Determination of the Osmotic Resistance Changes in Mouse Spermatozoa Using the Classic Osmotic Resistance Test, Short Osmotic Resistance Test and Modified Short Osmotic Resistance Test

Abstract

The acrosome is a very important structure on the sperm head. Made up of the outer and inner acrosomal membrane, the acrosome contained enzymes important to digest the zona pellucida of the ovum upon fertilization. Therefore, evaluation of the functional membrane integrity of the acrosome is one of the methods to recognize fertilizing capacity of the sperm. The method commonly used is the osmotic resistance test apart from the hypoosmotic test. Males can be better donors for sperm cryopreservation and this classification can be determine if osmotic resistance test showed excellent results. The traditional or classic osmotic resistance test (ORT) has a longer incubation time of two hours therefore a modified ORT was proposed with an incubation time of 5 minutes referred to as short osmotic resistance test (SORT), and later the modified SORT was introduced where samples were incubated in only 75 mOsmol solution for 5 minutes. As results vary for ejaculated sperm and cauda epididymal sperm, the aim of this study is to detect acrosomal membrane functional integrity in mouse sperm when incubated in classic ORT, short ORT and modified short ORT solutions. Cauda epididymal sperm from twenty mice were collected into Krebs Ringer's Buffer medium and divided into three parts: one for each test. Sperm were pooled, discarded of cell debris and centrifuged on a 2-step discontinuous Ficoll gradient. The sperm was collected from the pellet and incubated for 60 minutes in classic ORT buffer and 5 minutes in short ORT buffer and modified short ORT buffer. Tail swelling, proportion of normal acrosomes (%NAR) and viability was observed from smears taken at intervals of 10 minutes for classic ORT and 1 minute for SORT and modified SORT. Results were subjected to calculation for SORT and modified SORT only which is: SORT (5 min) =1/2[%NAR in 300mOsmol solution (5 min) + % NAR in 75mOsmol solution (5 min)] while modified SORT (5 min) =1/2[%NAR in samples from sperm morphology + % NAR in 75mOsmol solution (5 min)]. The results showed that classic ORT and modified SORT were found to yield the same findings however classic ORT and SORT showed different findings. The results suggest that the modified SORT can be apply to detect changes in acrosomal integrity to replace classic ORT as it is a rapid test and yields the same results as the classic ORT.