OPTIMIZATION OF FACS SORTING FOR THE IMPROVEMENT OF LIVESTOCK SEMEN QUALITY

J. Vašíček, A. Svoradová, A. Baláži, R. Jurčík, Marián Macháč, A. Ostró, P. Chrenek
{"title":"OPTIMIZATION OF FACS SORTING FOR THE IMPROVEMENT OF LIVESTOCK SEMEN QUALITY","authors":"J. Vašíček, A. Svoradová, A. Baláži, R. Jurčík, Marián Macháč, A. Ostró, P. Chrenek","doi":"10.15414/JMBFS.2021.10.4.697-705","DOIUrl":null,"url":null,"abstract":"Fluorescence-activated cell sorting (FACS) of spermatozoa was mainly used for sex sorting. Recently, FACS has been used to eliminate DNA-damaged human spermatozoa using YO-PRO-1 staining. Fragmentation of sperm DNA is considered as one of the reasons of male infertility. Moreover, YO-PRO-1 can effectively mark apoptotic as well as dead spermatozoa. Till now, only two FACS instruments were used for the spermatozoa sorting. However, both of them are not more commercially available from different reasons. In this study, we used novel FACSMelody Cell Sorter for the elimination of apoptotic and dead cells from the rabbit and ram semen samples in order to improve their overall quality. Briefly, semen samples were stained using YO-PRO-1 dye (apoptotic and dead cells) and/or propidium iodide (PI; only dead cells). Three different sorting experiments were performed: E1 – YO-PRO-1 and PI stained rabbit sperm cells were sorted into the tubes containing 1 ml of PBS; E2 – PI stained rabbit sperm cells were sorted into tubes that were washed with FBS prior adding PBS; and E3 – YO-PRO-1 and PI stained ram sperm cells were sorted into tubes washed with FBS prior adding PBS. As a sheath fluid sterile PBS was used. All samples, control (before sorting), negatively and positively sorted fractions, were analysed using CASA for motility assessment. Moreover, all sorted samples were re-stained with PI for viability assessment. In conclusion, elimination of dead (PI+) sperm from rabbit samples might improve their quality, since their progressive motility increased significantly (P<0.001) after sorting from 40 to 65%. However, ram spermatozoa seem to be sensitive to sorting procedure thus further optimisation of this procedure is required.","PeriodicalId":22746,"journal":{"name":"The Journal of Microbiology, Biotechnology and Food Sciences","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Journal of Microbiology, Biotechnology and Food Sciences","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.15414/JMBFS.2021.10.4.697-705","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

Fluorescence-activated cell sorting (FACS) of spermatozoa was mainly used for sex sorting. Recently, FACS has been used to eliminate DNA-damaged human spermatozoa using YO-PRO-1 staining. Fragmentation of sperm DNA is considered as one of the reasons of male infertility. Moreover, YO-PRO-1 can effectively mark apoptotic as well as dead spermatozoa. Till now, only two FACS instruments were used for the spermatozoa sorting. However, both of them are not more commercially available from different reasons. In this study, we used novel FACSMelody Cell Sorter for the elimination of apoptotic and dead cells from the rabbit and ram semen samples in order to improve their overall quality. Briefly, semen samples were stained using YO-PRO-1 dye (apoptotic and dead cells) and/or propidium iodide (PI; only dead cells). Three different sorting experiments were performed: E1 – YO-PRO-1 and PI stained rabbit sperm cells were sorted into the tubes containing 1 ml of PBS; E2 – PI stained rabbit sperm cells were sorted into tubes that were washed with FBS prior adding PBS; and E3 – YO-PRO-1 and PI stained ram sperm cells were sorted into tubes washed with FBS prior adding PBS. As a sheath fluid sterile PBS was used. All samples, control (before sorting), negatively and positively sorted fractions, were analysed using CASA for motility assessment. Moreover, all sorted samples were re-stained with PI for viability assessment. In conclusion, elimination of dead (PI+) sperm from rabbit samples might improve their quality, since their progressive motility increased significantly (P<0.001) after sorting from 40 to 65%. However, ram spermatozoa seem to be sensitive to sorting procedure thus further optimisation of this procedure is required.
提高畜禽精液质量的分型优化
精子荧光活化细胞分选(FACS)主要用于性别分选。最近,FACS已被用于通过YO-PRO-1染色清除dna损伤的人类精子。精子DNA断裂被认为是男性不育的原因之一。此外,YO-PRO-1可以有效地标记凋亡和死亡精子。迄今为止,仅有两台FACS仪器用于精子分选。然而,由于不同的原因,它们都不是更商业化。本研究采用新型FACSMelody细胞分选仪对兔和公羊精液进行凋亡细胞和死亡细胞的清除,以提高精液的整体质量。简单地说,精液样本用YO-PRO-1染色(凋亡细胞和死细胞)和/或碘化丙啶(PI;只有死细胞)。进行三种不同的分选实验:E1 - YO-PRO-1和PI染色的兔精子细胞分选到含有1ml PBS的试管中;E2 - PI染色的兔精子细胞分选到试管中,在加入PBS之前用FBS洗涤;将E3 - YO-PRO-1和PI染色的公羊精细胞分选到加入PBS之前用FBS洗涤的试管中。使用无菌PBS作为鞘液。所有样本,对照(分选前),阴性和阳性分选分数,使用CASA分析运动性评估。此外,所有分选的样品都用PI重新染色进行活力评估。综上所述,去除兔样品中的死精子(PI+)可能会提高兔样品的质量,因为在分选后,兔样品的进行性活力从40%显著增加(P<0.001)至65%。然而,公羊精子似乎对分选程序很敏感,因此需要进一步优化这一程序。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信