Sperm Membrane Integrity Research Articles

Synthesis of green zinc-oxide nanoparticles and its dose-dependent beneficial effect on spermatozoa during preservation: sperm functional integrity, fertility and antimicrobial activity.

Introduction: The development of an effective extender is important for semen preservation and the artificial insemination (AI) industry. This study demonstrates the beneficial effect of zinc oxide nanoparticles (ZnO-NPs) as an additive to semen extenders to improve semen quality, fertility, and antibacterial activity during liquid preservation in a boar model. Methods: Initially, to find out the safe concentration of ZnO-NPs in sperm cells, a wide range of ZnO-NP concentrations (0, 5, 10, 50, 100, 500, and 1,000μM) were co-incubated with sperm at 37°C for a cytotoxic study. These NP concentrations were compared to their salt control zinc acetate (ZA) at the same concentrations and to a control group. The effect of the different concentrations of ZnO-NPs on sperm motility, membrane integrity, mitochondrial membrane potential (MMP), and apoptosis was assessed. Accordingly, the non-toxic dose was selected and supplemented in MODENA extender to determine its beneficial effect on the boar semen parameters mentioned and the lipid peroxidation (LPO) levels during liquid preservation at 16°C for 6days. The non-cytotoxic dosage was subsequently chosen for AI, fertility investigations, and the evaluation of the antibacterial efficacy of ZnO-NPs during preservation hours. An antibacterial study of ZnO-NPs and its salt control at doses of 10μM and 50μM was carried out by the colony forming unit (CFU) method. Results and discussion: The cytotoxic study revealed that 5, 10, and 50μM of ZnO-NPs are safe. Consequently, semen preserved in the MODENA extender, incorporating the non-toxic dose, exhibited 10 and 50μM ZnO-NPs as the optimal concentrations for beneficial outcomes during liquid preservation at 16°C. ZnO-NPs of 10μM concentration resulted in a significantly (p < 0.05) improved conception rate of 86.95% compared to the control of 73.13%. ZnO-NPs of 10 and 50μM concentrations exhibit potent antimicrobial action by reducing the number of colonies formed with days of preservation in comparison to the negative control. The investigation concluded that the incorporation of 10μM ZnO-NPs led to enhancements in sperm motility, membrane integrity, and MMP, attributed to a reduction in the malondialdehyde (MDA) levels. This improvement was accompanied by a concurrent increase in fertility rates, including farrowing rate and litter size, during the liquid preservation process. Furthermore, ZnO-NPs exhibited an antimicrobial effect, resulting in decreased bacterial growth while preserving boar semen at 16°C for 6days. These findings suggest that ZnO-NPs could serve as a viable alternative to antibiotics, potentially mitigating antibiotic resistance concerns within the food chain.

Exosomes from porcine serum as endogenous additive maintain function of boar sperm during liquid preservation at 17 °C in vitro

The supplementation of sperm culture media with serum is quite common, and improves both sperm survival and motility. However, the link between serum and sperm remains poorly understood. The present study is the first investigation of the effects on sperm quality and function of endogenous porcine serum exosomes in medium used for culturing boar sperm. Scanning electron microscopy (SEM) confirmed that serum-derived exosomes from both castrated boars (cbsExos) and sows (ssExos) exhibited typical nanostructural morphology and expressed CD63, CD9, and Alix, as shown by Western blotting. At 17 °C, the progressive motility and membrane integrity of sperm were significantly increased after incubation of fresh boar semen for 7 days with cbsExos-4 (8 × 1010 particles/mL) or ssExos-16 (32 × 1010 particles/mL). Moreover, cbsExos-4 and ssExos-16 were found to be effective sperm additives, improving mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) content, total antioxidant activity (T-AOC), superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity while reducing reactive oxygen species (ROS) levels, and malondialdehyde (MDA) content following preservation at 17 °C after a 5-day incubation. Both fluorescence and SEM showed that the serum exosomes bound directly to the sperm membrane, suggesting an interaction that could influence sperm-zona pellucida binding. Overall, this study provides new insights into the potential benefits of adding cbsExos and ssExos to enhance the quality of boar sperm during ambient temperature preservation, which may lead to advancements in sperm preservation strategies.

Fresh Seminal Characteristics of Marwari Stallions in Breeding Season

Preserving the sperm of stallions with significant genetic worth is a widely accepted procedure. Thus, the species' typical reproductive behaviour is altered to facilitate the retrieval of sperm for the use of assisted reproductive biotechnologies, like artificial insemination and cryopreservation of sperm. As the Marwari horse breed is a native breed of our country, the present experiment was designed to define the basic semen parameters of Marwari stallions. The experiment was conducted on six healthy Marwari horses aged between 50 and 140 months for three weeks with a semen collection frequency of twice a week. A total of thirty-six semen samples (six from each horse) were collected using an artificial vagina. Following a macroscopic examination, fresh semen samples underwent a microscopic evaluation. The appearance of fresh semen was milky white or creamy white and the consistency was thin to thick. Fresh seminal pH was 7.54±0.03. Total ejaculated semen volume, gel volume and gel-free semen volume were 67.22±5.77 ml, 14.08±1.98 ml and 53.14±4.16 ml, respectively. Progressive sperm motility of sperm was 78.99±0.65%. Sperm concentration was 195.50±5.67 million/ml. Viability and total morphological abnormalities of sperm were 85.82±0.48% and 4.42±0.23%, respectively. Plasma membrane integrity (HOST), acrosome integrity and DNA integrity of sperm were 53.65±0.85%, 87.36±0.36% and 95.83±0.24%, respectively. A significant difference was observed among Marwari stallions for semen volume (total ejaculated, gel and gel-free) (P&lt;0.05), progressive motility (P&lt;0.05), viability (P&lt;0.05), concentration (P&lt;0.05), plasma membrane integrity (HOST) (P&lt;0.05), acrosome integrity (P&lt;0.05), and DNA integrity (P&lt;0.05) of sperm. Non-significant difference was observed for seminal pH (P&gt;0.05) and total sperm morphological abnormalities (P&gt;0.05).

Mitochonic Acid 5 Increases Ram Sperm Quality by Improving Mitochondrial Function during Storage at 4 °C

Semen preservation involves lengthening sperm’s fertile lifespan without any detrimental effects on its biochemical, functional, and ultrastructural properties. Liquid storage at 4 °C is a ram sperm preservation method. However, this method of storage causes irreversible damage due to cold shocks, osmotic stresses, oxidative stresses, and reductions in sperm metabolism. The present study aims to investigate whether the supplementation of mitochonic acid 5 (MA-5) in a sperm extender could improve chilled ram sperm quality and elucidate its mechanism of action. Ram sperm were diluted with a tris-citrate-glucose extender containing different concentrations of MA-5 (0, 0.1, 1, 10, and 100 nM) and stored at 4 °C for up to 48 h. Sperm motility, membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) level, ATP content, and the expression of NADPH dehydrogenase subunits 1 (MT-ND1) and NADPH dehydrogenase subunits 6 (MT-ND6) were evaluated. It was observed that compared to the control, the 10 nM MA-5 treatment significantly (p &lt; 0.05) increased total motility (82 ± 3.5% vs. 76 ± 5.9%), progressive motility (67.6 ± 8.2% vs. 51 ± 8.3%), and other parameters (straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL)). In addition, 10 nM MA-5 supplementation also improved ram sperm membrane integrity and acrosomal integrity as well increased mitochondrial membrane potential (51.1 ± 0.7% vs. 37.7 ± 1.3%), reduced ROS levels, and elevated adenosine triphosphate (ATP) contents. Furthermore, a Western blot analysis demonstrated that the addition of MA-5 significantly (p &lt; 0.05) increased the expression of MT-ND1 and MT-ND6 proteins in ram sperm, with the 10 nM MA-5 treatment resulting in the highest expression level. These results suggest that MA-5 improves ram sperm quality by maintaining high sperm mitochondrial function during liquid storage at 4 °C.

Effects of organic and inorganic selenium supplementation on frozen-thawed ram semen at two cooling periods.

The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38°C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2h and the other for 4h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1μg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37°C for 20s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5μg/mL ORG, subjected to 4h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5μg/mL SeNa group, under the 2h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Insights into alternative cryoprotectants to freeze sperm of domestic cats.

Globalization and habitat destruction pose a significant threat to wildlife felids. Even though conservation banks for genetic materials have been created, the sperm cryopreservation with minimal cell damage is still a great challenge. Thus, this study aimed to compare the effects of two commercial extenders with different concentrations of alternative cryoprotectants on thawed sperm quality of domestic cats. Five adult cats were anaesthetized (using a combination of 40 μg/kg medetomidine associated to 5 mg/kg ketamine), and the semen was collected by electroejaculation (electrical stimulation of 2-3 V). Semen samples were evaluated for sperm characteristics (kinetics, morphology, membrane integrity and morphometry). Subsequently, they were sorted into two aliquots and centrifuged. The aliquots were added to a commercial extender containing 3% glycerol and 2% methylformamide (extender I) or 2% glycerol and 3% methylformamide (extender II), frozen, thawed (37°C/30 s) and reevaluated. Comparatively, the sperm kinetics and membrane integrity of fresh semen were higher (p < .002) than frozen samples in extender I and II. Total and progressive motility were lowest in the thawed samples. However, the subjective analysis indicated high sperm motility, since the kinetics evaluation was impaired by the low cell number in the thawed samples. There were no differences in sperm morphology between the groups. In the sperm morphometric analysis, a significant difference (p = .04) was identified in the length of the intermediate piece in extender II samples compared with fresh and extender I. Thus, it can be concluded that although the concentrations tested did not maintain the kinetic parameters and membrane integrity of spermatozoa after thawing, the extender with a lower concentration of glycerol was less toxic for maintaining the midpiece length.

Sperm quality and in vitro fertilizing ability of boar spermatozoa stored at 4 °C versus conventional storage for 1 week.

Since boar spermatozoa show a marked deterioration in sperm quality when cooled, insemination doses are usually stored at 16-18 °C. However, maintaining this temperature during transport of semen doses is challenging, particularly during the summer months. An alternative could be to store the doses at 4 °C if cold-shock to the sperm could be prevented. The objective of this study was to evaluate boar sperm quality and fertility in in vitro fertilization after storage in AndroStar Premium at 4 °C for 1 week. Insemination doses (n = 9) in AndroStar Premium from a commercial boar semen collection station were transported to the laboratory at approximately 20 °C. At the laboratory, sperm quality evaluation and was preformed and each dose was split; half of each ejaculate was stored in a climate-controlled box at 16-18 °C, the other was slowly cooled to 4 °C. Both samples were stored for 1 week before further sperm quality evaluation and in vitro fertilization (IVF) were performed. Mean values were tested using generalized linear regression, with treatment and boar as fixed factors; p ≤ 0.05 was considered significant. Sperm membrane integrity (mean ± sem: 91 ± 0.05 and 83 ± 0.09% for 16 and 4 °C, respectively) and superoxide production (6.79 ± 2.37 and 13.54 ± 6.23% for 16 and 4 °C, respectively), were different between treatments. The DNA fragmentation index was lower in cold-stored samples than in conventionally stored samples (3.74 ± 2.25 and 7.40 ± 3.36% for 4 and 16 °C, respectively). The numbers of oocytes developing to blastocyst on Day 6 (mean ± sd: 9.0 ± 8.0 and 6.0 ± 5.0%, for storage at 16 and 4 °C, respectively) were not different between treatments. Therefore, storage of boar semen doses in AndroStar Premium at 4 °C for up to 7 days would be a viable alternative to current praxis.

Viability duration of pacu (Piaractus mesopotamicus) milt stored under refrigeration.

Cooling milt conserves viable spermatozoa to extend the period available for artificial fertilization and avoids the robust protocols and high costs associated with cryopreservation. Yet, the sperm quality curves of fresh and refrigerated milt have not yet been compared for pacu (Piaractus mesopotamicus), which is often used as a biological model. This study aimed to analyze the milt quality of male P. mesopotamicus across 24 h of refrigeration. Six adult males were induced with carp pituitary extract. Sperm movement, membrane integrity, and morphology was compared between extruded milt samples stored for 24 h under either ambient temperature or under refrigeration at 12.63 °C. Sperm motility differed significantly over time. After 24 h of storage, motility values were higher in refrigerated spermatozoa than in those kept at ambient temperature. Sperm cell survival rates did not differ 4-8 h post collection. After 16 h, refrigerated cells showed superior membrane integrity (82.05 ± 4.23%) compared to those stored at ambient temperature (66.98 ± 6.45%), maintaining this pattern up to 24 h. In terms of sperm morphology rate, milt from both treatment groups was still viable for use 8 h after collection. However, after 16 h of storage, both groups exhibited a large reduction in normality rates, and at 24 h, all milt were unfeasible. In conclusion, P. mesopotamicus milt can be stored up to 8 h after collection when refrigerated at 12.63 °C, without the use of extenders and/or cryoprotectants, maintaining enough quality for egg fertilization.

Adding chitosan nanoparticles of green tea extract in diluent and thawing temperatures ameliorate the post-thawed quality of Boer buck semen

Objective: To improve the quality of post-thawing Boer buck semen for artificial insemination by adding green tea extract chitosan nanoparticles to skimmed egg yolk diluent, and the proper thawing temperature. Methods: The ejaculate of Boer buck was added to skimmed egg yolk diluent without (the control group) and with adding 1 μg of chitosan nanoparticles of green tea extract per mL of diluent (the treatment group). Then, the diluted semen was filled in French mini straws containing 60× 106 live sperm per straw, frozen in a standard protocol, and stored as frozen semen at -196 °C for a week. Six replicates from each group were diluted for 30 s at 37 °C or 39 °C sterile water to evaluate the semen quality. Results: Post-thawing (at 37 °C or 39 °C) of live sperm, progressive motility, and plasma membrane integrity were lower compared to those of the pre-freezing stage (P&lt;0.05). Thawing at 37 °C resulted in no significant difference in live sperm, progressive motility, and plasma membrane between the control group and the treatment group (P&gt;0.05). The live sperm, progressive motility, and plasma membrane of the treatment group in the pre-freezing stage, and post-thawed at 39 C were higher compared to those of the control group (P&lt;0.05). There was no significant difference in malondialdehyde (MDA) concentration, DNA fragmentation, and catalase concentration of thawing at 37 °C compared to those of 39 °C in the same group. The MDA concentration and DNA fragmentation in thawing at 37 °C and 39 °C of the treatment group were significantly lower than those of the control group (P&lt;0.05). However, the catalase concentration in thawing at 37 °C and 39 °C of the treatment group was not significantly different than the control group (P&gt;0.05). Conclusions: Higher quality post-thawing Boer buck semen is achieved by adding 1 μg/mL of chitosan nanoparticles of green tea extract to the skimmed egg yolk diluent and thawing at 39 °C.

The effect of different concentrations of laminarin on the quality of cryopreserved ram semen

BACKGROUND: Increasingly, sheep breeders are using artificial insemination to produce lambs, so finding methods that preserve ram sperm can be useful. OBJECTIVE: To determine the protective effects of different concentrations of laminarin on ram sperm motility, viability, abnormalities, membrane, and DNA integrity, superoxide dismutase enzyme (SOD) activity, and malondialdehyde (MDA) production after freeze-thawing. MATERIALS AND METHODS: The ejaculates of four rams were collected and stored at 35 °C. Semen samples were diluted with a tris-base extender containing 100, 200, 400, and 800 μg/mL of laminarin and a control extender containing no laminarin, then frozen in liquid nitrogen after 4 h in the refrigerator. RESULTS: In the treatment of frozen-thawed spermatozoa with 800 μg/mL laminarin, motility, viability, membrane integrity, and DNA integrity were significantly higher than in the control. In spermatozoa that were exposed to 800 μg/mL laminarin after thawing, MDA production was significantly lower than in the control group. The percentage of abnormal spermatozoa in 800 μg/mL laminarin was significantly lower than that in the control. CONCLUSION: The addition of 800 μg/mL laminarin to the freezing extender increases motility, viability, SOD activity, and plasma membrane integrity, while reducing abnormality and MDA production in freeze-thawed ram semen.

Effects of liquid storage of buck semen at refrigeration temperatures on sperm viability and fertility to develop ready to use goat semen diluent

It is generally acknowledged that lower pH throughout liquid storage can decrease sperm motility and energy production, which may be advantageous for maintaining sperm viability during cryopreservation. Hence, the purpose of this study was to see how pH affected sperm viability, motility, membrane integrity, and oxidative changes after 5 days of storage in chemically specified extenders at refrigerator temperatures to formulate a ready-to-use goat semen diluent. Ejaculates were extended with tris-citric acid-fructose diluent with 20% or without egg yolk and stored at 5°C for 5 days. Sperm motility, live count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, pH, and malondialdehyde all substantially differed significantly (P &lt; 0.05) during different days of liquid storage. The pH of diluted semen decreased considerably (P &lt; 0.05) from its original pH of 6.46 on 0 day to pH 6.06 on the 5th day of liquid storage at refrigeration temperature. Diluted semen at refrigeration temperature having 20% egg yolk had significantly higher (P &lt; 0.05) seminal characteristics compared to liquid semen without egg yolk. The liquid storage of semen quality became worse day by day due to a drop in pH and egg yolks acted as a cryoprotectant against the harmful effects of chilling. Hence, 20% egg yolk may be used to enhance the keeping quality of diluted buck semen at refrigeration temperature for up to 5 days. Based on this result, we formulated a ready to use goat semen diluent for liquid semen storage at room temperature and refrigeration temperature.

Effect of Microfluidic and Density gradient techniques to select Frozen-thawed Epididymal Sperm from Bighorn rams.

Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and BovipureTM (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/ml) than DCG (68.42 ±29.18 ×106/ml) and MFD (52.94 ±28.76 ×106/ml). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.

Integrity of Sperm Cell Bali Bull Preserved at 5°C Using a Diluent Formulation of Extracted Red Fruit and Coconut Water

This study aims to determine the role of the best diluent in protecting the integrity of the spermatozoa membrane after storage using an alternative diluent formulation of red fruit extract (Pandanus Conoideus Lamk) and coconut water. An alternative diluent formulation featuring red fruit extract (Pandanus Conoideus Lamk) and coconut water was utilized. Fresh semen from Bali Bull was evaluated in the Integrated Field Laboratory, Department of Animal Science of Universitas Muhammadiyah Gorontalo. Bali bull sperm of high quality was diluted using a combination diluent containing red fruit extract (Pandanus Conoideus Lamk), green coconut water, and CEP-2 mixed with 10% egg yolk. Technical term abbreviations were explained at their initial use. Spermatozoa membrane integrity was assessed using the hypoosmotic swelling test (HOST), while chlortetracycline (CTC) fluorescent dye was used to evaluate spermatozoa capacitation and acrosome reaction. The randomized group design (RAK) was used in the research, consisting of four conditions and 10 replications. P0 served as the control group, which received CEP-2 plus 10% egg yolk. P1 was administered EBM diluent AK enriched with 15% KT, while P2 received EBM diluent AK plus 15% KT and fructose 1 mg/ml. P3 was given EBM diluent AK plus 15% KT and fructose 2 mg/ml. Analysis of variance was used to examine the resulting data. The findings indicated that CEP-2 plus 10% egg yolk diluent was more effective than the diluent formulations containing red fruit extract and coconut water. Spermatozoa with intact membranes and no capacitation remain high, while those with capacitation and acrosome reaction remain low.

Effect of Adenosine 5´ Triphosphate (ATP) and Incubation Time on Frozen Thawed Buffalo Bull Spermatozoa

The present study was aimed to determine the effect of ATP dose rate on longevity, motility, and viability of frozen-thawed buffalo bull’s sperm. For this purpose, 20 frozen semen straws of four different buffalo bulls were obtained from a private semen production unit (Al-Hiawan Sires, Sahiwal). The experiment was replicated four times. In each replicate, four frozen straws of the same bulls were thawed and pooled. After evaluating the motility, the pooled frozen-thawed sample was aliquoted into three separate tubes containing 0 (control), 1-, or 2-mM concentrations of ATP. Afterward, each aliquot was incubated at 37oC for 4 hrs. The sperm motility, viability, and membrane integrity were observed at various time points (10 min., 1hr, 2hr, and 4hr) during the incubation period of four hrs. It was observed that progressive motility and most of the motion kinematic parameters were greater (P&lt;0.05) in control. A significant (P&lt;0.05) interaction was observed between ATP dose and incubation time. The progressive motility, total motility, VCL, VAP, ALH, and BCF decreased (P&lt;0.05) both by incorporation of ATP (1mM or 2mM) and increasing incubation time (from 10 min to 4hrs) which indicate the ATP and incubation time of 4 hours negatively affect the motion kinetics of buffalo bull sperm. In conclusion, it was witnessed that the motility and plasma membrane integrity of sperm declined by the supplementation of ATP in frozen-thawed buffalo bull semen.

Magnetotactic Sperm Cells for Assisted Reproduction.

Biohybrid micromotors are active microscopic agents consisting of biological and synthetic components that are being developed as novel tools for biomedical applications. By capturing motile sperm cells within engineered microstructures, they can be controlled remotely while being propelled forward by the flagellar beat. This makes them an interesting tool for reproductive medicine that can enable minimally invasive sperm cell delivery to the oocyte in vivo, as a treatment for infertility. The generation of sperm-based micromotors in sufficiently large numbers, as they are required in biomedical applications has been challenging, either due to the employed fabrication techniques or the stability of the microstructure-sperm coupling. Here, biohybrid micromotors, which can be assembled in a fast and simple process using magnetic microparticles, are presented. These magnetotactic sperm cells show a high motility and swimming speed and can be transferred between different environments without large detrimental effects on sperm motility and membrane integrity. Furthermore, clusters of micromotors are assembled magnetically and visualized using dual ultrasound (US)/photoacoustic (PA) imaging. Finally, a protocol for the scaled-up assembly of micromotors and their purification for use in in vitro fertilization (IVF) is presented, bringing them closer to their biomedical implementation.

Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol.

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 106 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 106 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 106 (49.9% and 19.7%) and 35 × 106 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

MitoQ preserves the quality and fertility of liquid-preserved ram sperm

Supplementing the semen extender with some antioxidants may preserve sperm quality following liquid preservation. The aim of the current study was to evaluate the influence of the use of MitoQ in the semen extender on quality parameters and fertility of liquid-preserved ram semen. In this study, diluted semen samples were divided into five parts and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ. The prepared samples were stored at 3–5 °C for up to 50 h. Motility, viability, mitochondrial activity, membrane integrity, and malondialdehyde concentration of the chilled sperm were assessed at 0, 25, and 50 h. To evaluate reproductive performance, artificial insemination was performed with semen liquid-preserved for 25 h. In results, at 0 h, no difference between the groups was observed. The use of 10 and 100 nM MitoQ resulted in higher (P ≤ 0.05) total motility, progressive motility, membrane integrity, mitochondrial activity, viability, and lower malondialdehyde concentration than the other groups after 25- and 50-h storage. Pregnancy, parturition and lambing rates were higher (P ≤ 0.05) when ewes were inseminated with 25-h chilled semen samples containing 10 and 100 nM MitoQ compared to the control. Therefore, supplementing the semen extender with MitoQ (10 and 100 nM) could be an efficient method to improve the quality and fertility rate of liquid-preserved ram semen.

Successful cryopreservation in biodegradable containers of sperm from aquaculture Mediterranean fishes

We aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as biodegradable alternative containers to plastic straws in European eel (Anguilla anguilla), gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) sperm cryopreservation. Sperm samples from each European eel (n = 12) were diluted 1:8:1 (sperm: extender P1+5 % egg yolk: methanol). Gilthead seabream (n = 12) samples were individually diluted in a cryoprotectant solution of 5 % Me2SO + NaCl 1 % plus BSA (10 mg mL−1) at a ratio of 1:6 (sperm: cryoprotectant solution). European sea bass (n = 10) sperm from each male was diluted in non-activating medium (NAM) at a ratio of 1:5.7 (sperm: NAM), and 5 % of Me2SO was added. The diluted European eel and sea bass sperm aliquots (0.5 mL) were individually filled in plastic straws (0.5 mL), hard-gelatin, and HPMC capsules (0.68 mL). Gilthead seabream diluted sperm (0.25 mL) were filled in plastic straws (0.25 mL) and identical capsules described. All samples were frozen in liquid nitrogen vapor and stored in a liquid nitrogen tank. Sperm kinetic parameters were evaluated by CASA-Mot software. Sperm membrane integrity was performed using a Live and Dead KIT and an epifluorescence microscope. To quantify DNA damage, the alkaline comet assay was performed and TailDNA (TD-%) and Olive Tail Moment (OTM) were evaluated by CaspLab software. Sperm cryopreservation of the three Mediterranean species in straws, gelatin, or HPMC capsules reduced the kinetic parameters and cell membrane integrity. Generally, the post-thawing samples cryopreserved in straws and capsules did not differ for the kinetic parameters and cell membrane integrity, except for European sea bass sperm, where the samples stored in gelatin capsules showed higher velocities (VCL - 100; VSL - 76; VAP - 90 μm s−1) than the sperm stored in HPMC capsules (VCL - 87; VSL - 59; VAP - 73 μm s−1). The cryopreservation process did not damage the sperm DNA of European eel and European sea bass, regardless of the containers used. On the other hand, gilthead seabream sperm cryopreserved in gelatin (TD - 9.8 %; OTM - 9.7) and HPMC (TD - 11.1 %; OTM - 11.2) capsules showed higher DNA damage than fresh samples (TD - 3.6 %; OTM - 2.7) and the sperm stored in straws (TD - 4.4 %; OTM - 5.2). The hard-gelatin and HPMC biodegradable capsules can be used as an alternative to straws for European eel, gilthead seabream, and European sea bass sperm cryopreservation.

Cushioned and high-speed centrifugation improve sperm recovery rate but affect the quality of fresh and cryopreserved feline spermatozoa

The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints.The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.