Artificial insemination in cattle relies on laboratory semen evaluation, yet field fertility often diverges from laboratory-based expectations. This study compared spermatological and computer-assisted sperm analysis (CASA) kinematic traits of commercially frozen–thawed semen from dairy and beef bulls and examined their associations with pregnancy outcome under commercial conditions. Frozen semen from 11 bulls (beef: Angus, n = 5; dairy: Holstein, n = 6) was obtained through the Amasya Cattle Breeders’ Association; six straws from the same batch per bull were analysed and averaged at the bull level. After thawing (37 °C, 30 s), motility, sperm concentration, and kinematic parameters were assessed using CASA under standardized settings. Field inseminations were performed by a single veterinarian in healthy multiparous cows showing spontaneous estrus. A total of 413 inseminations were conducted (beef semen: 194; dairy semen: 219); pregnancy diagnosis was available for 409 inseminations at day 60. Mean daily milk yield (days 5–305) was derived from association records (beef females, n = 194; dairy females, n = 219). Semen from beef bulls showed higher total motility (83.59 ± 2.08% vs. 56.24 ± 2.42%; p < 0.0001) and higher progressive motility (58.54 ± 3.01% vs. 48.25 ± 9.57%; p = 0.0475) than semen from dairy bulls. Dairy bulls had higher STR and LIN and lower ALH, whereas velocity descriptors differed modestly and did not show consistent between-group separation. Mean bull-level pregnancy rate was numerically higher for beef than dairy bulls (68.06% vs. 63.71%), without a significant between-group difference at the insemination level. Daily milk yield differed markedly between female groups (18.47 ± 0.38 vs. 6.21 ± 0.19 kg/d; p < 0.001). Within-group Pearson correlations indicated that motility was positively associated with pregnancy rate in both production types, while several kinematic descriptors exhibited production-type-specific correlation directions. These findings suggest that production context may modulate how CASA phenotypes relate to field fertility, supporting cautious interpretation of single-parameter semen metrics in commercial AI systems.

Cryopreservation of ram semen is an essential tool in assisted reproductive technology; however, oxidative stress generated during the freezing process may compromise sperm quality. This study evaluated the effects of Se and SeNPs on post-thaw sperm quality, PRDX5 expression, and oxidative status in cryopreserved ram semen. In this study, semen samples collected from five mature rams (three collections at 2-week intervals, yielding a total of 15 ejaculates) were frozen in liquid nitrogen using extenders supplemented with selenium (1 μg/mL, S1; 10 μg/mL, S2) or selenium nanoparticles (SeNPs; 1 μg/mL, N1; 2 μg/mL, N2) alongside a nonsupplemented control extender. Post-thaw sperm quality was evaluated using computer-assisted sperm analysis (CASA) for motility, kinematic parameters, viability, membrane integrity (HOST) assays, chromatin condensation assessment, and morphological analysis. Total oxidant status (TOS) measurements and PRDX5 gene expression analysis were performed separately. Low-dose SeNPs (1 µg/mL) significantly improved total motility (55.73 ± 19.01%), progressive motility (25.05 ± 15.34%), viability (57.27 ± 19.30%), HOST-positive spermatozoa (50.87 ± 18.91%), and morphologically normal spermatozoa (88.27 ± 4.10%) compared with the control and high-dose sodium selenite groups (p < 0.05). Chromatin condensation abnormalities were lowest in the SeNP-treated group. S1 and N2 also improved motility and morphology compared with the control; however, the increases were numerically smaller than those observed in the N1 group. In contrast, S2 supplementation showed limited benefit, with values that were similar to those of the control. Morphologically normal spermatozoa were highest in N1, followed by S1 and N2, while S2 and the control exhibited the lowest values (p < 0.05). In contrast, no significant differences were detected in TOS or PRDX5 gene expression among the experimental groups (p > 0.05). These findings indicate that low-dose SeNPs enhance post-thaw sperm functional integrity and cryotolerance without inducing measurable changes in bulk oxidative markers or gene transcription.

In this study, the lack of standardized freezing protocols for sperm cryopreservation in dogs and the limited research on using exosomes in cryopreservation were considered. Additionally, unlike previous studies on sperm cryopreservation, we introduced an innovative approach using the Aqueous Two-Phase System (ATPS) method for exosome isolation. This study aimed to evaluate the sperm-protective effects of adipose tissue-derived mesenchymal stem cell exosomes (MSC-exo) and seminal plasma exosomes (SP-exo) in dog sperm cryopreservation. Ejaculates from six dogs were processed with Tris-based diluents and divided into four groups: MSC-exo, 1.5 % SP-exo, 2 % SP-exo, and control, and frozen. After thawing, sperm motility, viability, membrane integrity, chromatin integrity, morphological integrity, and gene expression levels were analyzed. The results showed that the MSC-exo group had significantly higher total motility (%60.31 ± 6.12), progressive motility (%22.09 ± 3.34), plasma membrane integrity (%66.94 ± 2.24), and viability (%70.88 ± 1.95) compared to the other groups (P < 0.05). Additionally, the normal chromatin packaging rate was highest in the MSC-exo group (%91.33 ± 0.61, P < 0.05). While some improvements were observed in the SP-exo groups, they were not as pronounced as in the MSC-exo group. No significant differences were found in gene expression levels, although an improvement trend was observed in the MSC-exo group. In conclusion, MSC-exo reduced cryopreservation-induced sperm damage and provided overall protection in sperm parameters. These findings suggest that MSC-exo could be a potential biological additive in dog sperm freezing protocols.

Cryopreservation of ram sperm is a cornerstone in reproductive biotechnology but is often accompanied by oxidative damage that compromises post-thaw sperm quality. Curcumin, a natural polyphenol with potent antioxidant properties, may offer protection against cryo-induced injuries. This study evaluated the effects of curcumin supplementation at two concentrations (10% and 20%) in semen extenders on the functional and molecular characteristics of ram spermatozoa. A total of 14 ejaculates were collected from healthy adult rams and randomly allocated into three groups: control (no curcumin), C1 (10% curcumin) and C2 (20% curcumin). Semen samples were cryopreserved using a Tris-based extender, and post-thaw evaluations included total motility, viability (eosin-nigrosin), morphology (head, midpiece, tail abnormalities), membrane integrity (HOST), chromatin integrity (toluidine blue) and PRDX5 gene expression via qPCR. Curcumin supplementation, particularly in the C2 group, significantly improved total motility and viability (p < 0.01), while reducing head and tail morphological abnormalities and chromatin damage (p < 0.05), compared to the control. No significant difference was observed in PRDX5 gene expression among groups (p > 0.05). These improvements are likely attributed to curcumin's antioxidant activity, including ROS scavenging and membrane stabilisation. Curcumin supplementation in semen extenders enhances post-thaw sperm quality in rams by improving functional parameters and maintaining chromatin integrity, without modulating PRDX5 gene expression. These findings support the potential of curcumin as a safe and effective cryoprotective additive in ovine artificial insemination and genetic conservation protocols. Further research combining transcriptomic and proteomic analyses (or approaches), along with validation of post-thaw sperm fertility, is needed to clarify curcumin's effects and its potential in livestock breeding.

Significant deterioration in semen quality is associated with increased male infertility. Alterations in the sperm proteome resulting from infertility are not well understood. The aim of this study was to examine the connection between the proteome and the quality of spermatozoa in fertile and infertile dogs. The research was conducted on 11 male dogs, including 3 German shepherds, 3 Turkish Kangal, 2 Cane Corso, and 3 Golden Retrievers, aged 4 and 6 years (4.81 +/- 0.75). Four of the 11 dogs constituted the infertile study group because of conception failure observed in at least 3 (3-5) matings with different fertile females within 1 year, and seven dogs from different owners constituted the fertile control group. Semen was manually manipulated and collected from all dogs in the presence of a female dog in heat. Ejaculates were used for spermatological examination and protein analysis. Computer-assisted semen analysis (CASA) was used to evaluate semen samples. In spermatological parameters, motility, kinematic parameters, morphology and plasma membrane integrity were evaluated. Gel electrophoresis (SDS-PAGE) was used to analyze proteins in sperm. The semen quality of infertile dogs was significantly lower. Motility, progressive motility, viability and plasma membrane integrity were statistically significantly lower in the infertile group than in the fertile group (P < 0.05). In morphological parameters, acrosome, head, middle part, tail and total defects were found to be statistically significantly higher in sterile dogs than in fertile dogs (P < 0.05). In the study, a total of 8 protein bands with molecular weights (MW) ranging from 11.0 to 245.0 kDa were identified in the protein analysis of semen. Variations in the sperm proteome composition were shown to be dependent on fertility. These proteins were associated with important metabolite pathways. Additionally, correlations of these bands with various spermatological parameters were revealed. The result of the study suggests that seminal plasma proteins play a role in semen quality and may be potential fertility biomarkers.

Sperm cryopreservation is a cornerstone of assisted reproduction and genetic conservation, yet the freeze-thaw process induces substantial structural and functional damage to spermatozoa. Recent progress in the field has positioned extracellular vesicles (EVs) as potential therapeutic tools, primarily because of their capacity to transport functional biomolecules that help counteract the cellular damage caused by cryopreservation. We then examine the types of cellular and molecular damages encountered during sperm cryopreservation and synthesize current evidence on the protective effects of EVs in this context. Particular attention is given to the mechanisms by which EVs may preserve sperm integrity, including membrane stabilization, antioxidant delivery, and modulation of signaling pathways. Despite encouraging findings, challenges remain, including the standardization of EVs isolation methods, optimal dosing, and delivery strategies. We also discuss the need for mechanism-of-action studies and multi-omics approaches to better understand EV-sperm interactions. Finally, future directions are outlined, including EVs engineering and clinical translation. Overall, EVs emerge as promising modulators in cryobiology, with potential to improve sperm preservation outcomes and fertility restoration strategies. This review offers an in-depth examination of EVs, covering the processes involved in their formation, various strategies employed for their isolation, and the techniques used to assess their properties. It also highlights their sources and functional significance in the male reproductive system.

This study investigated the effects of cryopreservation of testicular tissue and epididymal sperm in bulls on spermatological parameters, cell viability, and caspase-3 gene expression, a marker of apoptosis. Epididymal sperm and testicular tissue pieces obtained from testes collected from bulls (n: 15) were cryopreserved and stored in liquid nitrogen (-196°C). Epididymal sperm were frozen in liquid nitrogen vapor using the straw method, while testicular tissue pieces were frozen in cryotubes using a slow freezing protocol with Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) as cryoprotectants. Fresh semen exhibited significantly higher motility, progressive motility, kinematic parameters, and viability values compared to those after thawing (P < 0.05). Higher cell viability was achieved in DMSO-cryopreserved testicular tissues (53.72 ± 6.36) compared to EG-cryopreserved testicular tissues (41.83 ± 7.22) (P < 0.05). Caspase-3 gene expression was determined to be 42.66 ± 98.26-fold increased in DMSO-cryopreserved tissues and 65.05 ± 106.03 fold increased in EG-cryopreserved tissues after thawing. In epididymal sperm, gene expression levels increased by an average of 12.22 ± 34.60 fold compared to fresh samples. These findings suggest that cryopreservation affects cellular integrity and triggers apoptotic pathways and repair mechanisms, highlighting the need to optimize protocols to reduce cryo-induced damage and increase post-thaw viability.

This study aimed to investigate the protective effects of nanoparticle selenium (SeNP) and sodium selenite (SS) on preventing oxidative stress during the freezing process of dog semen. A total of six dogs were used in the study. The ejaculate was collected from dogs three times at different times by massage method. A total of 18 ejaculates were used and each ejaculate was divided in five experimental groups. The experimental groups were designed to tris extender containing no antioxidants control, 1 μg/mL SeNP1, 2 μg/mL SeNP2, and 1 μg/mL SS1 and 2 μg/mL SS2. Extended semen were equilibrated for 1 h at 4°C, then frozen in liquid nitrogen vapour and stored in liquid nitrogen (~−196°C). After thawing, semen samples were evaluated in terms of CASA motility and kinematic parameters, spermatozoa plasma membrane integrity and viability (HE Test), spermatozoa morphology (SpermBlue) and DNA fragmentation (GoldCyto). Antioxidant enzyme activity (glutathione peroxidase; GPX, superoxide dismutase; SOD, catalase; CAT) and lipid peroxidation (malondialdehyde; MDA) were evaluated in frozen–thawed dog sperm. When the results were evaluated statistically, the progressive motility, VCL, and VAP kinematic parameters in the SeNP1 group were significantly higher than the control group after thawing (p < .05). The highest ratio of plasma membrane integrity and viable spermatozoa was observed in the SeNP1 group, but there was no statistical difference found between the groups (p > .05). Although the ratio of total morphological abnormality was observed to be lower in all groups to which different selenium forms were added, compared to the control group, no statistical difference was found. Spermatozoa tail abnormality was significantly lower in the SeNP1 group than in the control and SS2 group (p < .05). The lowest ratio of fragmented DNA was observed in the SeNP1 group, but there was no statistical difference was found between the groups (p > .05). Although there was no statistical difference between the groups in the evaluation of sperm antioxidant profile, the highest GPX, SOD and CAT values and the lowest lipid peroxidation values were obtained in the SeNP1 group. As a result, it was determined that 1 μg/mL dose of SeNP added to the tris‐based extender in dog semen was beneficial on spermatological parameters, especially sperm kinematic properties and sperm morphology, and therefore nanoparticle selenium, a nanotechnology product, made a significant contribution to the freezing of dog semen.

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (-196 degrees C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (-196 degrees C). The total motility (TM) (85.89 +/- 12.83 %), progressive motility (PM) (54.02 +/- 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 +/- 13.13 %), PM (29.60 +/- 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 +/- 10.00) were statistically significantly lower than those frozen with DMSO (51.20 +/- 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 +/- 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 +/- 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 +/- 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

Our objectives were to investigate the effects of exogenous melatonin on testicular volume (TV), testicular blood flow (TBF), and semen quality in Bafra rams during the non-breeding season. One group of rams (MEL, n = 5) received a 36 mg melatonin implant twice, with 30 days in between, while the other group (CON, n = 5) served as the control. TBF, TV, and semen quality parameters were determined at three-week intervals starting three weeks before until twelve weeks after the first melatonin implant. Testicular blood flow was determined in the supratesticular (STA) and marginal testicular artery (MA) using color Doppler ultrasound. Semen was collected and evaluated, and the total oxidative status (TOS) and total antioxidative status (TAS) was determined using an ELISA. The MEL group had increased (p < 0.05) TV between the sixth and twelfth week after the start of treatment. Overall, the MEL group had lower resistance and pulsatility indexes (p < 0.05) between the third and ninth week, although there was no difference (p > 0.05) between the two groups in most semen quality parameters. However, TAS concentrations increased (p < 0.05) in the MEL group compared with the CON. The results of this study show that exogenous melatonin in the non-breeding season significantly increased both TBF and TV in Bafra rams. Therefore, giving rams implants with 36 mg melatonin twice at least one month prior to the non-breeding season is expected to improve testicular size and function and reproductive capacity.

In this study, we aim to show the effect of exogenous melatonin administration on the testis size, testicular hemodynamics, and semen quality in the non-breeding season of Bafra rams. Melatonin implant (36mg/ram) was applied to one group of rams (n=5) and compared to another control group (n=5) with similar sexual maturity (age≌2). Six different measurements and analyses were performed respectively; before melatonin application, the day of drug implementation (wk0) and after exposure in every three weeks (wk3,6,9 &12) for the rest of the study. Testicular morphometric changes were observed and testicular hemodynamics was measured in sub-sections of testicular artery (supra-testicular (STA), marginal (M), intra-testicular (IM) regions) by colored doppler ultrasound in this process. Moreover, semen collection and evaluation were performed at specified time intervals. Total oxidant and antioxidant measurements were determined using ELISA method. Our results showed no significant difference between melatonin treated ram semen volume & concentration and the control group. In contrast, the melatonin group illustrated a significant increase in testicular volume at wk 6, 9, and 12 after melatonin implication (P<0.05). Overall, the weekly difference effect (control group versus melatonin group) showed notable differences in PSV values in STA. Although, the correlation effect between (treatment x time) each group where only statistically significant in EDV values in STA (P<0.05). In conclusion, increasing the potential of exogenous melatonin to benefit from rams out of the breeding season supported the existence of its ability to improve sperm production as well as its contribution to a longer-term benefit for breeders.

Th e purpose of this study was to observe the eff ect of diff erent thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining.A total of 20 frozen sperm straws from the same Holstein bull were used.While the 30 sec thawing protocol at 37°C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70°C constitutes our fast-thawing group (n=10).Th e motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated.Th ere was a significant diff erence (P<0.05) in sperm plasma membrane integrity, head defect, and total abnormal sperm morphology.Th e chromatin decondensation rate detected by TB in bull semen thawed in the slow thaw system, and the decondensation rate in the fast thaw system, diff ered significantly from each other in line with the literature data (P<0.05).According to the evaluations made in terms of chromatin decondensation rate, the rate obtained in slow thawing (7±0.39)shows an increase up to two times compared to the fast-thawing rate (3.3±0.33)(P<0.05).Th e TB staining procedure can be used to evaluate infertility and chromatin integrity, especially in cases that are suspicious and require rapid evaluation.

In this study, 19 mares brought to the clinic at different times with the complaint of not getting pregnant were followed up. For this purpose, changes in the uterine and ovarian dynamics of mares and behavioral changes in estrus were followed and evaluated. In particular, it was attempted to increase the chance and rate of pregnancy by providing ovulation with ovulation follow-up followed by a single dose of hCG administration (Chorulon, 3.000 IU). All manipulations and applications in the study were carried out between March and July, that is, during the breeding season of the mares. During the study, a B-mode real-time ultrasonography device was used for ovary and uterus examinations of the mares. For the insemination of the mares, fresh semen was obtained from breeding stallions by the artificial vagina method and analyzed fresh semen was used. The intracornual insemination method was used as an artificial insemination method. Pregnancy controls were performed twice on the 15th and 30th days after insemination and the pregnancy status was evaluated according to the criteria of the presence of an embryonic sac in cornu uteri. While the average follicle diameter was around 43.7 millimeter (mm) at the stage when estrus symptoms were at their peak, the diameter of the follicle was 36.2 mm in the USG controls performed at the beginning of estrus. Uterine oedema was measured as 1.78 in the initial phase of estrus, 3.84 in the most intense phase of estrus, and 2.36 during insemination during the same periods. The pregnancy rate was found to be 68% (13/19). As a result, it is recommended to administer gonadotropic hormones such as hCG at an appropriate dose (3000 IU) to mares who have trouble conceiving, especially in cases of delayed ovulation.

The purpose of this study was to observe the effect of different thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining. A total of 20 frozen sperm straws from the same Holstein bull were used. While the 30 sec thawing protocol at 37??C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70??C constitutes our fast-thawing group (n=10). The motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated. There was a significant difference (P<0.05) in sperm plasma membrane integrity, head defect, and total abnormal sperm morphology. The chromatin decondensation rate detected by TB in bull semen thawed in the slow thaw system, and the decondensation rate in the fast thaw system, differed significantly from each other in line with the literature data (P<0.05). According to the evaluations made in terms of chromatin decondensation rate, the rate obtained in slow thawing (7??0.39) shows an increase up to two times compared to the fast-thawing rate (3.3??0.33) (P<0.05). The TB staining procedure can be used to evaluate infertility and chromatin integrity, especially in cases that are suspicious and require rapid evaluation.

Sperm morphology evaluation is an important parameter for determining the quality of semen and predicting fertility in rams. Different staining methods have been developed to detect the morphological status of sperm, but there is no optimized protocol, especially for animals yet. This study was designed to compare the results using SpermBlue®, Diff-Quick, and Coomassie Blue stain in the morphological evaluation of epididymal ram semen. In the study, samples collected from a Bafra ram (epididymal sperm/ram) known to have a good breeding history were diluted with Trisbased diluent and frozen. After thawing for each straw, three semen smears were made and stained with SpermBlue®, Diff-Quick, and Coomassie Blue. All morphological parameters were evaluated using a light microscope. 100 spermatozoa were examined randomly and classified according to their characteristics for each slide. In identifying morphological abnormalities, the staining protocols have compared amongst themselves, and no significant difference between DiffQuick and SpermBlue® staining methods was observed. However, significant differences were observed in midpiece abnormalities when SpermBlue® and Coomassie-Blue staining methods were compared, while significant difference was found in total abnormality in SpermBlue® and DiffQuick staining comparison (P <0.05). As a result, all staining methods evaluated can be easily optimized for laboratory conditions and used in the morphological analysis of ram semen.

Embryonic diapause or temporary cessation of embryonic development is a common phenomenon in the plant and animal species. Embryonic diapause is a temporary cessation of the development of embryogenesis in the blastocyst stage and is a reproductive strategy characterized by delayed implantation in the uterus. It is defined in over 130 species of mammals. It occurs obligate or facultative in cases where the development of the embryo from the blastocyst stage to later stages is not appropriate (eg, during environmental conditions or lactation). The embryonic diapause begins with the blastocyst entering the metabolic and proliferative state of silence so reduction or interruption of mitosis in the embryo. When exit from the diapause, reactivation, and blastocyst returns to active metabolism, mitotic activity restarts and with cell proliferation, the implantation process begins in the uterus. Embryonic diapause is a protective phenomenon, it represents an important developmental advantage for species survival and should be evolutionarily maintained.

The freezing and storage of the sperm are used cryopreservation of germplasm in livestock breeding, genetic improvement of indigenous species, preservation of rare races, successful tolerance to environmental changes and international germplasm exchanges. Both the freezing and thawing process causes large changes in the volume of the cell fluid. Spermatozoon removes most of its cytoplasm at differentiation stages and lacks the cytoplasmic component that contains antioxidants that counteract the harmful effect of reactive oxygen species and lipid peroxidation. Therefore, the sensitivity of spermatozoa to lipid peroxidation increases during the freezing and thawing of the sperm, which creates a significant mechanical stress on the cell membrane. Oxidative stress is caused by oxygen and oxygen-derived oxidants, commonly known as ROS, and is known as an imbalance between the ability of biological systems to easily detoxify or repair damaged reagents. Uncontrolled ROS production, which exceeds the antioxidant capacity of seminal plasma, causes oxidative stress that is harmful to spermatozoa. All cellular components, including lipids, proteins, nucleic acids, and sugars, are potential targets of oxidative stress. Antioxidants control the chemical degradation of the substrate caused by oxidation, neutralizing free radicals, thereby it is used to minimize the risk of damage to spermatozoa during cryopreservation.