A New Preparation Protocol for Measurement of Testicular Sperm Production

Abstract

Abstract. In studies of male reproductive toxicity, measuring daily sperm production is a quite important criterion. However, the accuracy of the values measured by the basic protocol is still controversial. In order to enhance the homogeneity of countable testicular sperm/spermatid heads, this report introduces a new enzymatic method with a subsequent detergent treatment. The testis of rat was firstly homogenized in phosphate-buffered saline. The homogenate (buffer mix) was then treated with collagenase and trypsin, and then sodium dodecyl sulfate (SDS) was added to produce detergent-resistant sperm/spermatid heads (detergent mix). After examination by hemocytometer, the coefficient of variation (CV) of the number of sperm/spermatid heads was compared with that obtained from the buffer mix. In addition, a MicroCell chamber was applied to the examination, and the CV was compared with other cases. In both examinations, homogeneity was improved by the detergent mix preparation. Counting with the hemocytometer showed an increased number of sperm/spermatid heads compared with that of the buffer mix (P<0.001), and the CV was decreased (P<0.05). In addition, when the MicroCell chamber was applied, the numbers increased about 3-hold compared with that of the buffer mix (P<0.001). The CV of the detergent mix was 23.7%, while that of the buffer mix was 38.9%. These results clearly demonstrate that the new preparation protocol generated in this study can provide more actual and accurate values when measuring daily sperm production. Key words: Agglutination, Collagenase, Daily sperm production, Homogeneity, Rat, Sodium dodecyl sulfate (SDS) (J. Reprod. Dev. 54: [90][91][92][93] 2008) perm analysis is an essential item for evaluation of male reproductive toxicity. This evaluation is generally categorized into sperm count, motility and morphology [1]. For sperm counting, methods using counting a chamber, computer-assisted sperm analysis systems (CASA) and automated systems (e.g., IVOS HTM-IDENT; HTM Integrated Visual Optical System semen analyzer) are widely used [2][3][4][5][6]. Among these methods, use of a counting chamber is problematic because of variation. CASA and HTM-IDENT are also inapplicable because of dependence on technical skill and high installation costs [2][3][4][5][6][7][8][9][10][11][12]. In addition to the problems concerning these measurement systems, the viscosity and agglutination of the sample (e.g., semen), the inhomogeneity of the sperm suspension distribution and the technician's carefulness, fatigue and skill in pipetting can cause problematic variations in analyses [1,[13][14][15][16].In animal experiments, epididymal and testicular sperm are usually used for sperm counting [17]. However, in the case of epididymal sperm, the condition and position (caput or cauda) of the sperm collection may cause variation [3]. In measurement of daily sperm production, testicular homogenization-resistant sperm/ spermatids, which contain mature spermatozoa after spermiation and step 17-19 spermatids in case of...

Cypermethrin-induced reproductive toxicity in the rat is prevented by resveratrol

“…Sperm head counts were performed using hemocytometer as described by Choi[12] with necessary modifications. The testis was removed and weighted.…”

Homogenization‐resistant spermatids in testicular tissue stored under suboptimal conditions—Effect of time and temperature

“…The method used in this study had a low variability, as shown by the low CV. Other methods for lysis of cells have been published (Choi, Tsunekawa, Kanai, & Kurohmaru, 2008;Pacheco, Anderson, & Boekelheide, 2012). These protocols may offer improvements to that may be required if computer-assisted counting is to be used.…”