Journal of Rafsanjan University of Medical Sciences

  Comparison of Viable Human Sperm Count Using Three Different Methods Hypo-Osmotic Swelling Test, Viability/Cytotoxicity Assay, and Eosin-Nigrosin Technique: A Laboratory Study

  H. Hakimi [1]

  Received: 14/10/08 Sent for Revision: 16/05/09 Received Revised Manuscript: 15/10/09 Accepted: 21/11/09

  Background and Objectives: Sperm viability is one of the semen parameters that should be noted in male infertility approaches. Depending on the laboratory’s facilities and especially the purpose of the experiment, i.e. diagnostic or therapeutic, one or more of the diagnostic techniques are employed. In the present study, three methods Hypo-Osmotic Swelling Test (HOST), Viability/Cytotoxicity Assay (VCA), and Eosin-Nigrosin Technique (ENT) have been compared in terms of cost, required equipments, diagnostic value, and rapidity.

  Materials and Methods: In this laboratory study semen samples from six healthy volunteers referred to the Andrology laboratory, Hallamshire hospital, Sheffield, UK were investigated in 2002. In 4 ependorfs, 180µl of 5 x10⁶ sperm/ml of prepared sperm using percoll gradient method were decanted. Three of the ependorfs were treated with 20µl of Chlamydia LPS at final concentration of 0.1 µ g/ml for 6 h at 37 ºC in 5% CO₂ for 6 h and the last ependorf was treated with 20µl of 1 x EBSS as control. After 6h incubation the sperm viability was measured using HOST, VCA, and ENT methods . The data was then analysed using t-test and One-way ANOVA.

  Results: The findings of HOST, VCA, and ENT indicated that sperm mortality rate enhanced markedly in the presence of lipopolysaccharide from Chlamydia trachomatis at�0.1μg/ml [35.5±4.1% (HOST) , 33.8±3.3% (VCA), 34.4±3.9% (ENT), p < 0.05). However, the differences between the test groups were not statistically significant in the inter-group comparison .

  Conclusion: If sperm viability assessment is required only for diagnostic purposes, ENT is suggested as a preferred method, because it is cheap, easy, fast, handy, and needs no specific facilities.

  Key words : Sperm, Viability, Cytotoxicity , Hypo- O smotic Swelling, Eosin-Nigrosin

  Funding: This research was funded by Rafsanjan University of Medical Sciences and conducted at the University of Sheffield, UK.

  Conflict of interest: None declared.

  Ethical approval : The Ethical Committee of Rafsanjan University of Medical Sciences and Sheffield University jointly approved the study.

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  [1] - Assistant Prof., Dept of Microbiology, Faculty of Medicine, University of Medical Sciences, Rafsanjan, Iran

  (Corresponding Author) Tel: (0391)5234003, Fax: (0391) 5225209, E-mail: hamid.hakimi@gmail.com