Pradieé J, Sánchez-Calabuig M J, Castaño C, O'Brien E, Esteso M C, Beltrán-Breña P, Maillo V, Santiago-Moreno J, Rizos D
Departamento de Reproducción Animal, SGIT-INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), SHIS QI 1, Lago Sul, Brasília DF, 71605-001, Brazil.
Departamento de Reproducción Animal, SGIT-INIA, Avda. Puerta de Hierro km 5.9, 28040 Madrid, Spain; Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro, s/n, 28040 Madrid, Spain.
Theriogenology. 2018 Mar 1;108:314-320. doi: 10.1016/j.theriogenology.2017.11.021. Epub 2017 Dec 20.
In this study, we successfully described for the first time a vitrification of epididymal Iberian ibex spermatozoa. Spermatozoa from epididymis were obtained from 15 Iberian ibex. The right epididymis' semen sample was vitrified and the left one was frozen. After thawing/warming, samples were selected by density gradient. Sperm characteristics from each treatment were evaluated. To test the spermatozoa fertilization ability, heterologous IVF was carried out using bovine oocytes. Despite of the observation of a decrease of about 40% for motility sperm between pre-freezing and post-thawing (75.0 ± 5.2 and 45.0 ± 6.0) and pre-vitrification and post-warming (78.2 ± 5.2 and 33.9 ± 6.2) (P < .05), after the washing, an improvement of sperm motility was found when using the vitrification treatment compared to frozen-thawed. Heterologous IVF showed that Iberian Ibex spermatozoa, either frozen-thawed or vitrified-warmed, were equally capable of penetrating ZP intact bovine oocytes, leading to pronuclear formation (%) and hybrid embryo cleavage (%), (31.3 ± 27.2 and 45.1 ± 24.4, respectively). As expected, in the homologous IVF group, higher percentages of penetration, pronuclei formation and cleavage were found compared to heterologous groups using Iberian ibex frozen and vitrified sperm (P < 0,5). The highest pronuclei formation was found after 20 h post insemination in both heterologous IVF groups (30.2 ± 6.7 and 31.7 ± 21.5 thawed and vitrified group). Consequently, the cleavage rate (48 h) followed the same results to homologous and thawed and vitrified groups (76.1 ± 15.9; 31.3 ± 27.2 and 45.1 ± 24.4, respectively) (P < .05). In conclusion, Iberian ibex sperm vitrification is a promising and useful alternative to conventional methods resulting in good quality spermatozoa post-thaw, and an adequate in vitro fertilizing ability.
在本研究中,我们首次成功描述了伊比利亚羱羊附睾精子的玻璃化冷冻方法。从15只伊比利亚羱羊获取附睾精子。右侧附睾的精液样本进行玻璃化冷冻,左侧的则进行常规冷冻。解冻/复温后,通过密度梯度法对样本进行筛选。评估每种处理方式下精子的特征。为测试精子的受精能力,使用牛卵母细胞进行了异源体外受精。尽管观察到冷冻前与解冻后(75.0±5.2和45.0±6.0)以及玻璃化冷冻前与复温后(78.2±5.2和33.9±6.2)精子活力下降约40%(P<0.05),但洗涤后发现,与冷冻解冻处理相比,玻璃化冷冻处理后的精子活力有所提高。异源体外受精显示,冷冻解冻或玻璃化冷冻复温后的伊比利亚羱羊精子同样能够穿透完整透明带的牛卵母细胞,导致原核形成率(%)和杂交胚胎分裂率(%)分别为(31.3±27.2和45.1±24.4)。正如预期的那样,在同源体外受精组中,与使用伊比利亚羱羊冷冻和玻璃化冷冻精子的异源组相比,发现更高的穿透率、原核形成率和分裂率(P<0.5)。在两个异源体外受精组中,授精后20小时发现最高的原核形成率(解冻组和玻璃化冷冻组分别为30.2±6.7和31.7±21.5)。因此,分裂率(48小时)在同源组、解冻组和玻璃化冷冻组中呈现相同结果(分别为76.1±15.9;31.3±27.2和45.1±24.4)(P<0.05)。总之,伊比利亚羱羊精子玻璃化冷冻是一种有前景且有用的替代传统方法,解冻后能获得高质量精子,并具有足够的体外受精能力。