected from 4 stallions with a total of 28 ejaculates. Each ejaculate was divided into 4 aliquots, the samples were cryopreserved and thawed (Papa et al. 2008; Animal Reprod Sci 107:293-301). The evaluation of sperm kinetics, including total motility (TM), progressive motility (PM), percentage of sperm with rapid movement (RAP), and average path velocity (VAP), was performed using CASA (HTM-IVOS 12 Hamilton Thorne Research, Beverly, MA, USA). The integrity of the cytoplasmic membrane (IM) was evaluated using the technique of Harrison and Vickers (Reproduction. 1990; 88:342-352). Statistical analysis was performed using the Kolmogorov-Smirnov test, ANOVA, and Tukey, with P<0.05. After cryopreservation and thawing, the groups G3 (78.1 ± 1.5) and G4 (78.9 ± 1.3) showed higher total motility compared to G2 (73.9 ± 1.7). Progressive motility, percentage of rapid sperm, and plasma membrane integrity were higher in groups G3 (44.7 ± 2.3 and 64 ± 2.8) and G4 (45.1 ± 2.3 and 65 ± 2.5) compared to groups G1 (40.8 ± 2.6 and 60.1 ± 3.2) and G2 (41.6 ± 2.6 and 61.2 ± 2.8). The presence of cholesterol in the extenders G3 and G4 was essential for the preservation of membrane fluidity and stability. Cholesterol helps prevent membrane damage during cryopreservation, while the sodium caseinate present in G4 also contributes to the protection of sperm cells. On the other hand, the extender containing pentoxifylline (G2), which functions by inhibiting phosphodiesterase enzymatic activity, leading to an increase in intracellular cAMP, showed worse performance post-thawing, possibly due to rapid depletion of the energy substrate, generating an increase in ATP production, which can lead to cell death. The findings of this study indicate that extenders with the inclusion of cholesterol (G3 and G4) are more effective in cryopreservation of equine semen than extenders based solely on skim milk. It is known that kinetic parameters of total motility and membrane integrity have proven to be crucial components in fertility prognosis, as these characteristics are interrelated (Foster et al. Theriogenology. 2011; 75:1499-1505). Therefore, the incorporation of cholesterol into the sperm membrane is an effective strategy to increase the viability of cryopreserved semen, particularly in stallions with low resistance to thermal shock, ensuring better results in equine reproduction programs.
不同填充剂在马精液冷冻预离心优化中的作用
低温保存可能会对精子造成损伤,因此加入延长剂对于维持生殖道外的生存能力至关重要。本研究评价了脱脂牛奶(G1)、脱脂牛奶 + Pentoxifylline (G2)、脱脂牛奶 + 胆固醇(G3)和胆固醇 + 酪蛋白(G4) 4种添加物对马精子细胞质膜动力学和完整性的影响。采集了4匹公马的精液,共28次射精。每次射精被分成4等份,样本被冷冻保存并解冻(Papa et al. 2008;动物生殖科学(107:293-301)。使用CASA (HTM-IVOS 12 Hamilton Thorne Research, Beverly, MA, USA)对精子动力学进行评估,包括总运动力(TM)、渐进运动力(PM)、快速运动精子百分比(RAP)和平均路径速度(VAP)。采用Harrison and Vickers (Reproduction. 1990;88:342 - 352)。统计学分析采用Kolmogorov-Smirnov检验、ANOVA和Tukey, P<0.05。冷冻解冻后,G3组(78.1±1.5)和G4组(78.9±1.3)的总运动性明显高于G2组(73.9±1.7)。G3组(44.7±2.3和64±2.8)和G4组(45.1±2.3和65±2.5)的进行性活力、快速精子百分比和质膜完整性高于G1组(40.8±2.6和60.1±3.2)和G2组(41.6±2.6和61.2±2.8)。扩展剂G3和G4中胆固醇的存在对于保持膜的流动性和稳定性至关重要。胆固醇有助于在冷冻保存过程中防止膜损伤,而G4中存在的酪蛋白酸钠也有助于保护精子细胞。另一方面,含有己酮茶碱(G2)的扩展剂,其功能是抑制磷酸二酯酶的酶活性,导致细胞内cAMP的增加,在解冻后表现出更差的性能,可能是由于能量底物的快速消耗,导致ATP的产生增加,从而导致细胞死亡。本研究结果表明,含胆固醇(G3和G4)的填充剂比纯脱脂乳填充剂对马精液的冷冻保存效果更好。众所周知,总运动性和膜完整性的动力学参数已被证明是生育预后的关键组成部分,因为这些特征是相互关联的(Foster等)。以及动物生殖部。技术部2011;75:1499 - 1505)。因此,在精子膜中掺入胆固醇是一种有效的策略,可以提高冷冻保存精液的活力,特别是在对热休克抵抗力较低的种马中,从而确保马的生殖计划取得更好的结果。
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来源期刊
Journal of Equine Veterinary Science
Journal of Equine Veterinary Science 农林科学-兽医学
CiteScore
2.70
自引率
7.70%
发文量
249
审稿时长
77 days
期刊介绍: Journal of Equine Veterinary Science (JEVS) is an international publication designed for the practicing equine veterinarian, equine researcher, and other equine health care specialist. Published monthly, each issue of JEVS includes original research, reviews, case reports, short communications, and clinical techniques from leaders in the equine veterinary field, covering such topics as laminitis, reproduction, infectious disease, parasitology, behavior, podology, internal medicine, surgery and nutrition.
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