Elsevier

Theriogenology

Volume 74, Issue 5, 15 September 2010, Pages 741-748
Theriogenology

Research article
Effect of liquid storage on sorted boar spermatozoa

https://doi.org/10.1016/j.theriogenology.2010.03.027Get rights and content

Abstract

A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation.

The aim of this study was to evaluate the modification induced by 24–26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24–26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study.

Introduction

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method to pre-determine the gender of offspring before fertilization. Although this technique has reached a commercial level in the bovine species, a routine use of sexed semen in pigs is far from being a reality. In fact, in pigs, one of the most limiting factors is represented by the long sorting time necessary to obtain the adequate number of sexed spermatozoa for conventional artificial insemination. In order to overcome this problem, boar sexed semen has been successfully used for low dose insemination protocols by using laparoscopic insemination [1], non-surgical deep intrauterine insemination [2], [3], [4], or for both IVF and intracytoplasmic sperm injection followed by embryo transfer [5], [6], [7], [8], [9].

Another factor that contributes to limit the application of sperm sorting technology in swine is the susceptibility of boar spermatozoa to stresses induced by the sorting procedure, susceptibility that seems to be higher as compared with bull and ram [1], [10]. Chemical, physical, and electrical insults during the sexing process (Hoechst 33342 staining, high pressure, exposure to the UV laser beam, electrical charging of droplets containing spermatozoa, projection into the collection tube, high dilution, centrifugation) have been reported to induce capacitation-like changes, to shorten the lifespan after sorting and to reduce the fertilizing ability of sexed spermatozoa [11], [12], [13], [14], [15].

A further problem for the large scale use of boar sexed semen is represented by its high susceptibility to damages induced by cryopreservation. Sorted semen can either be stored at 15–17° or frozen, however this last method is still unsatisfactory in pigs as only one litter born has been obtained with non surgical inseminations of sexed frozen boar spermatozoa [16] while in a subsequent study, after achieving a conception rate of nearly 70%, all pregnancies were lost [17]. Until now, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. Before the use of sorted semen will become applicable in pigs under field conditions, sorted sperm survival after storage needs to be improved, defining the damages and then minimizing their sources.

The aim of this study was to evaluate the modification induced by 24–26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, and in vitro fertilizing ability. Hsp70 presence and immunolocalization were also evaluated as a decline of Hsp70 levels has been correlated with a decrease of quality traits of boar semen and, moreover, this chaperonin seems to undergo relocalization after capacitation in bull and pig spermatozoa and to have a role during gamete interaction in these species [18], [19], [20].

Section snippets

Methods

Animals were housed and handled according to EEC animal-care guidelines.

All the reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.

Plasma membrane integrity

Data on membrane integrity of sorted and unsorted spermatozoa pre and after storage are shown in Figure 1. The percentage of live cells was negatively affected (P < 0.05) by both sorting procedure and storage. Moreover the membrane integrity of sorted spermatozoa was significantly decreased after liquid storage for 24–26 h as compared to all the other groups.

Acrosome exocytosis

The results are summarized in Table 1. The percentage of live spermatozoa with intact acrosome (PNA−/PI−) and that of live cells with

Discussion

Storage of boar sexed spermatozoa would be necessary in order to ship it from sorting facilities to recipient females thus stimulating its use on a wider scale; however in this species the quality of frozen-thawed sexed semen is unsatisfactory [16], [17]. For this reason boar sexed semen is usually liquid stored even if sorted spermatozoa rapidly lose their fertilizing ability with prolonged intervals from sorting to insemination. In this study we prolonged liquid storage of boar sorted

Acknowledgements

Work supported by a Bologna University grant (ex 60%). The Authors wish to thank “Società Italiana Produttori Sementi.”

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