Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Effect of fumigation height and time on cryopreservation of ram semen.

The cooling rate is a crucial factor in the process of freezing semen, influencing the overall freezing effectiveness. The height and time of fumigation can significantly impact the rate of cooling. Appropriate cooling rates can help minimize the formation of ice crystals in spermatozoa and reduce potential damage to them. Therefore, the aim of this study was to evaluate the effect of different fumigation heights and time for the cryopreservation of Hu ram semen. Experiments I-IV assessed the effect of semen cryopreservation by testing the post-thawed spermatozoa total motility (TM), progressive motility (PM) and kinetic parameters fumigated at distances of 2, 4, 6 and 8 cm for durations of 5, 10, 15 and 20 min, respectively. Based on the results of experiments I to IV, experiment V evaluated the effect of semen cryopreservation by testing the post-thawed spermatozoa TM, PM, kinetic parameters, plasma membrane integrity, acrosome integrity and reactive oxygen species (ROS) level fumigated at distances of 2, 4, 6 and 8 cm for duration of 20 min. The results indicated that fumigation at 2 cm for 20 min significantly (P < 0.05) improved spermatozoa TM, PM, mean angular displacement (MAD), plasma membrane integrity and acrosome integrity compared to other groups. Additionally, it significantly (P < 0.05) reduced spermatozoa ROS level compared to the 6 and 8 cm groups. In conclusion, fumigation for 20 min at a distance of 2 cm from the liquid nitrogen surface is the most suitable cooling method for the cryopreservation of Hu ram semen.

The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Cryopreservation of bovine semen using extract of Cinnamomum zeylanicum.

BACKGROUND: Antioxidants minimise oxidative stress and enhance sperm quality in the process of cryopreservation. OBJECTIVE: To assess the impact of Cinnamomum zeylanicum extract as an additive during the post-dilution and post-thaw stages of Murrah buffalo semen cryopreservation. MATERIALS AND METHODS: The semen sample was diluted using Tris-Egg-Yolk-Citric-Acid-Fructose-Glycerol extender and subsequently divided into three groups: Group 1, TEYCAFG without any additives or controls (C); Group 2, TEYCAFG fortified with a 50 ug/mL aqueous extract of cinnamon (T1); and Group 3, TEYCAFG fortified with a 50 ug/mL ethanolic extract of cinnamon (T2). The evaluation included an assessment of progressive motility, live spermatozoa, sperm abnormalities, HOST, CMPT, and enzyme leakage (GOT and GPT) at both the post-dilution and post-thaw stages. RESULTS: The groups that received cinnamon supplementation demonstrated statistically significant improvements (p<0.05) in various parameters, including an increase in the progressive motility, live spermatozoa, and HOS-positive spermatozoa, as well as greater distance traveled by vanguard spermatozoa compared to the control group. Furthermore, the cinnamon-added groups exhibited a significant decrease (p<0.05) in the percentage of sperm abnormalities and lower enzyme leakage (GOT and GPT) in post-thawed semen. CONCLUSION: Aqueous extract of C. zeylanicum at a concentration of 50 µg/mL provides superior protection of sperm structures and functions as compared to both the ethanolic extract of C. zeylanicum at the same concentration and the control group. Doi.org/10.54680/fr24310110712.

Aquaporin expression in cryopreserved human sperm: exploring the capabilities of natural deep eutectic solvents (NADEs).

BACKGROUND: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells. OBJECTIVE: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. MATERIALS AND METHODS: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. RESULTS: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. CONCLUSION: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

Oral supplementation with Scabiosa artropurperea L. aqueous extract improves the in vivo sexual behaviour, sperm quality and androgen level in rams.

The effects of an aqueous extract of Scabiosa atropurpurea L. (AES) on the reproduction potential of Queue Fine de l'Ouest rams were evaluated over 9 weeks. Eighteen mature (4-6 years old) rams (52.8 ± 2.6 kg) were divided into three groups. The control (C) group was fed oat hay ad libitum with 700 g of concentrate and the other two groups were fed the same diet supplemented with AES at 1 and 2 mg/kg body weight (AES1 and AES2, respectively). Ram sperm was collected with an artificial vagina (2 × 2 days/week) to evaluate sperm production and quality, antioxidant activity, the adenosine triphosphate (ATP) and calcium concentrations. Sexual behaviour and plasma testosterone concentrations were also investigated. The administration of AES improved sexual behaviour (the duration of contact and the number of lateral approaches). The addition of AES also improved individual spermatozoa motility (C: 71.7% ± 6.3%; AES1: 78.3% ± 4.9%; AES2: 83.8% ± 4.4%), the sperm concentration (C: 5.6 ± 0.36; AES1: 6.4 ± 0.81; AES2: 6.7 ± 0.52 × 109 spermatozoa/mL), the ATP ratio (C: 1 ± 0.08; AES1: 2.1 ± 0.08; AES2: 3.3 ± 0.08) and the calcium concentration (C: 5.6 ± 0.24; AES1: 7.7 ± 0.21; AES2: 8.1 ± 0.24 mmol/L). AES treatment decreased the percentage of abnormal sperm (C: 18.5% ± 1.2%; AES1: 16.2% ± 1.1%; AES2: 14.8% ± 0.94%) and DNA damage (C: 62%; AES1: 27%; AES2: 33%) and was associated with elevated seminal fluid antioxidant activity (C: 22 ± 0.27; AES1: 27.1 ± 1.08 and AES2: 27.5 ± 0.36 mmol Trolox equivalents/L) and plasma testosterone (C: 8.3 ± 0.7; AES1: 11.7 ± 0.4; AES2: 15 ± 0.7 ng/L). In conclusion, our study suggests that S. atropurpurea may be potentially useful to enhance libido and sperm production and quality in ram.

[Association between long-term exposure to ambient ozone and sperm quality in Shandong Province].

Objective: To evaluate the association between long-term exposure to ambient ozone (O3) and sperm quality. Methods: From January 1, 2014, to December 31, 2019, healthy sperm donors were recruited through the Human Sperm Bank of Shandong University Affiliated Reproductive Hospital. A total of 37 977 sperm donation data from 2 971 healthy volunteers were analyzed. The average annual O3 concentration (0.01°× 0.01°) was matched according to household address. A multivariate mixed-effect model was used to analyze the exposure-response relationship between the average O3 exposure concentration and sperm quality in the previous year, with each donor as a random intercept. All results were presented as % changes with 95% confidence intervals (CIs) for all sperm parameters associated with 10 µg/m3 increases in O3. The effects of individual characteristics on the association between O3 and sperm quality were evaluated by stratified analysis. Results: The average O3 concentration in the year before semen collection was (107.09±7.50) µg/m3. Each 10 µg/m3 increase in O3 was associated with declined sperm concentration (-3.12%, 95%CI:-4.55%, -1.67%), total sperm count (-5.21%, 95%CI:-7.28%, -3.09%), total sperm motility (-1.49%, 95%CI:-2.37%, -0.61%), progressive motility (-2.53%, 95%CI:-3.78%, -1.26%), total motile sperm count (-5.82%, 95%CI:-8.17%, -3.41%), and progressively motile sperm count (-6.22%, 95%CI:-8.73%, -3.64%). Men aged 30 and above, obese, and with lower education levels might be more susceptible to the influence of O3 on sperm quality, but the difference was not statistically significant (P>0.05). Conclusion: Long-term exposure to O3 in Shandong Province is associated with a decrease in sperm quality.

Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Zinc alleviates high fat diet-induced spermatogenic dysfunction in Wistar rats: role of oxidative stress, HMGB1 and inflammasome.

Whether chronic inflammation in the genital tract induced by obesity shares in spermatogenic dysfunction is not clearly known. We aimed to study the effect of high fat diet (HFD) on spermatogenesis, seminal oxidative stress (malondialdehyde (MDA)) and inflammatory markers (high mobility group box 1 (HMGB1), nucleotide-binding oligomerization domain, leucine rich repeat and pyrin-3 domain containing (NLRP3)) in the rat testes and the role of zinc on testicular dysfunction and chronic inflammation in high fat diet (HFD) fed rat testes. This parallel group comparative experimental study included 36 male wistar rats divided into 3 groups: group A (fed on normal control diet); group B (fed on high fat diet (HFD) only); and group C (fed on HFD with zinc supplementation 3.2 mg/kg/day orally). At the end of the 12th week, sperm count, viability and motility were assessed by computer-assisted seemen analysis (CASA), seminal malondialdehyde measured by calorimetry and histopathological examination of testicular sections was done. Immunohistochemical staining was done for HMGB1 and NLRP3 evaluation. Sperm count was lowest in group B. Groups A and C showed statistically significant higher mean sperm vitality, total and progressive motility scores (p < 0.001), while no difference was found between the groups A and C (p > 0.05). Seminal malondialdehyde level was significantly highest in group B. Tubular diameter, epithelial height and Johnsen score were significantly lowest in group B. Significantly higher HMGB1 and NLRP3 levels were demonstrated in group B (p < 0.001). Obesity is associated with testicular dysfunction, testicular oxidative stress and increased testicular HMGB1 and NLRP3. We suggest a beneficial effect of zinc on testicular function in HFD-rats.

Therapeutic Potential of 4-Hexylresorcinol in Preserving Testicular Function in Streptozotocin-Induced Diabetic Rats.

It is known that many diabetic patients experience testicular atrophy. This study sought to investigate the effect of 4-hexylresorcinol (4HR) on testicular function in rats with streptozotocin (STZ)-induced diabetes, focusing on testicular weight, sperm motility, histological alterations, and serum testosterone levels to understand the efficacy of 4HR on testes. Our findings reveal that 4HR treatment significantly improves testicular health in diabetic rats. Notably, the STZ group exhibited a testicular weight of 1.22 ± 0.48 g, whereas the STZ/4HR group showed a significantly enhanced weight of 1.91 ± 0.26 g (p < 0.001), aligning closely with the control group's weight of 1.99 ± 0.17 g and the 4HR group's weight of 2.05 ± 0.24 g, indicating no significant difference between control and 4HR groups (p > 0.05). Furthermore, the STZ/4HR group demonstrated significantly improved sperm motility compared to the STZ group, with apoptotic indicators notably reduced in the STZ/4HR group relative to the STZ group (p < 0.05). These results underscore the therapeutic potential of 4HR for maintaining testicular function under diabetic conditions.

Effect of Aqueous Extract of Unripe Musa Paradisiaca Linn on Parameters Affecting Reproduction in Rats.

OBJECTIVE: Aqueous extract of unripe Musa paradisiaca fruit is commonly used for the treatment of ulcers in eastern Nigeria. This study aimed to assess the acute and subacute effects of an aqueous extract of unripe fruit on male and female fertility in rats. METHODS: Aqueous extracts obtained by maceration were analyzed for acute and subacute toxicity and for the presence of phytochemical constituents using standard procedures. The extract (100, 500, and 1000 mg/kg) was administered daily to rats of both sexes for 28 d. Blood samples collected on days 0 and 28 were assessed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA). Testes and ovaries were harvested for histopathological analysis. Sperm were also collected to determine the sperm count and motility. RESULTS: Phytochemical screening revealed the presence of saponins, tannins, alkaloids, and resins. After an oral dose of up to 5000 mg/kg, there were no deaths in the acute toxicity test. The extract (500 mg/kg) significantly (P < .05) enhanced sperm count and motility relative to the untreated control; significantly (P < .05) reduced SOD, CAT, and glutathione levels, while significantly (P < .05) elevated LH, FSH, and MDA levels in male and female rats. Histological examination revealed significant structural damage to the ovaries. CONCLUSION: Unripe Musa paradisiaca fruit exhibited an adverse toxicological profile following prolonged administration and caused oxidative stress in rodents.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-µg zinc oxide (ZnO), ZnONP50: Lake with 50-µg ZnONP, ZnONP100: Lake with 100-µg ZnONP and ZnONP200: Lake with 200-µg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-µg ZnO and 50- to 100-µg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Phoenix dactylifera L. pollen versus pentoxifylline on improvement of sperm parameters in idiopathic male infertility: A randomized clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Phoenix dactylifera L. pollen is the male reproductive dust of palm flowers known as a natural product that is considered a strong stimulant of sexual potency and fertility in Iranian traditional medicine (ITM). In this regard, no evidence-based medications are empirically prescribed to treat IMI. However, applying traditional medicine for the treatment of male infertility has attracted more attention in recent years. AIM OF THE STUDY: Phoenix dactylifera L. pollen was compared with pentoxifylline (PTX) to evaluate its efficacy on sperm parameters. MATERIALS AND METHODS: During this parallel randomized controlled trial, 80 adult men with asthenozoospermia, oligozoospermia, or teratozoospermia (age 20-35 years) were enrolled. In two separate groups of participants with a 1:1 ratio, participants received either 6 g of Phoenix dactylifera L. pollen powder daily or 400 mg of PTX tablets daily for 90 days. We measured the sperm parameters as well as the serum sex hormones in the sample. ANCOVA and t-tests were used to compare groups. RESULTS: There was no significant difference between the study groups in terms of baseline characteristics or demographic characteristics. According to the results, participants who took Phoenix dactylifera L. pollen powder had significantly improved sperm concentration (p = 0.016), morphology (p = 0.029), sperm counts (p = 0.012), progressive motility (p = 0.016), total motility (p = 0.018), and reduced immotile sperms (p = 0.014) compared to those who took PTX. CONCLUSIONS: In light of these results, Phoenix dactylifera L. pollen is recommended as a treatment factor for ameliorating IMI by enhancing sperm functional capacity and semen parameters.

Environmental pollutants and male infertility: Effects on CatSper.

Infertility is a growing health concern among many couples worldwide. Men account for half of infertility cases. CatSper, a sperm-specific Ca2+ channel, is expressed on the cell membrane of mammalian sperm. CatSper plays an important role in male fertility because it facilitates the entry of Ca2+ necessary for the rapid change in sperm motility, thereby allowing it to navigate the hurdles of the female reproductive tract and successfully locate the egg. Many pollutants present in the environment have been shown to affect the functions of CatSper and sperm, which is a matter of capital importance to understanding and solving male infertility issues. Environmental pollutants can act as partial agonists or inhibitors of CatSper or exhibit a synergistic effect. In this article, we briefly describe the structure, functions, and regulatory mechanisms of CatSper, and discuss the body of literature covering the effects of environmental pollutants on CatSper.

Disposables used cumulatively in routine IVF procedures could display toxicity.

STUDY QUESTION: Is there a cumulative toxicity of disposables used in IVF procedures? SUMMARY ANSWER: A toxicity may be detected when consumables are used cumulatively, while no toxicity is detected when the same consumables are used and tested individually. WHAT IS KNOWN ALREADY: Many components of items used in IVF laboratories may impair human embryonic development. Consequently, it is necessary to screen all reagents and materials which could be in contact with gametes and embryos. Toxicity tests, such as the mouse embryo assay and the human sperm motility assay (HSMA), are used by manufacturers as quality control tools to demonstrate the safety of their products. This evaluation is currently individually performed for each single consumable. However, during an IVF cycle, several devices are used sequentially, potentially creating a cumulative exposure to chemical contaminants, which could not be detected for individually tested consumables. STUDY DESIGN, SIZE, DURATION: The objective of this observational study conducted from March 2021 to October 2022 was to evaluate with the HSMA methodology if there was a cumulative toxicity when several disposables are sequentially used. Fourteen categories of consumables currently used in routine IVF procedures were studied, which included devices used for sperm and oocyte collection (cups, condoms, and oocyte aspiration needles), manipulation (flasks, tubes, tips, pipettes, embryo transfer catheters, syringes, and gloves), culture (dishes), and storage (straws). PARTICIPANTS/MATERIALS, SETTING, METHODS: After obtaining patient consent, the surplus semen assessed as having normal parameters according to the World Health Organization 2010 criteria were used to perform the HSMAs. First, each consumable was tested individually. Then, associations of three, four, and five consumables, previously validated as non-toxic when tested individually, were analyzed. HSMAs were conducted three times to ensure reproducibility, with a defined toxicity threshold of a sperm motility index (SMI) below 0.85 in at least two of three tests. MAIN RESULTS AND THE ROLE OF CHANCE: Thirty-six references of disposables were first individually tested across 53 lots. Forty-nine (92%) demonstrated compliance. However, four (8%) devices revealed toxicity: one lot of 1 ml syringes, two lots of sperm cups, and one lot of 25 cm2 flasks. These four references were excluded from the IVF routine procedures. A total of 48 combinations of consumables were assessed, involving 41 lots from 32 references that were previously individually tested. Among the evaluated combinations, 17 out of 48 (35%) associations exhibited toxicity with a SMI below 0.85 for two of the three tests (n = 8) or all the three tests (n = 9). Notably, three out of 17 (18%) of the three-consumable associations, five out of 16 (31%) of the four-consumable associations, and nine out of 15 (60%) of the five-consumable associations were found not compliant. The toxicity did not originate from a single consumable, because only consumables that were individually pre-validated as non-toxic were included in the combinations, but the toxicity had a cumulative origin. The risk of cumulative toxicity increased with the number of consumables included in the association (Cochran-Mantel-Haenszel statistic, P = 0.013). LIMITATIONS, REASONS FOR CAUTION: The high proportion of non-compliant combinations of disposables can be attributed directly to the extreme rigorous extraction conditions employed during the tests, which could deviate from the conditions encountered in routine clinical use. Also, the methodology employed in the HSMAs (e.g. toxicity extraction duration, sperm concentrations, and protein supplementation of the medium) can influence the sensitivity of the tests. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the significance of performing toxicity testing on devices before introducing them into clinical practice. Disposables should be tested individually to detect immediate toxicities and also in combination. Our results advocate rationalizing the number of consumables used in each IVF procedure and re-evaluating the use of glass consumables. STUDY FUNDING/COMPETING INTEREST(S): This study received fundings from GCS Ramsay Santé pour l'Enseignement et la Recherche (Paris, France) and the Centre de Biologie Médicale BIOGROUP (Le Chesnay-Rocquencourt, France). The authors declare that they have no conflict of interest that could be perceived as prejudicing the impartiality of the reported research. TRIAL REGISTRATION NUMBER: N/A.

Alpha-lipoic acid improves cryopreservation of rooster semen by reducing oxidative stress.

Inhibiting oxidative stress is key for ensuring sperm motility during semen cryopreservation. The aim of this study was to investigate the effect of adding alpha-lipoic acid (ALA) as an extender in rooster semen cryopreservation. Different concentrations of ALA were added to the frozen diluent of rooster semen; subsequently, computer-aided semen analysis was used to determine membrane functional integrity, acrosome integrity, antioxidant capacity (based on T-AOC, GSH-Px, SOD, CAT, and MDA contents), and mitochondrial integrity. The frozen sperm ultrastructure was observed using transmission electron microscopy. The results showed that the addition of different concentrations of ALA partially to greatly improved the quality of frozen sperm; in particular, 8 µg/mL ALA significantly improved multiple parameters of sperm quality, including sperm motility and antioxidant enzyme activity, after freeze-thaw. The results of this study provide empirical and theoretical support for effective rooster semen cryopreservation and can inform the development of new protective agents in the field of livestock reproduction.

Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations.

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

In vitro effects of the combination of serotonin, selenium, zinc, and vitamins D and E supplementation on human sperm motility and reactive oxygen species production.

Infertility affects 15% of all couples worldwide and 50% of cases of infertility are solely due to male factors. A decrease in motility in the semen is considered one of the main factors that is directly related to infertility. The use of supplementation to improve the overall sperm quality has become increasingly popular worldwide. The purpose of this study was to evaluate whether sperm motility was affected by the combination of serotonin (5-HT), selenium (Se), zinc (Zn), and vitamins D, and E supplementation. Semen samples were incubated for 75 min at 37°C in medium containing varying concentrations of 5-HT, Se, Zn, vitamin D, and E. 5-HT (200 µM), Se (2 µg/ml), Zn (10 µg/ml), vitamin D (100 nM), and vitamin E (2 mmol) have also been shown to increase progressive sperm motility. Three different mixtures of supplements were also tested for their combined effects on sperm motility and reactive oxygen species (ROS) production. While the total motility in the control group was 71.96%, this was found to increase to 82.85% in the first mixture. In contrast the average ROS level was 8.97% in the control group and decreased to 4.23% in the first mixture. Inclusion of a supplement cocktail (5-HT, Se, Zn, vitamins D and E) in sperm processing and culture medium could create an overall improvement in sperm motility while decreasing ROS levels during the incubation period. These molecules may enhance the success of assisted reproduction techniques when present in sperm preparation medium.