Research articleEffect of liquid storage on sorted boar spermatozoa
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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2016, TheriogenologyIntra- and interboar variability in flow cytometric sperm sex sorting
2014, TheriogenologyCitation Excerpt :The current staining protocol for boar spermatozoa calls for the incubation of semen samples (200 × 106 sperm/mL) in H-42 at a final concentration of 0.09 mM [6], similar to that customarily used in other species, such as equine [23], canine [13], and bovine [11] species. It has been well demonstrated that an H-42 concentration that is slightly or twofold higher than 0.09 mM is not deleterious to the quality and functionality of sorted boar sperm [24–27] and does not induce genotoxic effects in the sex-sorted spermatozoa [28]. Surprisingly, the increase from 0.09 to 0.135 mM H-42 did not improve the percentage of ejaculates exhibiting a well-defined split and, on the contrary, decreased the percentage due to overstaining, which was observed in some of the semen samples that exhibited a well-defined split using the more common H-42 concentration.