Campanholi Suzane Peres, Monteiro Fabio Morato, Ribeiro Dias Erika Aline, Mercadante Maria Eugênia Zerlotti, de Paz Claudia Cristina Paro, Dell'Aqua Junior José Antonio, Papa Frederico Ozanam, Dell'Aqua Camila de Paula Freitas, Vantini Roberta, Garcia Joaquim Mansano
Departamento de Reprodução Animal, Universidade Estadual Paulista, FCAV/UNESP, Jaboticabal, São Paulo, Brasil.
Centro APTA Bovinos de Corte, IZ-APTA, Sertãozinho, São Paulo, Brasil.
Theriogenology. 2017 Feb;89:114-121. doi: 10.1016/j.theriogenology.2016.10.008. Epub 2016 Oct 19.
Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 10 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.
牛精液的冷冻保存是养牛业中一种常见的生物技术程序。然而，当通过电刺激采精时，精子/精浆（SP）比例会出现很大差异，这可能会影响该物种精液的冷冻能力。去除精浆可能会提高冷冻牛精液的质量。本研究的目的是评估从射精中去除精浆对38头通过电刺激采集的内洛尔公牛精液冷冻保存的影响。采集后，将射精分为三等份：（1）对照组（N）稀释至浓度为60×10⁶个精子/mL并与精浆一起冷冻；（2）以600g离心（C）10分钟，将沉淀重悬并以与N相同的浓度冷冻；（3）通过精子过滤器过滤（F），回收精子并以与N相同的浓度冷冻。解冻后，评估精子活力、质膜和顶体膜完整性、线粒体膜电位、氧化应激和体外受精能力。使用SAS 9.2软件包进行统计分析，当P<0.05时差异被认为具有统计学意义。与对照组相比，去除精浆的组观察到更高的平均路径速度和直线速度（P<0.01）。相比之下，过滤后的样本与其他组相比表现出更高的拍频交叉频率、直线性和线性度。去除精浆时质膜完整性降低，但与N组（57.39±2.95%）相比，C组和F组（分别为34.91±2.95%和31.63±2.95%）观察到较低的氧化应激。然而，N组和F组的囊胚孵化率相似（分别为21.22±1.05%和24.00±1.05%），且高于C组（18.83±1.05%）。总之，通过离心去除精浆用于牛精液冷冻会降低体外产生胚胎的比率，而预冷冻精液的过滤在精液冷冻能力和牛胚胎体外生产方面被发现是一种有效的替代方法。
