Quantitative holographic analysis in stallion spermatozoa following cryopreservation

Abstract

This study employs Holographic tomography (HT) to examine structural and biophysical changes occurring during the cryopreservation of stallion sperm. HT is an advanced imaging technique that integrates digital holography with tomography to achieve three-dimensional, quantitative reconstructions of objects without the need for treatment or reporter dyes. By using refractive index (RI) intervals to represent specific structural regions of sperm cells, variations in optical density, surface area, volume, and dry mass across different cryopreservation extenders and donors have been quantified. Three main sperm components, (i) nuclear region, (ii) post-acrosomal region and midpiece and (iii) whole cell were identified and discriminated based on different RI. Our results revealed significant differences in volume of post-acrosomal region and midpiece among stallions as well as between fresh and frozen/thawed sperm, whereas no significant differences were observed between freezing extenders, aligning with our findings on sperm kinetics. A significant stallion-extender interaction underscores the need to personalize the sperm freezing process.

Keywords: Acrosomal integrity; Freezing extender; Holographic tomography; Horse; Refractive index; Sperm freezing.

Fig. 1

Comparison of Kinematic Parameters in Fresch Stallion Sperm. Kinematic parameters of spermatozoa before freezing, including total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm s⁻¹), straight-line velocity (VSL, µm s⁻¹), and average path velocity (VAP, µm s⁻¹), in the eleven stallions enrolled in this study.

Fig. 2

Comparison of Kinematic Parameters Using Different Cryopreservation Extenders. Kinematic parameters of spermatozoa after cryopreservation, including total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm s⁻¹), straight-line velocity (VSL, µm s⁻¹), and average path velocity (VAP, µm s⁻¹), in the eleven stallions studied. Each ejaculate was divided and frozen with four extenders: Spectrum Duo Red (A), BotuCrio (B), INRA Freeze (C), and HF-20 (D).

Fig. 3

Holographic Tomographic Analysis of Stallion Spermatozoon Substructures Based on Refractive Index Values. (a) Whole cell, (b) post-acrosomal region and midpiece, (c) nuclear region and (d) merged tomogram with subcellular structures corresponding to different RI ranges reported with different colors.

Fig. 4

Sperm volume assessment. Sperm volume assessment conducted for three analyzed regions corresponding to different refractive index ranges: (a) Post-acrosomal region and Midpiece, (b) Nuclear region, and (c) Whole cell. The box plots on the left illustrate individual variations in fresh stallion sperm, while those on the right depict extender-related variations in fresh and frozen/thawed sperm, independent of individual variation.

Fig. 5

Distribution of HT Freezability index. FI_HT evaluated for the three analyzed regions corresponding to different refractive index ranges: (a) Post-acrosomal region and Midpiece; (b) Nuclear region; (c) Whole cell. Box plots on the left side represent the analysis across different donors. Box plots on the right side represent the results obtained for all donors under different freezing extenders, compared to the fresh sperm extender (INRA 96).