Awan Muhammad Amjad, Mehmood Abid, Sultana Tasawar, Shahzad Qaisar, Ansari Muhammad Sajjad, Rakha Bushra Allah, Saqlan Naqvi S M, Akhter Shamim
Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, 46300, Pakistan.
Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, 46300, Pakistan.
Anim Reprod Sci. 2017 Jul;182:69-76. doi: 10.1016/j.anireprosci.2017.04.011. Epub 2017 May 6.
Sperm sexing through flow-sorting technology is relatively expensive, requires considerable technical support and is actually not practicable in many developing countries. The aim of this study was to investigate the feasibility of producing enriched pools of X or Y chromosome-bearing sperm by a modified swim-up method. For this purpose semen was collected from five mature Nili-Ravi buffalo bulls for a period of six weeks. The qualifying ejaculates were divided into two aliquots for further processing through modified swim-up or control (untreated). After processing, semen was cryopreserved in tris citric acid extender using standard techniques. Semen quality was assessed at pre dilution, post dilution and post thawing. Validation of technique was done by using SYBR green real time PCR using two sets of primers, PLP and SRY for X and Y chromosome of buffalo genes, respectively. Sperm recovery rates, pre freeze and post thaw sperm quality were found significantly higher in X chromosome bearing sperm fraction than Y chromosome bearing fraction and control. Mean fold relative expression of X bearing sperm was significantly higher (4-5 fold) in X chromosome bearing fraction of supernatant than Y chromosome bearing fraction (0.06 fold), similarly mean fold relative expression of Y chromosome bearing sperm was significantly higher in Y chromosome bearing fraction (4 fold) of supernatant than X chromosome bearing fraction (0.15 fold) compared to control (1.00). In conclusion, a modified swim up method proved to be an effective method for Nili-Ravi buffalo sperm sexing as validated by real time PCR.
通过流式细胞分选技术进行精子性别分选相对昂贵，需要大量技术支持，并且在许多发展中国家实际上并不可行。本研究的目的是探讨通过改良的上浮法制备富含X或Y染色体精子库的可行性。为此，从5头成熟的尼里-拉菲水牛公牛采集精液，为期6周。合格的射精样本分为两份，分别通过改良上浮法或对照（未处理）进行进一步处理。处理后，精液采用标准技术在柠檬酸三羟甲基氨基甲烷稀释液中冷冻保存。在稀释前、稀释后和解冻后评估精液质量。分别使用针对水牛基因X和Y染色体的两组引物PLP和SRY，通过SYBR绿实时荧光定量PCR对该技术进行验证。发现X染色体精子部分的精子回收率、冷冻前和冷冻后精子质量显著高于Y染色体精子部分和对照组。与对照组（1.00）相比，上清液中X染色体精子部分的X染色体精子平均相对表达倍数显著更高（4-5倍），高于Y染色体精子部分（0.06倍）；同样，上清液中Y染色体精子部分的Y染色体精子平均相对表达倍数显著更高（4倍），高于X染色体精子部分（0.15倍）。总之，经实时荧光定量PCR验证，改良上浮法被证明是一种有效的尼里-拉菲水牛精子性别分选方法。
