Biology, Medicine, & Natural Product Chemistry  ISSN 2089-6514 (paper) 
Volume 9, Number 2, October 2020 | Pages: 105-107 | DOI: 10.14421/biomedich.2020.92.105-107 ISSN 2540-9328 (online) 
 

 

 

 

A Simple Technique for Rapid Assessment of  

Rat (Rattus norvegicus) Sperm Motility 

 
Muhammad Ja’far Luthfi1,*, Mahanem Mat Noor2 

1Department of Biological Education, Faculty of Tarbiyah and Education, Universitas Islam Negeri Sunan Kalijaga Yogryakarta, Indonesia 
2School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Malaysia 

 

Corresponding author* 
jafarluthfi@yahoo.com 

 

 
Manuscript received: 20 September, 2020. Revision accepted: 19 February, 2021. Published: 25 February, 2021. 

 

 

Abstract 

 

Assesment of rat sperm motility can be carried out on sperm sample from epididimal source using minimal amount of equipment. This 

method will aid researcher and practitioner working in the field of sperm quality to determined rat sperm motility rapidly and efficiently. 

 

Keywords: sperm quality; sperm motility; rat; haemocytometer. 

 

 

INTRODUCTION 
 

Sperm motility is one of the most important parameters 

determining sperm quality (Martinez et al., 2000; 

Talarczyk-Desole et al., 2017). The results of sperm 

motility assessment can be used more directly to address 

problems affecting male reproductive organs (Björndahl, 

2010). The other parameters are sperm count and sperm 

morphology (Freund & Carrol 1964). These three 

components (sperm motility, sperm count, and sperm 

morphology) compose of sperm quality (Perreault & 

Cancel 2001). The most frequently used techniques to 

assess sperm qualiy are counting chambers (e.g. 

haemocytometer and Makler chamber). These 

techniques, however, are subject to a high variability 

(Yang et al., 2019). 

Much effort has been made to speed up the objective 

measurement of sperm motility. CASA (Computer 

Aided Sperm Analysis) and IVOS (Integrated Visual 

Optical System) are automatic methods of sperm quality 

testing. The techniques, however, need sophisticated 

equipment not available in many laboratories. They are 

too technically complex and too expensive for routine 

use. Manual observation is still the most widely used 

method for testing sperm motility (Das 1985; Kuster 

2005; Macpherson, 2001). 

The aim of this study was to determine the optimal 

manual technique for assessment of rat sperm motility 

using arbitrary scale. The technique was based on years 

of practices in our laboratories. It provides a practical 

and simple means for assessment of rat sperm motility 

for the purpose of determining the fertility status the 

animal. 

 

MATERIALS AND METHODS 
 
Equipments and Materials 
The equipment needed in the analysis of rat sperm 

motility were as follows: a light microscope, 

micropipette, petri dishes, Improved Neubauer 

Haemocytometer, CO2 incubator, small animal surgical 

instruments, and hand counter. 

The materials needed in the analysis of rat sperm 

motility were as follows: three adult male Sprague-

Dawley rats (Rattus norvegicus), Biggers, Whitten, & 

Whittingham (BWW) medium, and cover glasses. The 

study was done in the Zoology Laboratory of University 

Kebangsaan Malaysia and the Zoology Laboratory of 

UIN Sunan Kalijaga Yogyakarta. 
 
Preparation of Sperm Suspension 
Sperm samples were taken from the left cauda 

epididymis. The cauda epididymis was separated based 

on the epididymal division by Hamilton (1975), then 

placed in a petri dish, minced and incubated in 15 mL of 

Biggers, Whitten & Whittingham (BWW) media 

solution (Biggers et al. 1971) for 30 minutes in 37°C in 

an incubator of 5% CO2 to let sperm swim in the media 

(swim-up technique) (Luthfi, 2015; Luthfi & Noor, 

2015). 
 
Procedure for Assessing Sperm Motility 
Sperm motility were determined using Improve 

Neubauer Haemocytometer as described previously 

(Prasad et al., 1972; WHO, 1999) with modification. 

The cover glass was attached firmly to the Improved 

Neubauer Haemocytometer counting grid area. The 

interference pattern (> 10 newton's rings/fringes) should 

be seen between the glass surfaces of the two areas 

https://doi.org/10.14421/biomedich.2020.92.105-107
https://www.ncbi.nlm.nih.gov/pubmed/?term=Bj%26%23x000f6%3Brndahl%20L%5BAuthor%5D&cauthor=true&cauthor_uid=20111079


 

 

 

106 Biology, Medicine, & Natural Product Chemistry 9 (2), 2020: 105-107 
 

 

where the glass cover is attached to the haemocytometer. 

The line/newton's rings that appear too few indicate that 

the distance between the cover glass and the 

haemocytometer is widened, therefore the counting 

chamber volume becomes larger and the assessment 

would be incorrect. The center of the haemocytometer 

counting grid area is different from the other ones 

because it consists of 25 larger squares bounded by 

triple lines each, while the ones in the corners consist of 

16 larger squares. In addition, the larger squares inside 

the central counting grid are subdivided into 16 even 

smaller squares each (Figure 1). 

A total of 10 μL of sperm suspension from the 

preparation of sperm samples were taken with a 

micropipette, then inserted into the space between the 

cover glass and the haemocytometer in one of its 

counting chamber. The other counting chamber was also 

filled in the same way. Let the sperm settled in the 

chambers for 5 minutes. Each counting chamber must be 

filled correctly. The suspension was inserted slowly to 

let the liquid evenly distributed by capillary force. If the 

filling on to the counting chamber was uneven, wipe the 

slide and then the process of filling the chamber must be 

repeat. The removal of excessive volume from the 

counting chamber must not be done because it will 

change the density of sperm in the counting chamber.  

 
Figure 1. Haemocytometer. A. Side view. B. Top view. C. One of the two 

haemocytometer counting chambers. D. Insert of iv. Legend: i. Cover 

glass; ii. Counting chamber; iii. Counting grid area; iv. One of the 25 
larger squares. 

 

After both counting chambers were filled, 

assessment of sperm motility was carried out. The sperm 

motility was observed at 200x or 400x magnification 

using a light microscope. Systematically scan the center 

of the haemocytometer counting grid area, started with 

the uppermost left square of the 25 large squares, 

continued to the next three squares to the right, and the 

squares at the row below, and so on. Assessment were 

done only for intact sperm (defined as having a head and 

a tail). Approximately 100 sperm in each counting 

chamber were assigned to minimize deviation. Sperm 

motility is estimated to follow the criteria as defined by 

WHO (1999; 2010) and is denoted by the letters a, b, c 

(from the fastest to the slowest) as follows: 

a: progressive motility; ≥ 25 μm/s where 25 μm is the 
same as the length of 2 squares (smallest square) of 

the haemocytometer (see red rectangle in figure 1D) 

b: non-progressive motility; <5 μm/s 
c: no motility  
 
 

RESULT AND DISCUSSION 
 

Determination of sperm motility is an important step in 

the analysis of sperm quality. However, there are 

various methods of assessing sperm motility in terms of 

equipment, technicality and practicality. Different 

laboratories use different methods. This study was use 

haemocytometer to determine sperm motility. The 

determination of the method is mostly based on the 

practice and experience of testing rat sperm samples 

carried out at the Zoology Laboratory of the Faculty of 

Science and Technology, UIN Sunan Kalijaga and 

Zoology Laboratory of University Kebangsaan Malaysia 

(data not shown). 

The sperm motility of the laboratory animals can be 

determined from ejaculate or epididymal samples. The 

determination of epididymal sperm motility is using 

only sperm from the caudal part (Clegg et al., 2001). 

Changes in the progression/rate of sperm motility after 

substance or drug treatment can provide important clues 

as to the effect of a substance on sperm fertilizing 

capacity (Working, 1988). 

Motility pattern is one of the most important and 

simplest variables in determining the fertility potential 

of sperm because immotile sperm will not be able to 

penetrate the mucus layer of the cervix to reach the 

ovum and fertilization to occur. Sperm motility patterns 

is an important factor in fertilization, as a strong swing 

of the sperm tail is necessary for the sperm head to 

penetrate the lining of the ovum (Partodihardjo, 1992). 

Progressive motility is a normal sperm movement. 

Circular motion and reversion movements are often 

signs of cold shock or due to abnormal sperm 

morphology. Meanwhile, wavy motion and vibrating 

motion occur more frequently in old men (Atherton, 

1977). 



 

 

 

 Luthfi & Noor – A Simple Technique for Rapid Assessment of … 107 
 

 

Sperm motility is observed with either physiological 

saline or other nutrient medium. Sperm were observed 

under a microscope slide with/without a cover glass or 

with a capillary tube. CASA (Computer Aided Sperm 

Analysis) and IVOS (Integrated Visual Optical System) 

are automatic objective methods for testing sperm 

motility. However, manual observation is still the most 

widely used method for testing sperm motility using an 

arbitrary scale (Das 1985; Kuster 2005; Strader 1996). 

This study revealed that using this method, 25-45 

percent of rat sperm would fall into category of 

progresssive motility, 45-65 percent were of non-

progressive motility category, and 15-35 were of no 

motility category. Aproximately, those numbers could 

be used as a reference for normal sperm motility of rat 

sperm. Several things need to be considered in order to 

achieve consistency in using this method. First, we must 

ensure that the cutting of cauda epididymis was done 

consistently in every replication. Second, micropipette 

insertion of samples in each counting chamber must be 

perfect. Third, the room temperature used in determining 

the sperm motility must be the same for all sperm 

sample assessed. Fourth, the time needed to assess the 

sperm motility should consistent from slide to slide.  All 

these things affect the consistency of the sperm motility 

assesment. The procedures that are followed strictly will 

ensure the accuracy of the assessment. 
 
 

CONCLUSION 
 

Using this method, researchers can determine the rat 

sperm motility in a practical way. Most of the materials 

and equipments used in this method are widely 

available. The increasing importance of sperm motility 

analysis will be consistent with the adoption and 

development of this simple and rapid method. 
 
 

Conflict of interest: The author declares that there are 
no conflicts of interest concerning the publication of this 

article. 
 
 

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