Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls.

Inmaculada Palacín,Carolina Tamargo,Jesús L Yániz,Miguel A Silvestre,Carlos Olegario Hidalgo,Pilar Santolaria

doi:10.3390/biology10111135

Inmaculada Palacín, Carolina Tamargo + Show 4 more

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https://doi.org/10.3390/biology10111135

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Abstract

Simple SummaryThe objective of the present study was to investigate whether differences in bull fertility are associated with variations of sperm quality. Differences between high- and low-fertility bulls were found mainly in parameters related to sperm acrosome integrity when using a new fluorescence method that allows clear and precise detection of the sperm plasma membrane and acrosome: the ISAS3Fun method. It was concluded that the simultaneous assessment of sperm viability and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than the more conventional in vitro sperm quality test. These results may help to predict the breeding soundness of bulls used in artificial insemination, which is important for the dairy industry.The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane–acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Highlights

The prediction of breeding soundness of bulls and of the fertilising ability of semen used in artificial insemination (AI) is important for the dairy industry [1]
Significant differences between high- and low-fertility bulls were observed in acrosome integrity, the proportion of intactintact acroacrosomes and damaged plasma membranes (IADM) sperm and the head width of the IAIM sperm subpopulation (Table 1)
No statistical differences were found for the other sperm quality parameters analysed

Summary

Introduction

The prediction of breeding soundness of bulls and of the fertilising ability of semen used in artificial insemination (AI) is important for the dairy industry [1]. Achieving satisfactory predictions based on the in vitro evaluation of sperm quality remains a challenge and still heavily relies on the simple assessment of sperm motility and morphology [2] Beyond these important parameters, a motile sperm may not be able to fertilise an oocyte if it does not have an intact acrosome or may give rise to a viable embryo if its DNA is damaged. Pisum sativum (edible pea) agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA) are the most commonly used lectins for this purpose [5] They bind to glycosidic residues in different parts of the acrosomal membrane and, being conjugated with a fluorochrome such as fluorescein isothiocyanate (FITC), sperm with damaged acrosomes may be visualised. The use of acidotropic probes, with an affinity for organelles such as the acrosome, has been shown to be ineffective due to non-specific (LysoSensorTM Green DND-189) or heterogeneous

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