J Vet Sci.  2009 Sep;10(3):249-255. 10.4142/jvs.2009.10.3.249.

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

• Lee HL
• Kim SH
• Ji DB
• Kim YJ

• KMID: 1726925
• DOI: http://doi.org/10.4142/jvs.2009.10.3.249

Abstract

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.

Keyword

glass wool; in-vitro fertilization (IVF); Percoll; Sephadex

MeSH Terms

Animals
Cattle
Cryopreservation/methods/*veterinary
Cytological Techniques/methods/*veterinary
*DEAE-Dextran
Female
Fertilization in Vitro/methods/*veterinary
*Glass
Male
Semen Preservation/methods/*veterinary
Spermatozoa/*physiology
Zygote/cytology

Figure

• 

  Fig. 1 Sperm motility in treated and control samples. Data are presented as mean ± SE. a,bDifferent superscripts indicate significant differences among treatments (p < 0.05, n = 6).

• 

  Fig. 2 Sperm plasma-membrane integrity evaluated by carboxyfluoresceindiacetate/propidium iodide (CFDA/PI) fluorescent staining and hypo-osmotic swelling test (HOST) in treated and control samples. Data are presented as mean ± SE. a,b,c,dDifferent superscripts indicate significant differences among treatments within an evaluation method (p < 0.05, n = 6).

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