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. 2021 Nov 4;10(11):1135.
doi: 10.3390/biology10111135.

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

Jesús L Yániz  1 , Inmaculada Palacín  1 , Miguel A Silvestre  2 , Carlos Olegario Hidalgo  3 , Carolina Tamargo  3 , Pilar Santolaria  1
Affiliations

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

Jesús L Yániz et al. Biology (Basel). .

Abstract

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.

Keywords: Bos taurus; acrosome reaction; capacitation; male fertility; spermiogram.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fluorescence image of bull spermatozoa stained with ISAS3Fun kit (1) and fluorescence-phase contrast image of bull spermatozoa stained with PI/PNA. (2): a, Intact plasma membrane and acrosome; b, intact plasma membrane and damaged acrosome; c, damaged plasma membrane and intact acrosome; d, damaged plasma membrane and acrosome. Scale bar = 10 µm.
Figure 2
Figure 2
Time evolution of sperm membrane and acrosome integrity using the PNA/PI and the ISAS3Fun fluorescence methods after capacitation (4 h) and ionomycin (5 h) treatment of frozen/thawed bull semen samples (a–f).
See this image and copyright information in PMC

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