Barros Mothé Gabriele, Scott Caroline, Sicherle Carmen Cecília, de Freitas Guaitolini Carlos Renato, de Paula Freitas Dell'aqua Camila, Dantas Malossi Camila, Araújo-Júnior João Pessoa, de Souza Fabiana Ferreira
Department of Animal Reproduction and Veterinary Radiology, College of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil.
Department of Animal Reproduction and Veterinary Radiology, College of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, São Paulo, Brazil.
Anim Reprod Sci. 2018 Dec;199:84-92. doi: 10.1016/j.anireprosci.2018.11.003. Epub 2018 Nov 10.
Sexed sperm in dogs is of interest because of being polytocous, and as a result, the greatest number of offspring of the same sex can improve the market, although few studies assessing sperm sexing have been performed in this species. The present study, therefore, was conducted to evaluate the effects on sperm quality and the effectiveness of three discontinuous density gradients to separate dog sperm containing X and Y chromosomes. Thirty ejaculates from ten adult dogs were collected by digital manipulation of the penis. Cells were separated using gradients of Percoll® and Percoll® associated with Nycodenz® or Ficoll. The cells were evaluated for motility by the CASA system (Computer-Aided Semen Analyzer) and for concentration and recovered sperm concentration (after centrifugation), sperm morphology, plasma and acrosomal membrane integrity, and mitochondrial function pre- and post-centrifugation. The percentage of sperm containing X and Y chromosomes was also evaluated pre- and post-centrifugation by quantitative real-time PCR (qPCR). The use of the Ficoll gradient resulted in the greatest sperm quality after centrifugation; however, no sperm enhancement containing X or Y chromosome occurred with use of any of the methods (Percoll 54.8 ± 1.9 compared with 45.2 ± 1.9; Percoll associated with Nycodenz 53.2 ± 2.0 compared with 46.8 ± 2.0; and Percoll associated with Ficoll 55.0 ± 1.5 compared with 45.0 ± 1.5 for the percentages of cells containing the X and Y chromosomes, respectively). Thus, it was concluded that the technique of sexing dog sperm using density gradients was not effective for commercial application.
由于犬类是多胎动物，因此对犬类的性别分选精子很有意义，这样一来，同一性别后代数量最多就能提升市场价值，不过针对该物种评估精子性别分选的研究很少。因此，本研究旨在评估三种不连续密度梯度对犬类精子质量的影响以及分离含X和Y染色体犬类精子的有效性。通过对阴茎进行手法按摩，从10只成年犬收集了30份射精样本。使用Percoll®梯度以及与Nycodenz®或Ficoll相关联的Percoll®梯度对细胞进行分离。通过CASA系统（计算机辅助精液分析仪）评估细胞活力，并评估离心前后的浓度、回收精子浓度、精子形态、质膜和顶体膜完整性以及线粒体功能。还通过定量实时PCR（qPCR）评估离心前后含X和Y染色体精子的百分比。使用Ficoll梯度离心后精子质量最佳；然而，使用任何一种方法（Percoll：含X染色体细胞百分比54.8±1.9，含Y染色体细胞百分比45.2±1.9；与Nycodenz相关联的Percoll：含X染色体细胞百分比53.2±2.0，含Y染色体细胞百分比46.8±2.0；与Ficoll相关联的Percoll：含X染色体细胞百分比55.0±1.5，含Y染色体细胞百分比45.0±1.5）均未提高含X或Y染色体的精子比例。因此，得出结论：使用密度梯度对犬类精子进行性别分选的技术在商业应用中无效。
