Pentoxifylline treatment as a safe method for selecting viable testicular spermatozoa before cryopreservation of a small numbers of spermatozoa in azoospermia individuals.

BACKGROUND: Single sperm cryopreservation (SSC) is a specific technique especially used in individuals with small numbers of sperm who suffered from non-obstructive azoospermia (NOA). Testicular specimens possess poor motility and low population of viable spermatozoa. Therefore, sperm selection methods such as applying pentoxifylline (PTX) may improve motility in these cases. The main aim of this study was to evaluate the protective effects of PTX on testicular spermatozoa before and after performing SSC. METHODS: Thirty testicular samples were obtained from men with azoospermia. This study was conducted in two phases. Phase 1 evaluated the effect of PTX for sperm selection before SSC. Twenty testicular samples were divided to two experimental groups: SSC without (I) and with PTX treatment (II). For PTX treatment spermatozoa were incubated with PTX at 37°C for 30 min and only motile spermatozoa were selected for SSC. In phase 2, ten testicular samples were cryopreserved with SSC and warming procedure was carried out in droplet with and without PTX. Motility and viability rates, morphology by motile sperm organelle morphology examination (MSOME), DNA fragmentation by sperm chromatin dispersion test (SCD) and mitochondrial membrane potential (MMP) were evaluated. RESULTS: In phase 1, post warm motility rate was higher in PTX exposed group compared to the unexposed group (25.6 ± 8.13 vs. 0.85 ± 2.1) (p > 0.00). Recovery rate, viability and morphology were not significantly different between groups. DNA integrity and MMP were also similar between both groups. In phase 2 although motility increased in PTX group compared to without PTX group (29.30 ± 12.73 vs. 1.90 ± 2.64) (p > 0.00), the viability rate was not different (70.40 ± 12.12 vs. 65.30 ± 11.87). All above mentioned parameters were similar between the two SSC groups. CONCLUSIONS: Supplementation of testicular spermatozoa with PTX before cryopreservation increases motility and did not have adverse effects on viability, morphology, DNA integrity and MMP. PTX could be used as sperm selection method before single sperm cryopreservation, but PTX could not maintain motile the most of viable testicular sperms.

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes).

Japanese medaka (Oryzias latipes) has been used as a model organism in different research fields, including reproductive physiology. Sperm motility is the most important marker for male fertility in fish and, thus, reproduction success. However, because of small volume of ejaculate and short motility duration, it is still challenging to manage the sperm collection and analysis in small model fish. In the present study, we aimed to investigate sperm motility and to optimize sperm collection, short-term sperm storage, and cryopreservation in Japanese medaka (Oryzias latipes). Using two different approaches for sperm collection: testes dissection and abdominal massage, different housing conditions and activating the sperm with different activation solutions, we investigated immediate sperm motility. In the second part of this study, we used different osmolalities of immobilization solution, Hank's Balanced Salt Solution (HBSS) for sperm storage at 0, 2 and 3 h after sperm collection. Finally, the sperm were cryopreserved using methanol as cryoprotectant and HBSS as extender at two different osmolalities, and post-thaw sperm motility was investigated. The highest post-activating sperm motility was achieved in the groups activated by the extender at 300 mOsm/kg. The quality of sperm remained unaffected by co-housing with females or with males only. Furthermore, Hanks' Balanced Salt Solution (HBSS) with an osmolality of 600 mOsm/kg demonstrated its efficacy as a suitable extender for sperm storage, preserving motility and progressivity for 3 h. The highest post-thaw motility was around 35%. There were no significant differences between post-thaw motility in different groups. We also found that post-thaw incubation on ice can maintain the motility of the sperm for up to one hour after thawing.

Investigation of the protective effects of different forms of selenium in freezing dog semen: Comparison of nanoparticle selenium and sodium selenite.

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.

Effect of autologous platelet-rich plasma on the fertility and quality of cryopreserved buffalo bull semen: a comparative study using OptiXcell® and tris egg yolk extenders.

BACKGROUND: Buffalo spermatozoa have a distinct membrane structure that makes them more vulnerable to cryopreservation, resulting in lower-quality post-thawed sperm. This decreases the success rate of artificial insemination in buffaloes. Understanding and addressing these specific vulnerabilities are essential for improving reproductive techniques in buffalo populations. The properties of cryopreserved buffalo bull semen were examined in this study regarding the impact of adding autologous platelet-rich plasma (PRP) to OptiXcell® or Tris egg yolk-based extenders. Ten buffalo bulls were used to collect semen. Each bull's ejaculate was separated into two main equal amounts, each of which was then diluted with either OptiXcell® or Tris egg yolk-based extender, supplemented with various PRP concentrations (5%, 10%, and 15%), and the control (0%), before being cryopreserved according to established protocols. Following equilibration and thawing, the quality and functionality of the sperm were evaluated, along with the antioxidant enzyme activities (GSH and TAC), malondialdehyde (MDA) content, and in vivo fertilization rate of the thawed semen. RESULTS: All PRP concentrations in both extenders, particularly 10% PRP, improved the quality and functionality of the sperm in both equilibrated and frozen-thawed semen. Additionally, the antioxidant enzyme activities in both extenders were higher in the PRP-supplemented groups compared to the control group in thawed semen (P < 0.05). All post-thaw sperm quality, antioxidant enzyme activities, and functionality aside from DNA integrity were higher (P < 0.05) in the PRP-supplemented OptiXcell® than in the PRP-supplemented Tris egg yolk-based extender. The fertility of cryopreserved semen in the extenders supplemented with 10% and 15% PRP increased (P < 0.05) significantly more than that of the control extenders, with 10% PRP being the optimum concentration in OptiXcell® (80%) compared to that of Tris egg yolk-based extender (66.67%) and control of two extenders (53.33% and 46.67%, respectively). CONCLUSIONS: Even though autologous PRP-supplemented extenders have a protective impact on equilibrated and cryopreserved semen, 10% PRP-supplemented OptiXcell® extenders are more effective at preserving post-thaw semen quality, functionality, and antioxidant capacity, which increases the in vivo fertility of buffalo bulls.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Impact of papain on the treatment of raw diluted dromedary semen.

A variety of parameters, including liquefaction and semen viscosity, affect the sperm's ability to travel and reach the egg for fertilization and conception. Given that the details behind the viscosity of the semen in male camels have not yet been fully clarified, the purpose of this study was to ascertain how the addition of papain affected the viscosity of fresh diluted camel semen. The study examined semen samples derived from camels that had distinct viscosities. Sperm motility, viability, abnormal sperm percentage, concentration, viscosity, morphometry, acrosome integrity and liquefaction were among the evaluations following 0, 5, 10, 20 or 30 min of incubation at 37°C with papain (0.004 mg/mL, 0.04 mg/mL or 0.4 mg/mL; a semen sample without papain was used as a control). A statistically significant interaction between the effects of papain concentrations and incubation time was found (F = 41.68, p = .0001). Papain concentrations (p = .0001) and incubation times (p = .0001) both had a statistically significant impact on viscosity, according to a simple main effects analysis. A lower viscosity was found (p < .05) at 0.04 mg/mL (0.1 ± 0.0) after 10 min of incubation. A simple main effects analysis showed that papain concentrations and incubation time have a statistically significant effect on sperm motility (p = .0001). At 0.04 mg/mL papain, the sperm motility % was higher (p < .05) after 10 min (64.4 ± 4.8), 20 min (68.4 ± 6.2), and 30 min incubation (72.2 ± 6.6) compared to 0, 5 min (38.3 ± 4.1 and 51.6 ± 5.0, respectively). In conclusion, the fresh diluted camel semen had the lowest viscosity properties after 10 min of incubation with 0.04 mg/mL papain, without compromising sperm motility, viability, acrosome integrity and sperm morphology.

Quality and fertilizing ability of frozen-thawed porcine sperm separated using a migration sedimentation method.

We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.

The effects of MitoQ as a mitochondrial-targeted antioxidant in a plant-based extender on buck sperm quality parameters during cryopreservation.

Sperm cryopreservation plays an important role in the artificial insemination (AI) industry of small ruminants. It, however the use of frozen-thawed goat semen is limited due to the insufficient number of sperm with good biological functions. Mitochondria are the most sensitive organelles to cryopreservation damage in sperm. This study was conducted to determine the effects of MitoQ, the mitochondrial-targeted antioxidant, in a plant-based extender on the quality parameters of Markhoz goat sperm after the freezing and thawing process. Semen samples were collected and diluted in the extender, divided into five equal aliquots and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, lipid peroxidation (LPO), DNA fragmentation, reactive oxygen species (ROS) concentration, viability and apoptotic-like changes were measured. The use of 10 and 100 nM MitoQ resulted in higher (P≤0.05) total motility (TM), progressive motility (PM), viability, membrane functionality, mitochondrial activity, and acrosome integrity compared to the other groups. On the other hand, LPO, apoptotic-like changes, DNA fragmentation and ROS concentration were lower (P≤0.05) in MQ10 and MQ100 groups compared to the other groups. MitoQ has no effect (P>0.05) on sperm abnormal morphology and velocity parameters. In conclusion, MitoQ can reduce oxidative stress by regulating mitochondrial function during the cryopreservation process of buck sperm and could be an effective additive in the cryopreservation media to protect sperm quality.

The Effect of Cryopreserved Sperm on the Early Development, Survival, and Growth of Intergeneric Sterbel Hybrids (Acipenser ruthenus × Huso huso).

The aim of this study was to analyze the survival and growth of intergeneric (Acispenser ruthenus × Huso huso L.) sterbel hybrids obtained by fertilizing sterlet eggs with cryopreserved beluga semen. The rate of embryonic development did not differ between sterbel hybrids (experimental groups) and sterlets (control groups), and the hatching period was identical in all groups. The survival rate of hybrid larvae was higher in the experimental groups than in the control groups. Body weight and body length measurements revealed that sterbel hybrids grew at a faster rate than the control group sterlets. The hybrid origin of sterbels produced with the use of cryopreserved beluga semen was confirmed in a genetic analysis based on species-specific DNA fragments. To the best of the authors' knowledge, this is the first study to analyze the growth of sterbel hybrids derived from cryopreserved semen. The research findings indicate that this type of intergeneric hybridization delivers satisfactory results and can be applied in sturgeon aquaculture.

Heat shock protein 70 kDa (HSP70) is involved in the maintenance of pig sperm function throughout liquid storage at 17 °C.

At present, liquid storage is the most efficient method for pig semen preservation. This approach relies upon reducing sperm metabolism, allowing for the maintenance of cell lifespan. In this context, the study of proteins that could protect sperm during liquid storage is of high relevance. The 70 kDa Heat Shock Protein (HSP70) is an anti-apoptotic protein that has been reported to be relevant to sperm survival. Thus, we explored the role of HSP70 during prolonged storage of pig semen at 17 °C. Six semen pools were incubated with YM-1 (0, 0.05, 0.1 and 0.2 µM), an HSP70 inhibitor, and stored at 17 °C for 21 days. On days 0, 4, 10, 14 and 21, sperm quality and function were evaluated through flow cytometry and Computer-Assisted Sperm Analysis (CASA), and HSP70 activity and chromatin condensation were also determined. While inhibition of HSP70 increased progressive motility, Ca2+ and Reactive Oxygen Species (ROS) levels, and mitochondrial activity during the first 10 days of storage, it had a detrimental effect on sperm motility after 14 and 21 days. In spite of this, sperm viability was not altered. We can conclude that HSP70 contributes to the liquid storage of pig semen because it keeps mitochondrial activity low, which is needed for the maintenance of sperm function.

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture.

Coral reefs are facing a crisis as the frequency of bleaching events caused by ocean warming increases, resulting in the death of corals on reefs around the world. The subsequent loss of genetic diversity and biodiversity can diminish the ability of coral to adapt to the changing climate, so efforts to preserve existing diversity are essential to maximize the resources available for reef restoration now and in the future. The most effective approach to secure genetics long-term is cryopreservation and biobanking, which permits the frozen storage of living samples at cryogenic temperatures in liquid nitrogen indefinitely. Cryopreservation of coral sperm has been possible since 2012, but the seasonal nature of coral reproduction means that biobanking activities are restricted to just a few nights per year when spawning occurs. Improving the efficiency of coral sperm processing and cryopreservation workflows is therefore essential to maximizing these limited biobanking opportunities. To this end, we set out to optimize cryopreservation processing pathways for coral sperm by building on existing technologies and creating a semi-automated approach to streamline the assessment, handling, and cryopreservation of coral sperm. The process, which combines computer-assisted sperm analysis, barcoded cryovials, and a series of linked auto-datasheets for simultaneous editing by multiple users, improves the efficiency of both sample processing and metadata management in the field. Through integration with cross-cutting research programs such as the Reef Restoration and Adaptation Program in Australia, cryopreservation can play a crucial role in large-scale reef restoration programs by facilitating the genetic management of aquaculture populations, supporting research to enhance thermal tolerance, and preventing the extinction of coral species. The described procedures will be utilized for coral cryopreservation and biobanking practitioners on reefs worldwide and will provide a model for the transition of cryopreservation technologies from research laboratories to large-scale applications.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Evaluation of predictive ability of linear type gonadal traits on reproductive capacity of breeding dairy bulls.

Dimensions of linear type traits facilitate selection of livestock for breeding and rearing. To date, use of linear type traits for selection of breeding bulls is highly concentric to scrotal circumference (SC), with probable overlook to other important traits. Present study reported the importance of various gonadal linear type traits on spermatozoa production, age-related changes in gonadal linear type traits of bulls and predictive ability of these traits on bulls' reproductive potentials. Among all gonadal traits, testicular density (TD), scrotal volume (SV), paired testicular weight (PWT) and SC were found most important predictor variables in order, which can discriminate between good/poor breeding bulls, that is, produced frozen semen doses (FSD) or not. Dimensions of gonadal traits increased significantly up to 36 months age and thereafter, development became slow and negligible. In contrast, TD decreased by 30%, 51%, 64%, 68% and 71% at 12, 24, 36, 48 and >49 months age, respectively, from its base value at 6 months. Bulls of lower TD (≤0.88 g/cm3) had significantly higher ejaculate volume (+9%), sperm motility, sperm concentration (+100 million/mL) and sperm output (+26%)/ejaculate as compared to bulls of higher TD (>0.88 g/cm3). Discriminant function was developed using TD, SV, PWT and SC to identify bulls of superior reproductive potentials. It was concluded that among the investigated traits, TD was the strongest to discriminate between FSD and Non-FSD bulls. Therefore, our findings suggested that TD could be more potential trait than SC for dairy bulls' breeding soundness evaluation and assessment of reproductive ability.

Update of the cooling protocol for antibiotic-free storage of boar semen at 5°C improves sperm quality and maintains low bacterial counts.

Preserving boar semen at 5°C instead of the conventional storage temperature of 17°C would enable a reduction of antibiotic use in pig insemination. To protect the chilling-sensitive boar spermatozoa, holding the extended semen at a higher temperature before cooling could be beneficial and facilitate the implementation of the innovative preservation concept in practice, provided that bacterial growth is kept at a low level. The aim of this study was to introduce a holding time (HT) at 17°C before cooling and to examine the effect on sperm quality and bacterial growth compared to the original cooling protocol for antibiotic-free 5°C semen storage. A series of experiments with semen doses from eight boars extended in Androstar® Premium without conventional antibiotics revealed that sperm kinematics and the integrity of sperm plasma membranes and acrosomes were improved with HT between 16 and 24 h followed by delayed cooling with 0.04°C/min when compared to the original protocol for semen preservation at 5°C (p < 0.05). Both a shorter HT of 6 h and a faster cooling rate of 0.07°C/min reduced sperm quality (p < 0.05). The HT for 24 h did not compromise the inhibitory effect on bacterial growth during long-term semen storage at 5°C, not even in semen doses spiked with Serratia marcescens. In conclusion, semen storage at 5°C with the modified cooling protocol improved sperm quality and is antimicrobially efficient. It thus presents a ready-to-use tool for a reduction or replacement of antibiotics in pig insemination.

Voltage-dependent anion channels are involved in the maintenance of pig sperm quality during liquid preservation.

Pigs are usually bred through artificial insemination with liquid semen preserved at 15-20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 µM and 50 µM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 µM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 µM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.

Low concentrations of soybean lecithin nanoparticles had a positive impact on Holstein bulls' cryopreserved semen.

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p < .05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p < .05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p < .05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p > .05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p < .05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages.

A longer period of epididymal sperm interaction with extender components during cryopreservation improves sperm quality, decreases the size of sperm distal cytoplasmic droplets, and changes the number of nanoparticles in the extender.

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.

Applying a heat transfer mathematical model for the cryopreservation of rainbow trout (Oncorhynchus mykiss) sperm: How straw location over liquid nitrogen level affects freezing rate and fertilization yield.

Cryopreservation of rainbow trout semen under field conditions was analyzed. Straw location over liquid nitrogen level is a crucial variable that affects freezing rate and fertilization yield due to changes in nitrogen vapor external temperature. The objectives were: to analyze cryopreservation protocols by experimentally measuring the cooling rates and fertilization yield of 0.5 ml plastic straws located in nitrogen vapor at different heights corresponding to different external temperatures; to numerically simulate the freezing process, by solving the heat transfer partial differential equations with the corresponding thermo-physical properties of the biological system and the plastic straw; to evaluate and analyze the surface heat transfer coefficient (h) during the freezing process of the straws; to introduce a new variable, the characteristic freezing time (tc), that enables comparison between protocols; this variable was defined as the elapsed period between the initial freezing temperature and a final reference temperature of -40 °C (temperature in which more than 80 % of the water is in a frozen state). The mathematical model predicted the temperature distribution inside the straw, showing a low effect of straw plastic materials (polyethylene-terephthalate glycol, polyvinyl-chloride, and polypropylene) on freezing rates. The average h value obtained from numerical simulations was 25.5 W/m2 K, close to that obtained from the analytical Nusselt correlation for natural convection. An improvement on fertilization trials was observed when the average external nitrogen temperature was -129.6 °C (temperature range: -94 to -171 °C) with an average tc of 56.8 s (ranging between 47 and 72 s). These results corresponded to a height above the level of liquid nitrogen of 2 cm. Comparison with literature reported data showed satisfactory results. Applying mathematical models in the cryobiology field achieved results that are relevant for cryopreservation activities.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats.

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.