Peña A I, Barrio M, Becerra J J, Quintela L A, Herradón P G
Unit of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Santiago de Compostela, Lugo, Spain.
Reprod Domest Anim. 2007 Oct;42(5):471-8. doi: 10.1111/j.1439-0531.2006.00809.x.
A 4-year-old Basque Shepherd male dog was presented for breeding soundness evaluation after the dog failed to impregnate the three bitches he had mated. Clinical examination showed no anomaly of the reproductive system. Semen evaluation showed normal sperm count (640 x 10(6)), 80% had progressively motile spermatozoa, and 96% had morphologically abnormal sperm of which 84% had proximal cytoplasmic droplet and 12% had proximal droplet plus other anomaly. A zona pellucida-binding assay, using canine oocytes derived from frozen-thawed ovaries, was performed in order to investigate the zona-binding ability of dog spermatozoa with proximal cytoplasmic droplets. For the zona pellucida-binding assay, ovaries were thawed and minced in phosphate-buffered saline + 0.4% bovine serum albumin, the oocytes recovered were divided into two groups of 35-40 oocytes to be, respectively, used with the infertile dog and with a control fertile dog. Spermatozoa were capacitated in Canine Capacitating Medium (CCM) at 38.5 degrees C and 5% CO(2) in air for 2 h before oocyte insemination. Groups of five to six oocytes placed in 45 microl droplets of CCM were incubated for 1 h. Afterwards, 5 microl of CCM containing 25,000 spermatozoa were added to each droplet and co-incubated for 2 h before fixation and evaluation of the complexes. After oocyte insemination, sperm motility and viability were evaluated: the sample from the infertile dog had 85% sperm motility with fast and linear progressive movement, and sperm viability of 92%. The sample from the control dog showed 40% sperm motility with fast and highly curvilinear and erratic movement, high degree of sperm agglutination and sperm viability of 32%. For the infertile dog the mean number of bound spermatozoa/oocyte was 0.33 whereas for the control dog it was 1.80. It was concluded that dog sperm with proximal cytoplasmic droplets seem to lack normal capacitating ability in vitro, and consequently, they may have reduced capacity to bind to the zona pellucida of canine oocytes.
一只4岁的巴斯克牧羊犬公犬在与它交配的三只母犬均未受孕后，前来接受繁殖健全性评估。临床检查显示生殖系统无异常。精液评估显示精子计数正常（640×10⁶），80%的精子具有进行性运动能力，96%的精子形态异常，其中84%有近端细胞质滴，12%有近端滴加其他异常。为了研究带有近端细胞质滴的犬精子与透明带的结合能力，使用从冻融卵巢获得的犬卵母细胞进行了透明带结合试验。对于透明带结合试验，将卵巢解冻并在磷酸盐缓冲盐水 + 0.4%牛血清白蛋白中切碎，回收的卵母细胞分成两组，每组35 - 40个卵母细胞，分别与不育犬和对照可育犬的精子一起使用。在卵母细胞授精前，精子在犬获能培养基（CCM）中于38.5℃、5%二氧化碳的空气中获能2小时。将五到六个卵母细胞放入45微升CCM液滴中孵育1小时。之后，向每个液滴中加入5微升含有25000个精子的CCM，共同孵育2小时，然后固定并评估复合物。卵母细胞授精后，评估精子活力和生存力：不育犬的样本有85%的精子活力，呈快速直线前进运动，精子生存力为92%。对照犬的样本显示40%的精子活力，呈快速高度曲线和不规则运动，精子凝集程度高，精子生存力为32%。对于不育犬，每个卵母细胞结合的精子平均数为0.33，而对照犬为1.80。结论是带有近端细胞质滴的犬精子在体外似乎缺乏正常的获能能力，因此，它们与犬卵母细胞透明带结合的能力可能降低。
