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. 2023 Dec 26;14(1):87.
doi: 10.3390/ani14010087.

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation

Junwei Li  1   2 , Juncheng Li  1   2 , Shuaibiao Wang  3   4 , Huiming Ju  1   2 , Shufang Chen  5 , Athina Basioura  6 , Graça Ferreira-Dias  7   8 , Zongping Liu  1   2 , Jiaqiao Zhu  1   2
Affiliations

Post-Thaw Storage Temperature Influenced Boar Sperm Quality and Lifespan through Apoptosis and Lipid Peroxidation

Junwei Li et al. Animals (Basel). .

Abstract

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Keywords: apoptosis; boar semen cryopreservation; oxidative stress; post-thaw storage; sperm survival.

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Conflict of interest statement

Author Shuaibiao Wang was employed by the company DanAg Agritech Consulting (Zhengzhou) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; the collection, analysis, or interpretation of data; writing the manuscript; or the decision to publish the results.

Figures

Figure 1
Figure 1
The line graph shows total sperm motility (TM) and progressive sperm motility (PM) changes of frozen-thawed boar semen affected by post-thaw storage time and temperature. General linear analysis showed interaction between post-thaw storage time and temperature affecting TM and PM. Values of TM and PM were compared among different storage times and temperatures. Data are expressed as the mean ± SEM. The letters a, b, and c denote significant differences between storage times at each storage temperature, p < 0.05. * Indicates differences among storage temperatures at each storage timepoint, p < 0.05.
Figure 2
Figure 2
The line graph shows changes in sperm velocity parameter VCL of frozen-thawed boar semen affected by post-thaw storage time and temperature. General linear analysis showed interaction between post-thaw storage time and temperature affecting VCL. Histograms show changes in sperm velocity parameters (VSL and VAP) and linearity parameters (LIN). Because no interaction between post-thaw storage time and temperature was observed in these parameters, the data were combined to analyze the effect of storage time. VCL: curvilinear velocity (µm/s), VSL: straight-line velocity (µm/s), VAP: average path velocity (µm/s), LIN: linearity of sperm movement (%). Data are expressed as the mean ± SEM. The letters a, b, and c denote significant differences between storage temperatures at each storage timepoint or between storage timepoints, p < 0.05. * Indicates differences among storage temperatures at each storage timepoint in line graph, p < 0.05.
Figure 3
Figure 3
Histograms show changes in sperm linearity parameters (STR, WOB) and vigor parameters (ALH, BCF) during post-thaw storage. Because no interaction between post-thaw storage time and temperature was observed in these parameters, the data were combined to analyze the effect of storage temperature. STR: straightness of the average path (%), WOB: wobble coefficient (%), ALH: amplitude of lateral head displacement (µm), BCF: beat cross frequency (Hz). Data are expressed as the mean ± SEM. The letters a and b denote significant differences between storage temperatures or timepoints, p < 0.05.
Figure 4
Figure 4
The line graphs show changes in sperm viability, percentage of acrosome membrane damage, and mitochondrial membrane potential of frozen-thawed boar semen affected by post-thaw storage time and temperature. General linear analysis showed interaction between post-thaw storage time and temperature in sperm viability and percentage of acrosome membrane damage but not in sperm mitochondrial membrane potential. Data are expressed as the mean ± SEM. The letters a, b, and c denote significant differences between storage timepoints at each storage temperature, p < 0.05. * Indicates differences among storage temperatures at each storage timepoint, p < 0.05.
Figure 5
Figure 5
The line graphs show the changes in early and late sperm apoptosis of frozen-thawed boar semen affected by post-thaw storage time and temperature. General linear analysis showed interaction between post-thaw storage time and temperature affecting both early and late sperm apoptosis. Data are expressed as the mean ± SEM. The letters a, b, and c denote significant differences between storage timepoints at each storage temperature, p < 0.05. * Indicates differences among storage temperatures at each storage timepoint, p < 0.05.
Figure 6
Figure 6
The line graphs show the changes in SPZ MDA, SN MDA, and intracellular ROS levels of frozen-thawed boar semen affected by post-thaw storage time and temperature. General linear analysis showed interaction between post-thaw storage time and temperature affecting SPZ MDA and SN MDA but not intracellular ROS levels. SPZ: frozen-thawed boar sperm, SN: the surrounding environment of frozen-thawed boar sperm, MDA: malondialchehyche. Data are expressed as the mean ± SEM. The letters a, b, and c denote significant differences between storage timepoints at each storage temperature, p < 0.05. * Indicates differences among storage temperatures at each storage timepoint, p < 0.05.
Figure 7
Figure 7
Histograms show the changes in SPZ TOS, SN TOS, SPZ TAC, and SN TAC levels of frozen-thawed boar semen. Because no interaction between post-thaw storage time and temperature was observed in both parameters, the data were combined to analyze the effect of storage time and temperature, respectively. SPZ: frozen-thawed boar sperm, SN: the surrounding environment of frozen-thawed boar sperm, TOS: total oxidant status, TAC: total antioxidant capacity. Data are expressed as the mean ± SEM. The letters a and b denote significant differences between storage timepoints, p < 0.05.
Figure 8
Figure 8
Histograms show the changes in SPZ SOD, SN SOD, SPZ GPX5, and SN GPX5 levels of frozen-thawed boar semen. Because no interaction between post-thaw storage time and temperature was observed in these parameters, the data were combined to analyze the effect of storage time and temperature, respectively. SPZ: frozen-thawed boar sperm, SN: the surrounding environment of frozen-thawed boar sperm, SOD: superoxide dismutase, GPX5: glutathione peroxidase 5. Data are expressed as the mean ± SEM. The letters a and b denote significant differences between storage timepoints, p < 0.05.
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