Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender

Abstract

In the past years a series of functional assays has been developed to determine the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. Cell volume regulation is an important physiological function crucial for the success of cryopreservation. In this study, the effects induced by freezing-thawing were judged by evaluating the functional characteristics of frozen-thawed semen samples submitted to secondary stress such as osmotic challenge or incubation under capacitating conditions, following cryopreservation. Prior to freezing, dog semen samples were diluted in the presence or absence of Equex STM Paste, which contains sodium dodecyl sulphate (SDS) as the active ingredient. Cell volume regulation and capacitation and calcium ionophore-induced membrane dynamics were assessed in freshly diluted and frozen-thawed semen samples by electronic volume measurement and flow cytometry. Cryopreservation led to a disturbance of the volume regulatory function and to a rapid decrease in the proportion of acrosome-reacted live spermaotozoa. Extender containing Equex STM Paste had a protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome-reacted cells. The evaluation of the functional state of sperm submitted to secondary stress after freezing-thawing leads to a more subtle characterization of sperm function and helps improve the cryoprotective efficiency of the extender.

Introduction

Cryopreservation is widely used for long-term preservation of dog sperm. However, dog spermatozoa respond to cryopreservation very differently than that of other species of domestic animals. Dog spermatoza vary widely in the loss of progressive motility, in acrosomal integrity, and in viability [1]. Differences in semen of individual dogs in the tolerance to freezing is one source of fluctuation in pregnancy rates (between 40% and 60% [2]). Clearly, there is a need for freezing protocols and composition of extenders to be modifed and optimized.

Appropriate, sensitive, and rapid methods of assessment are necessary for adequate evaluation of sperm function [3]. Conventional sperm parameters are not sufficient to identify animals known as “poor freezers”, whose sperm quality is greatly impaired by cryoconservation. Thus, we applied recently established functional evaluation techniques in a sequential study to characterize the effects of different diluents on post-thaw sperm quality.

In the last decade a series of functional assays has been developed to detect the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. While flow cytometry is commonly used to assess the integrity of the plasma membrane, a variety of techniques are used to assess the state of the acrosomal membrane. There are essentially two main approaches, either to record the levels of spontaneous acrosome reaction or to induce the acrosome reaction with calcium ionophore. In the past few years, the level of the acrosome-reacted cells after cryopreservation has been used as a parameter to estimate sperm function and response to cryopreservation in human, canine, bovine, and equine species [4], [5], [6], [7], [8], [9], [10]. Szasz et al. [11] evaluated the response of spermatozoa to calcium ionophore to characterize the freezability of canine ejaculates. Those investigators observed a correlation between cryopreservation-induced membrane deterioration and loss of acrosome integrity and motility and the response of fresh sperm to calcium ionophore. Considering these correlations and the similarity of capacitation and cryopreservation-induced changes, they concluded that the detection of such changes is a simple and rapid approach to predicting the quality of frozen-thawed dog sperm [11].

Volumetric analysis is a method that is physiologically and mathematically appropriate to detect the functional membrane changes in live cell populations characterized by high sensitivity to differences in individual ejaculates [12], [13], [14]. The cells must be able to regulate changes in the cell volume in order to withstand considerable osmotic changes in their environment, which are most extreme within the epididymis and at ejaculation [15]. Moreover, major local osmotic gradients are generated across the sperm membranes during the freeze-thaw cycle, and cell death occurs mainly during thawing [16], [17], [18]. Therefore, sperm response to osmotic challenge (measured as cell volume) may be indicative of sperm survival and functional membrane integrity after cryopreservation.

In a recent study, we established the methodology for recording the cell volume in dog spermatozoa and showed that the volume control of canine spermatozoa depends on the functionality of the quinine-sensitive channels of the plasma membrane and on cytoskeletal integrity [14]. Although volumetric measurements appeared to be sufficiently sensitive to detect individual changes, it was not clear whether volumetric cell response could characterize the subtle differences caused by different ways of processing semen, i.e. with different extenders. Equex STM Paste, which contains sodium dodecyl sulfate as its active component, has been shown to improve the-post thawing characteristics of canine spermatozoa. Sperm diluted in egg yolk-Tris extender in the presence of Equex have been shown to have good survival and motility rates after long-term post-thaw incubation [19], better thermoresistance [20], higher rates of live acrosome intact cells [7], and higher intracellular calcium-levels [7]. The aims of the present study were (1) to compare the sperm cell volumes and volume regulatory ability in dog semen diluted in the presence or absence of Equex STM Paste before and after cryopreservation; and (2) to evaluate and to compare acrosome reaction in live sperm suspensions incubated under capacitating conditions with calcium ionophore in freshly diluted and frozen-thawed samples in the presence or absence of Equex STM Paste.