Sperm Cryosurvival Research Articles

The effect of myo-inositol antioxidant activity on human sperm parameters and DNA damage in ultra-rapid and conventional freezing methods

Male fertility preservation is still challenged by cell damage induced during sperm cryopreservation and impaired sperm structure and function. Sperm ultra-rapid freezing, despite a higher protective effect compared to conventional freezing method, is still associated with suboptimal sperm cryosurvival and needs to be modified to increase its efficiency in sperm protection. Sperm freezing media supplemented with antioxidants can improve sperm parameters following freezing-warming process. In this study, we aimed to investigate the effect of employing ultra-rapid freezing and myo-inositol on sperm cryosurvival. Thirty semen samples with normal sperm parameters were collected and each one was divided into four portions to cryopreserve by conventional freezing, ultra-rapid freezing, conventional freezing + myo-inositol 2 mg/ml, and ultra-rapid freezing + myo-inositol 2 mg/ml. Sperm samples warmed after at least 24 h of freezing and sperm cryosurvival were analyzed by evaluation of sperm motility, viability, morphology and DNA fragmentation index (DFI). Freezing method had a significant influence on post-thaw sperm DFI and morphology (p < 0.05) and the interaction between freezing method and antioxidant supplementation significantly affected sperm morphology (p < 0.05). The highest percentage of sperm normal morphology and minimal DFI was achieved using ultra-rapid freezing supplemented by myo-inositol antioxidant compared to other groups (P < 0.05). The highest sperm DNA damage after freezing-warming was observed following the conventional freezing method. In conclusion, sperm freezing method was identified as factor strongly influencing sperm DFI and morphology after thawing/warming. Sperm samples can be rapidly frozen using the modified freezing media supplemented by myo-inositol without impacting sperm DNA and morphology.

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.

Cooling rate modifies the location of aquaporin 3 in spermatozoa of sheep and goat

The freeze-thawing process induces osmotic changes that may affect the membrane domain location of aquaporins’ (AQP) in spermatozoa. Recent studies suggest that changes in AQP3 localization allows better sperm osmo-adaptation, improving the cryoresistance. Ultra-rapid freezing is an alternative cryopreservation technique that requires less equipment than conventional freezing, and it is faster, simpler and can be used in the field. This study aimed to determine the influence of freezing-thawing rates (slow (control) vs. ultra-rapid) on AQP3 expression and location in the spermatozoa from small ruminants (sheep and goats) and its relationship with sperm cryo-damage. Spermatozoa were collected from 10 Merino rams and 10 Murciano-Granadina bucks. The presence and distribution of AQP3 were assessed by Western blotting and immunocytochemistry (ICC), employing a commercial rabbit polyclonal antibody. Sperm motility was CASA system-analyzed, and membrane and acrosome integrity assessed by fluorescence (PI/PNA-FITC). Western blotting did not detect a significant effect of freezing-thawing rate on the amount of AQP3 while ICC found freezing-thawing rate affecting AQP3 location (P < 0.05). In both species, the percentages of spermatozoa showing AQP3 in the post-acrosome region, mid-piece, and principal piece of the tail were greater in samples cryopreserved by slow freezing-thawing (control) than ultra-rapid freezing-thawing rates (P < 0.05). Spermatozoa cryopreserved using ultra-rapid freezing-thawing showed decrease motility, plasma membrane, and acrosome integrity (P < 0.05), which might be related, at least in part, to a lower expression of AQP3. In conclusion, the cooling rate modifies the location of AQP3 in spermatozoa of sheep and goat, which might be associated with sperm cryosurvival.

Effect of storage and single layer centrifugation before cryopreservation on stallion sperm cryosurvival

The objectives of this study were to evaluate the effect of a short, cooled storage before cryopreservation on sperm progressive motility (PM) and compare the effect of different centrifugation methods on post-thaw PM of stored samples. Semen was diluted in chilling extender and aliquoted in 6 protocols: i) Standard centrifugation (SC) followed by freezing; ii) Single Layer Centrifugation (SLC) followed by freezing; iii) Storage for 8 h/5 °C before SC; iv) Storage for 8 h/5 °C before SLC; v) Storage for 8 h/15 °C before SC; and vi) Storage for 8 h/15 °C before SLC. PM was assessed before centrifugation, after centrifugation, and post-thawing. Stallions were classified as “good freezers” (GF) or “bad freezers” (BF). The PM in samples immediately frozen was greater than in the stored ones (71.98 ± 14.2, 52.91 ± 17.8, 53.93 ± 18.9 for no storage, 5 ºC storage and 15 ºC storage, respectively) (P˂ 0.0001). There was an effect of storage condition (p ˂ 0.0001), centrifugation method (p ˂ 0.0001), and freezability (P=0.0016), with an interaction between them (P= 0.0004), on PM after centrifugation. Post-thaw PM was greater in samples treated by SLC than in samples processed by SC, for all storage conditions (p ˂ 0.05). All BF stallions ‘showed post-thaw PM ˂ 30 % when samples were previously stored. Storage at 5 ºC or 15º C for 8 h maintains an appropriate quality in GF stallions. Applying a sperm selection technique as SLC is suggested to improve post-thaw motility, allowing GF straws to be frozen after storage, although BF semen should be prepared by SLC immediately after collection.

Apigenin supplementation substantially improves rooster sperm freezability and post-thaw function

This pioneering research investigated apigenin potential to augment rooster sperm cryosurvival in an extender model. Apigenin is a natural antioxidant flavonoid showing promise for improved post-thaw sperm function. However, its effects on avian semen cryopreservation remain unexplored. This first study supplemented rooster sperm Lake extender with 0, 50, 100, 200, 400 μmol/L apigenin to determine the optimal concentrations for post-thaw quality. Supplementation with 100 μmol/L apigenin resulted in significant enhancements in total motility (from 41.5% up to 71.5%), progressive motility (18.1% to 29.1%) (p < 0.05), membrane integrity (40% to 68%), mitochondrial function (p < 0.001), viability (37% to 62%) and total antioxidant capacity (p < 0.001) compared to the control. It also substantially reduced percentages of abnormal morphology, reactive oxygen species and apoptosis (p < 0.001). Although 200 μmol/L apigenin significantly enhanced some attributes, effects were markedly lower than 100 μmol/L. Higher doses did not improve cryoprotective parameters. This indicates 100 μmol/L as the optimal apigenin concentration. This represents the first report of apigenin protecting rooster sperm from cryodamage. The natural antioxidant improved post-thaw sperm quality, likely by suppressing oxidative stress and apoptosis. Apigenin shows promise for enhancing rooster sperm cryosurvival.

Freezing Stallion Semen-What Do We Need to Focus on for the Future?

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation.

Effects of Nobiletin supplementation on the freezing diluent on porcine sperm cryo-survival and subsequent in vitro embryo development

Nobiletin (NOB) is a bioflavonoid compound isolated from citrus fruit peels. The present study aimed to elucidate whether NOB facilitates the porcine sperm cryosurvival and embryo development after in vitro fertilization (IVF). To this end, spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 50, 100, 150, and 200 μM Nobiletin. The kinematic patterns of frozen-thawed (FT) sperm were assessed after 30 and 90 min incubation using a Sperm Class Analyzer (SCA). Viability, acrosome integrity, and mitochondrial membrane potential (MMP) were measured by fluorescence microscopy 30 min after thawing using SYBR-14/PI, PSA/FITC, and R123/PI, respectively. Lipid peroxidation was determined using MDA assay after incubation for 90 min. The addition of 100 μM and 150 μM NOB to the extender significantly improved sperm progressive motility, and acrosome integrity compared to the control group (P < 0.05). The proportion of viable spermatozoa was significantly higher in the 150 μM NOB group. MDA levels were less in 50 μM and 150 μM NOB treated groups compared to the control. In addition, IVF with FT sperm was used to assess the embryo developmental competence. Treatment with 150 μM NOB before cryopreservation increased the cleavage and blastocyst formation rates compared to the control group. Furthermore, the relative expression of POU5F1 and AMPK, genes related to pluripotency and cell differentiation were significantly upregulated in embryos resulting from NOB-treated sperm compared to the control group. These results suggest that Nobiletin is a functionally novel phytochemical to mitigate oxidative stress during the freezing-thawing of porcine spermatozoa as reflected by improved FT sperm quality and IVF outcome.

Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Soybean lecithin and cholesterol-loaded cyclodextrin in combination to enhances the cryosurvival of dairy goat semen

The objective of the study was to examine the effect of soy lecithin (SL) and cholesterol loaded cryclodestrin (CLC) on cryo-survival of sperm cryopreserved in the presence or absence of seminal plasma in Saanen dairy goats. Tris-based dilutions containing various concentrations of SL (0, 0.5%, 1.0% or 2.0%) and CLC (0, 2.0 g/L, 4.0 g/L or 6.0 g/L CLC) were used to cryopreserve Saanen dairy goat sperm. The quality of frozen-thawed sperm, including progressive motility, viability, acrosome and plasma membrane integrity, as well as fertility were detected. Results found that the optimal combination of the two cryoprotectants was 1.0% SL+4.0 g/L CLC, which significantly increased progressive motility, viability, acrosome and plasma membrane integrity of frozen thawed sperm. The impact of the two cryoprotectants in combination was not affected by the presence of seminal plasma. The conception rates obtained after artificial insemination using sperm cryopreserved with and without seminal plasma were 88.89% and 91.67% (P > 0.05), respectively. The respective values for average number of litter sizes were 1.55 ± 0.17 and 1.56 ± 0.21 (P > 0.05). Therefore, this study improved the cryopreservation efficiency of goat semen, enhanced the sperm cryosurvival, and layed a foundation for the wide application of frozen goat semen.

Evaluation of Post-Thaw Swim-Up Selection Combined With Glutathione Addition for Improvement of Sperm Chromatin Maturity in Normozoospermic Samples.

Cryopreservation of human semen alters the spermatozoal structure, resulting in a reduction in sperm function parameters. Various antioxidants may be able to slow or prevent this type of injury. Glutathione (GSH) has numerous antioxidant properties; supplementing the semen with GSH before freezing may assist in the restoration of post-thaw sperm functionality. To investigate the effect of adding 5mM of glutathione before freezing on human sperm cryosurvival. Semen samples were collected from 30 patients (22 normozoospermic and 8 asthenozoospermic) after 3-5 days of sexual abstinence. Following liquefaction, macro- and microscopic examinations were performed. The samples were then divided into two equal aliquots: the first aliquot received 5 mM of glutathione before freezing, and the second aliquot was considered the control (without glutathione). The samples were frozen using the rapid cryopreservation method and then preserved for 7-10 days in a liquid nitrogen tank before being thawed. Thawed samples from each group were examined microscopically according to the WHO 2010guidelines. Aniline blue was used to assess the maturity of the DNA. Then, the direct-swim-up technique was applied to thawed samples from both groups to select the best sperm quality. The cryopreservation process negatively impacts all sperm characteristics in both groups. Sperm DNA integrity decreased. Glutathione addition before freezing decreased sperm chromatin immaturity (SCI) percent compared to the control (22.98 ± 0.83, 20.79 ± 0.56). The post-thaw performance of the swim-up technique resulted in a select sperm population with high progressive motility (p =0.04) andan improvement in DNA integrity in the treated group versus the control group after thawing. The DNA integrityin normozoospermic samples was improved by adding 5 mM glutathione before freezing. Performing the swim-up technique after thawing had no effect on sperm quality in asthenozoospermic patients.

ROLE OF SELENIUM AND VITAMIN E IN LACTOSE BASED EXTENDER ON SEMEN CRYOPRESERVATION OF BUFFALO BULL (BUBALUS BUBALIS)

&#x0D; &#x0D; &#x0D; This study was designed to evaluate the supplementation of various concentrations of selenium and vitamin E and combinations in lactose egg-yolk-glycerol based extender on cryosurvival of buffalo bull semen. The semen of four healthy donor bulls and ejaculates (n= 80) were processed and pooled. The collected semen was extended with the experimental extenders and then frozen. The frozen straws of semen were thawed and analyzed for motility, viability, acrosomal integrity and functional integrity of sperm. The study indicated that combined use of selenium and vitamin E @ 2μg/ml + 0.75 mg/ml supplemented in extender had significantly (P&lt;0.05) increased sperm motility, viability, acrosomal integrity and functional membrane integrity. Selenium (2μg/ml) in freezing extender also improved semen cryosurvival. The provision of selenium (3μg/ml) alone and in combination with Vitamin E (1 mg/ml) did not provide any beneficial impact on sperm cryosurvival. It was concluded that addition of selenium and vitamin E alone in fructose lactose egg yolk glycerol extender increased the cryosurvival of buffalo sperms.&#x0D; &#x0D; &#x0D;

Cryopreservation of Yak Semen: A Comprehensive Review.

An urgent need to boost the sustainability and efficiency of animal production exists, owing to the growing global population. Enhancing the global fertility of animals, especially cattle, is essential to ameliorate this issue. Artificial insemination and sperm cryopreservation have a considerable and favorable influence on the quantity and quality of the cattle produced. Sperm cryopreservation is crucial for livestock production because it promotes and accelerates genetic diversity and the worldwide dispersion of animals with enhanced genetics. Owing to the importance of cryobiology in reproductive technologies, researchers are developing new approaches, and they are testing cryoprotectant drugs to enhance sperm cryosurvival. However, the viability of sperm after freezing is low and widely varies across breeding yaks. These faults are crucial because they impede advances in reproductive biotechnology and the study of mammalian gametes at a fundamental level. Using chemicals, researchers have developed and enhanced various extenders with varying degrees of efficiency to reduce cryodamage and oxidative stress. In this article, we review the cryopreservation of yak semen, the development of extenders, the difficulties faced during cryopreservation, and the evaluation of semen quality using various methodologies. This review might be helpful for researchers exploring semen cryopreservation in the future, as demand for enhanced cryopreservation exists to boost the post-thaw viability and fertility of sperm.

Time and dose-dependent effect of preconditioning with sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) on post-thaw semen quality of Karan-Fries (KF) bulls.

A novel strategy, focused on the induction of sub-lethal oxidative stress to optimize sperm cryosurvival, has been used before cryopreservation. The present study compared the effect of preconditioning with various concentrations of nitric oxide-donor (sodium nitroprusside, SNP) and peroxynitrite-generator (3-morpholinosydnonimine, SIN-1) on in vitro sperm functions and lipid peroxidation status (LPO) of cryopreserved Karan-Fries (KF) crossbred bull semen. To optimize the concentration of additives, spermatozoa obtained from 36 ejaculates were supplemented with different concentrations of SNP (0.01, 0.05, 0.1μM) and SIN-1 (80, 160, 200μM) versus control in the extender. The post-freezing sperm motility and viability were greater (p < 0.05) in 0.1μM SNP and 80μM SIN-1 in comparison to other concentrations used. Furthermore, the spermatozoa obtained from 48 ejaculates were supplemented with 0.1μM SNP and 80μM SIN-1 in the extender. A significant increase (p < 0.05) was observed in progressive motility, viability and membrane integrity in SNP and SIN-1 treated extender at 24h, 15days, and 2-month post-cryopreservation (PC) periods. There was no significant difference in sperm abnormality in the extended groups and the control group. The seminal plasma of SNP-treated extender had less (p < 0.05) lipid peroxidation as compared to SIN-1 treated and control groups. In post-thaw semen, both SNP and SIN-1 showed a higher (p < 0.05) proportion of acrosome intact (FITC-PNA) sperm with a greater decrease (p < 0.05) in membrane scrambling and lipid peroxidation. SNP and SIN-1 improved (p < 0.05) the proportion of sperm with higher mitochondrial membrane potential (Δψm) as compared to the control. In conclusion, it seems that the preconditioning of SNP and SIN-1 at lower doses may have beneficial effects on post-thawed crossbred bull sperm quality.

Effect of Melatonin and Caffeine Supplementation to Freezing Medium on Cryosurvival of Peruvian Paso Horse Sperm Using a Two-Step Accelerating Cooling Rate.

This research examined the antioxidant and cryoprotective effects of melatonin (ME) and caffeine (CAF) supplementation in freezing medium on the cryosurvival of Peruvian Paso horse sperm using a two-step accelerating cooling rate. Twenty ejaculates from four adult and fertile stallions were recovered, initially diluted with INRA-96®, and finally frozen with INRA-Freeze® with either no supplementation (as control), 1 μM ME, or 2 mM CAF using a two-ramp freezing system content inside a cryogenic-box and liquid nitrogen vapors. The sperm kinematic parameters and integrity of the plasma and acrosomal membranes of fresh semen and cryopreserved samples were evaluated using the CASA system (SCA-Evolution® 2018) and PI/fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin double fluorescent test, respectively. The oxidative stress of post-thaw sperm samples was also assessed using the CellRox Deep Red fluorescence test. The results showed that curvilinear velocity and average-path velocity were greater (p < 0.05) after freezing with CAF than the control group. In addition, there were significance differences (p < 0.01) between stallions (1-4) in post-thaw kinematic parameters regardless of ME or CAF addition. Both ME and CAF improved (p < 0.05) the proportion of sperm with intact plasma membranes and intact acrosomes. Nevertheless, neither CAF nor ME improved the oxidative stress after the cryopreservation process.

CRIOPRESERVACIÓN DE ESPERMATOZOIDES DE GALLO DE COMBATE: EFECTO DE DIFERENTES CONCENTRACIONES DE GLICEROL

Glycerol (Gly) is the cryoprotective agent (CPA) most widely used to cryopreserve rooster semen. However, its cryoprotection, toxicity, and efficacy may vary in different breeds of roosters (e.g.,fighting rooster). In this sense, this investigation evaluated the effect of different concentrations of Gly added to the Lake-Ravie extender on the kinetic variables and plasma membrane integrity (PMI, equivalent to viability) of rooster spermatozoa. A total of 42 semen ejaculates from 6 Spanish fighting roosters (collected in 7 weekly sessions) by dorsal massage technique were used to conform 7-pools. Each pool was divided into 6-aliquots and then 6 treatments were formed according to Gly concentrations: 0 (control), 2% (Gly-2), 4% (Gly-4), 6% (Gly-6), 8% (Gly-8), and 10% (Gly10). The samples were frozen in static liquid nitrogen vapors, and their post-thaw sperm quality was analyzed using the CASA system (SCA-2018®) and Fluorescence (PI). Results after thawing showed that total (MT, %) and progressive (MP, %) motilities were higher (P&lt;0.01) with the Gly-8 treatment compared to the control group and the Gly-4 and Gly-6 treatments. Regarding the postthaw kinetics, the oscillation (WOB, %) and the beat-cross frequency (BCF, Hz) were higher (P &lt; 0.05) in the Gly-6, Gly-8, and Gly-10 treatments compared to their control. Finally, the PMI (%) was greater with the Gly-8 treatment compared to the Gly-2 treatment (P&lt;0.05) and the control group (P&lt;0.01). In conclusion, the addition of 8% glycerol to the freezing medium produced better sperm cryosurvival based on higher kinetics and integrity of the plasma membrane of fighting rooster spermatozoa.

Alginate encapsulation of stallion sperm for increasing storage stability

The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calcium chloride, and hardening (10 min). The first procedure resulted in structures with sperm residing in a liquid core surrounded by a spherical alginate shell, whereas the second procedure resulted in sperm embedded in solid beads of alginate matrix. It was found that use of calcium for alginate gelation resulted in decreased sperm motility as compared to using barium, and that encapsulation in solid beads had a negative impact on sperm plasma membrane intactness. Percentages of membrane intact sperm in barium-alginate core-shell structures were similar as found for ordinary diluted sperm, and did not change during 4 d storage at 5 °C. Sperm motility was reduced after direct recovery from core-shell structures, however, remained stable during 4 d storage leading to similar values as found for un-encapsulated sperm at this time point. Cryosurvival of sperm encapsulated in solid beads or core-shell structures was found to be lower compared to that of ordinary diluted sperm.

Increasing the Yield and Cryosurvival of Spermatozoa from Rhinoceros Ejaculates Using the Enzyme Papain.

Simple SummaryEfficient collection and cryosurvival of semen from threatened wildlife species is key for the success of artificial reproductive technologies (ARTs). The viscous nature of ejaculates often collected from species such as rhinoceros, elephant, hippopotamus and primate, render the majority of spermatozoa collected useless and is therefore wasted. The enzyme papain has been used to reduce the viscosity of camelid semen but has yet to be tested in wildlife species. Therefore, the current study investigated the ability of papain to improve the yield and quality of spermatozoa collected from viscous fractions of the rhinoceros ejaculate during cryopreservation. Papain increased the quantity of useable spermatozoa collected from ejaculates, as well as the motility prior to freezing. It also improved the post-thaw motility, velocity, linearity and straightness of samples compared to spermatozoa frozen from the sperm-rich fraction of the ejaculate. There was no detrimental effect on membrane characteristics or DNA integrity. These results show that treating rhinoceros ejaculates with papain is able to salvage valuable spermatozoa and improve survival post-thaw, ultimately increasing the success of ARTs and creation of biobanks for the maintenance and survival of threatened species.The preservation of rhinoceros semen is vital for captive breeding programs. While successful collection and cryopreservation of rhinoceros semen has been reported, the volume and quality of semen produced is often low due to the high viscosity associated with ejaculates collected via electroejaculation. Reducing semen viscosity would enable access to previously unusable spermatozoa from viscous fractions and could improve quality post-thaw. The enzyme papain successfully reduced the viscosity of camelid semen but has yet to be tested in wildlife species. This study assessed the influence of papain on the in vitro quality of rhinoceros spermatozoa during cryopreservation using advanced semen assessment. In experiment 1, the motility of spermatozoa from the viscous fraction of an ejaculate, either untreated or treated with papain and its inhibitor E-64 prior to cryopreservation, was assessed post-thaw. In experiment 2, spermatozoa from papain-treated viscous fractions were compared to spermatozoa frozen from untreated sperm-rich fractions pre-freeze, as well as after 0, 1.5 and 3 h of incubation post-thaw (37 °C). Papain significantly increased the quantity of spermatozoa collected from ejaculates, as well as the motility prior to freezing. Papain also improved the post-thaw motility, velocity, linearity and straightness of samples compared to sperm-rich samples, with no detriment to sperm viability, lipid membrane disorder, production of ROS or DNA integrity (p < 0.05). Results show the benefit of supplementing rhinoceros spermatozoa with papain prior to cryopreservation on sperm cryosurvival and demonstrates the potential of using papain to improve the success of cryopreservation protocols, not only for the rhinoceros, but also for other wildlife species.

Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01μM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated thatthe abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa

Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen using three commercial semen extenders. Sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated before and after freezing for each aliquot. In addition, the percentage of ASA-bound sperm was evaluated post-thaw. In Experiment 2, semen from 22 stallions was frozen immediately after collection a single formulation of semen extender. Sperm motility and ASA binding were evaluated post-thaw. The results of both experiments showed similar findings. The frequency of ASA-positive samples was higher among stallions with poor sperm cryosurvival (Exp. 1 and 2 = 6/11, 54.5%) than for good sperm cryosurvival (Exp. 1 = 0/16, 0%; Exp. 2 = 1/11, 9.1%). The percentage of IgG- and IgA-bound sperm was also higher in stallions with poor sperm cryosurvival in both experiments (P < 0.05). Post-thaw sperm motility, velocity and distance parameters were lower in ASA-positive than ASA-negative stallions (P < 0.005). No effect of the semen extender used was observed. In addition, stallions with ASAs had a higher percentage of apoptotic sperm than stallions without ASAs. The presence of sperm-bound ASAs was associated with poor cryosurvival for stallion spermatozoa. Thus, it may be beneficial to evaluate stallions for binding of ASAs prior to freezing to offer and indicator of the prognosis for cryosurvival.

Effects of different ultrastructures of lecithin on cryosurvival of goat spermatozoa.

This study was to evaluate the effects of two different ultrastructures of lecithin including nanoparticles (NPE mostly nanomicelles) and lecithin nanoliposome (NLE) with egg yolk extender (EYE) on goat sperm cryopreservation. Semen samples were collected from 6 goats, then pooled, diluted and then frozen. Motility and motion parameters, plasma membrane integrity and functionality, morphology, apoptosis status (Annexin V-PI), acrosome integrity, DNA fragmentation and in vitro fertilisation were assessed. Total motility and most motion parameters were higher in EYE (p<.05) compared with the two lecithin extenders, while there were no significant differences between NLE and NPE. NLE and NPE had higher values for viable spermatozoa (Annexin V-PI) (p<.05) compared with EYE. The highest value for dead spermatozoa was observed in EYE (p=.08). A higher percentage of DNA fragmentation (p<.05) was detected in EYE compared with NPE. Plasma membrane integrity and functionality, morphology, acrosome integrity and fertility of spermatozoa indicated no significant differences between extenders. Data suggested that ultrastructural changes of lecithin (micelles versus. liposome) could not improve the sperm cryosurvival of goat spermatozoa. Moreover, we cannot also claim that lecithin-based diluent supplies better protection compared with the egg yolk in goat.