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.

High percentage of midpiece defects in Brangus bull sperm with no reduction in sperm kinematics.

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.

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.

Fatty acid composition and biophysical characteristics of the cell membrane of feline spermatozoa.

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

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.

A brief review of the methodology and cryoprotectants in selected fish and mammalian species.

Cryopreservation is a valuable technique used to assist in the genetic improvement of cultured stocks and provide a continuous supply of good-quality semen for artificial insemination. Conserving semen by cryopreservation serves several purposes (e.g. artificial reproductive technologies and species conservation) and is also used in the clinical treatment of human infertility. However, the lifespan of cryopreserved semen is influenced by a range of factors, including storage temperature, cooling rate, chemical composition of the extender, the concentration of cryoprotectant, reactive oxygen species, seminal plasma composition and hygienic control. The choice of cryoprotectant is a vital factor underlying the success of animal semen cryopreservation. In this regard, extensive research has been carried out on various cryoprotectants, such as egg yolk, dimethyl sulfoxide, methanol, ethylene glycol and dimethylacetamide. Recent studies have also described the use of a range of new cryoprotectants for cryopreservation, including compounds of plant origin (soy), amino acids, antifreeze proteins, carbohydrates and cyclodextrins. Moreover, semen cryopreservation and storage require the use of liquid nitrogen or ultralow refrigeration methods for both long- and short-term storage. This review summarizes the general methods used for freezing semen and discusses the use of traditional and newly emerging cryoprotectants (permeable and non-permeable) for the cryopreservation of semen in selected fish and mammalian species.

Factors influencing pregnancy rate and loss after laparoscopic artificial insemination with frozen-thawed semen in Lohi sheep under sub-tropical conditions.

This study evaluates factors influencing pregnancy rates per artificial insemination (P/AI) and pregnancy loss in Lohi ewes undergoing laparoscopic AI with frozen-thawed semen under sub-tropical conditions. Data from three experiments comprising ewes (n = 358) of mixed parity (nulliparous; NP and parous; P), various body condition score (BCS) and assigned to long-term (LTP, 11 days) and short-term (STP, 5 days) oestrus synchronization regimen across high breeding season (HBS) and low breeding season (LBS) were analysed. Laparoscopic insemination was conducted 54 h post-sponge removal. Pregnancy diagnosis and loss were evaluated on days 35 and 90 post-insemination via ultrasonography. Results showed parity significantly influenced P/AI, with nulliparous ewes achieving higher pregnancy ratios than parous ewes (p = .001). BCS significantly influenced P/AI (p < .05), with a quadratic relationship observed between BCS and season (BCS*BCS*Season; p = .07). Progestin treatment did not significantly influence the ratio of pregnant ewes (p = .07). Pregnancy losses were significantly higher during LBS than HBS (p < .05), irrespective of progestin treatment. In conclusion, parity and BCS significantly influenced P/AI, with BCS demonstrating a quadratic association with season. Ewes bred during LBS experienced higher pregnancy losses than HBS, irrespective of progestin treatment.

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.

Safety evaluation of single-sperm cryopreservation technique applied in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is a technique that directly injects a single sperm into the cytoplasm of mature oocytes. Here, we explored the safety of single-sperm cryopreservation applied in ICSI. This retrospective study enrolled 186 couples undergoing ICSI-assisted pregnancy. Subjects were allocated to the fresh sperm (group A)/single-sperm cryopreservation (group B) groups based on sperm type, with their clinical baseline/pathological data documented. We used ICSI-compliant sperm for subsequent in vitro fertilization and followed up on all subjects. The recovery rate/cryosurvival rate/sperm motility of both groups, the pregnancy/outcome of women receiving embryo transfer, and the delivery mode/neonatal-related information of women with successful deliveries were recorded. The clinical pregnancy rate, cumulative clinical pregnancy rate, abortion rate, ectopic pregnancy rate, premature delivery rate, live birth delivery rate, neonatal birth defect rate, and average birth weight were analyzed. The two groups showed no significant differences in age, body mass index, ovulation induction regimen, sex hormone [anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH)/luteinizing hormone (LH)] levels, or oocyte retrieval cycles. The sperm recovery rate (51.72%-100.00%) and resuscitation rate (62.09% ± 16.67%) in group B were higher; the sperm motility in the two groups demonstrated no significant difference and met the ICSI requirements. Group B exhibited an increased fertilization rate, decreased abortion rate, and increased safety versus group A. Compared with fresh sperm, the application of single-sperm cryopreservation in ICSI sensibly improved the fertilization rate and reduced the abortion rate, showing higher safety.

Effect of exogenous melatonin implant on post-thaw semen quality of buffalo bulls.

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.

Optimizing semen cryopreservation in Calomys laucha: a step forward in rodent reproductive research.

BACKGROUND: Examining semen cryopreservation in Calomys laucha offers valuable insights for reproductive research and species conservation. OBJECTIVE: To determine the most effective sugar for the cryopreservation of C. laucha semen. MATERIALS AND METHODS: Using 36 epididymides from C. laucha, semen samples were diluted in a 3% skimmed milk medium supplemented with one of four sugars (glucose, fructose, lactose, or sucrose) at a concentration of 0.3 M. These mixtures underwent a conditioning phase at 37 degree C for 10 min, cooled to -80 degree C for another 10 min, and were subsequently stored in liquid nitrogen. RESULTS: Upon thawing, samples treated with lactose and glucose solutions show superior sperm motility, achieving 8.2% and 10.0% respectively, in contrast to the fructose (2.0%) and sucrose (4.1%) mixtures. Furthermore, samples preserved in glucose registered the highest sperm penetration rates, reaching 44.9%. CONCLUSION: Our findings suggest that a cryopreservation medium containing 0.3 M glucose can contribute to the safeguarding C. laucha rodent semen. https://doi.org/10.54680/fr24210110612.

The kinetics of goat sperm is negatively affected after freezing in an extender including zinc oxide nanoparticles.

BACKGROUND: Nanotechnology can benefit livestock industries, especially through postharvest semen manipulation. Zinc oxide nanoparticles (Np-ZnO) are potentially an example. OBJECTIVE: To investigate how the addition of zinc oxide nanoparticles (Np-ZnO) affected the characteristics of post-thawed goat semen. MATERIALS AND METHODS: Seminal pools from four Saanen bucks were used. Semen was diluted in Tris-egg yolk extender, supplemented with Np-ZnO (0, 50, 100 or 200 ug/mL), frozen and stored in liquid nitrogen (-196 degree C), and thawed in a water bath (37 degree C / 30 s). Semen samples were evaluated for sperm kinetics by computer-assisted sperm analysis (CASA), and assessed for other functional properties by epifluorescence microscopy, such as plasma membrane integrity (PMi), acrosomal membrane integrity (ACi) and mitochondrial membrane potential (MMP). RESULTS: For total motility (TM), the group treated with 200 ug/mL Np-ZnO was superior to the control. In straight-line velocity (VSL), the control was better than the group containing 200 ug/mL of Np-ZnO. For average path velocity (VAP), the control was higher than with 100 ug/mL Np-ZnO. For linearity (LIN), the control was higher than with 200 µg/mL Np-ZnO. In straightness (STR), the control and 100 µg/mL Np-ZnO were higher than with 200 ug/mL Np-ZnO. In wobble (WOB), the control was better than the 50 µg/mL Np-ZnO treatment. In PMi, ACi and MMP no significant differences were found. CONCLUSION: The addition of Np-ZnO (200 ug/mL) to the goat semen freezing extender improved the total motility of cells, whilst negatively affecting sperm kinetics. https://doi.org/10.54680/fr24210110512.

Predicting the cryotolerance of boar sperm through antioxidant stress.

High sperm cryotolerance is crucial to the successful cryopreservation of boar sperm. Evaluating the cryotolerance of boar sperm by using a rapid and convenient technique can enhance the commercial viability of these sperm. This study investigated the correlation between sperm parameters for three sample subsets-fresh sperm, sperm with H2O2-induced oxidative damage (hereinafter referred to as H2O2-induced sperm), and frozen-thawed sperm-to identify the potential of these correlations to predict cryotolerance. A total of 64 sperm samples were obtained from 64 Duroc boars. The sperm parameters of the three subsets, where the frozen-thawed sperm were analysed at 30 or 180 min after thawing, were determined, and the coefficients of correlation between these parameters were calculated. The results indicated that H2O2-induced oxidative stress resulted in decreases in various sperm parameters-including total motility (TM), viability (VIA), mitochondrial membrane potential (MMP), and live sperm with MMP (LMP)-but increased their coefficients of variation. Receiver operating characteristic (ROC) curve analysis revealed that the kinematic parameters of the H2O2-induced sperm effectively predicted those of the frozen-thawed boar sperm at 30 min after thawing; the corresponding area under the ROC curve (AUC) was 0.8667 for TM and 0.8733 for progressive motility in the H2O2-induced sperm. For measurement at 180 min after thawing, the sperm membrane and mitochondrial parameters of the H2O2-induced sperm effectively predicted the LMP of the frozen-thawed boar sperm; the corresponding AUC was 0.8489 for VIA, 0.8289 for MMP, and 0.8444 for LMP. To our knowledge, this is the first study to directly establish a strong correlation between post-thaw boar sperm quality and H2O2-induced oxidative stress before freezing. Our proposed technique can serve as a valuable reference for the development of practical applications aimed at enhancing techniques for cryopreserving boar sperm.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Effects of Long-Term Cryopreservation on the Transcriptomes of Giant Grouper Sperm.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.

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.

The beneficial effects of quinoa seed extract supplementation on ram sperm quality following cryopreservation.

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500-1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H2O2 levels than the control group (P<0.001). The expressions of SOD1, CAT, GABPB1, and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene.