Effect of recombinant β-defensin 1 protein on human sperm motility and viability

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of “sperm maturation” during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is “β-defensin 1” which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO₂ atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.     

Rest days and storage of boar semen at 17°C: Effect on motility and sperm concentration

Boar fertility is an important factor in farm production; it is therefore of interest to determine factors which reduce the fertilising capacity of semen samples stored at 17°C for use in intrauterine insemination. This work evaluated the effect of the number of rest days between each mounting of the boar, and the number of days that the semen was stored at 17°C, on sperm motility and semen concentration. We also analysed whether the boar's age influenced the sperm concentration. The results showed that only the total motility diminished as the storage time at 17°C increased (p < .05). A low negative correlation was observed between the variables’ rest days and total and progressive motility. The sperm concentration presented no relation with rest days or the boar's age. The boars’ rest days had no effect on motility and sperm concentration in the males studied, allowing them to be used with the frequencies described with no effect on these parameters.     

Short‐term storage of salmonids semen in a sodium alginate‐based extender

Short‐term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate‐based extender on sperm function in the short‐term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish^® (Ext‐C) and Storfish^® supplemented with sodium alginate (Ext‐A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O₂^?) level and DNA fragmentation (DNA Frag) were assessed. Ext‐A had positive effect in preservation of sperm motility, viability, ΔΨmit, O₂^? level and DNA integrity in the three species analysed compared to control samples. In Ext‐A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O₂^? level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate‐based extender is effective for protecting sperm quality during 10 days of short‐term storage.     

Effect of short‐term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry

The short‐term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short‐term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short‐term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning – undiluted (SSD) and diluted (SD) (Storfish^® 1 : 2v/v; IMV AI solutions, France) – were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short‐term storage evaluated by flow cytometry.     

Response to cooling of pony stallion semen selected by glass wool filtration

The aim of this study was to compare the sperm separation technique using filtration through glass wool compared with just diluted cooled semen. Eighteen ejaculates were collected from 6 pony stallions of the Brazilian pony breed. Evaluations were done on pH, osmolarity, total motility, membrane functionality (HOST), membrane integrity (CFDA/PI), morphology and mitochondrial viability (MTT) in fresh, 24 and 48 h of cooled semen at 5°C. After dilution, the half of the extended semen was cooled (control group). The other half was cooled after filtration trough glass wool (filtered group). Retained semen was considered the portion of cells that did not transpose glass wool barrier. Total motility from the control, filtered and retained groups after 24 h of cooling was 35.5%, 43.3% and 10% (p < .0001) respectively. Sperm membrane integrity percentage at the CFDA/PI test was 37.9%, 44.8% and 14.8% (p < .0001), on the control, filtered and retained groups respectively. The results confirmed that the passage of spermatozoa through glass wool increased the selection of spermatozoa from pony stallions with higher motility, mitochondrial viability and membrane integrity for cooling in milk extender up to 24 h. Moreover, it was not obtained higher sperm parameters to control after cooling 48 h under the conditions that the study was conducted.     

Monitoring the reactive oxygen species in spermatozoa during liquid storage of boar semen and its correlation with sperm motility,free thiol content and seasonality

Oxidative stress is an important factor affecting the quality of spermatozoa during liquid storage of boar semen; however, monitoring of reactive oxygen species (ROS) that provides direct insight into the oxidative status is not yet attempted. This study aimed to monitor ROS in boar sperm during liquid semen storage to determine its correlation with sperm motility and free thiol (SH) content, and seasonality. Ejaculate was collected from mature Duroc boars in a commercial farm in autumn and spring, diluted in Mulberry III extender, stored at 15°C, and examined daily for sperm ROS level, SH content and motility. The ROS levels in spermatozoa prepared during autumn and spring were constantly low until days 4 and 5 of storage, respectively, which thereafter progressively increased in association with the loss of sperm motility. The increased sperm ROS level correlated with the higher SH level and lower motility, which was accentuated from day 4 of storage and was higher in September, or early autumn. This study indicates that increased sperm ROS levels during liquid storage results in oxidative damage, causing loss of sperm motility, presumably through decreased sperm viability, suggesting that sperm ROS monitoring effectively evaluates the quality of boar semen.     

Effect of addition of melatonin on liquid storage of ram semen at 4°C

Adding a certain amount of antioxidants to semen extender has been shown to improve semen quality. The aim of present study was to elucidate whether the supplementation of melatonin to the Tris‐based extender (CTR) could enhance the quality of ram spermatozoa during storage at 4°C. Ram semen samples were collected and diluted with CTR extender containing different concentrations (0, 0.05 (M 0.05), 0.1 (M 0.1), 0.2 (M 0.2) or 0.4 (M 0.4) mM) of melatonin. Sperm routine indicators, mitochondrial activity, total antioxidant capacity (T‐AOC) and malondialdehyde (MDA) content were analysed in control and melatonin treatment groups. The higher per cent of motility, plasma membrane integrity, mitochondrial activity and T‐AOC activity was observed in M 0.05, M 0.1 and M 0.2 groups compared to control group at 5 days of storage (p < 0.05), while lower percentage of MDA content was observed among these groups (p < 0.05). In addition, there were no significant differences in acrosome integrity among the control and M 0.05, M 0.1 and M 0.2 groups during the experiment. The above results show that the addition of 0.05, 0.1, 0.2 mM of melatonin is beneficial to the preservation of ram semen during liquid storage at 4°C mainly through antioxidative stress.     

Comparison of two cooling protocols for llama semen: with and without collagenase and seminal plasma in the medium

Seminal plasma (SP) of South American Camelids could interfere with the interaction of spermatozoa with the extenders; therefore it becomes necessary to improve semen management using enzymatic treatment. Our objective was to compare two cooling protocols for llama semen. Twelve ejaculates were incubated in 0.1% collagenase and then were divided into two aliquots. One was extended in lactose and egg yolk (LEY) (Protocol A: collagenase and SP present). The other aliquot was centrifuged, and the pellet was resuspended in LEY (Protocol B: collagenase and SP absent). Both samples were maintained at 5°C during 24 hr. Routine and DNA evaluations were carried out in raw and cooled semen. Both cooling protocols maintained sperm viability, membrane function and DNA fragmentation, with Protocol A showing a significantly lowered total and progressive motility (p < .05) and Protocol B showing a significant increase in chromatin decondensation (p < .05). Protocol A avoids centrifugation, reducing processing times and making application in the field simpler. However, as neither protocol showed a significant superiority over the other, studies should be carried out in vivo to evaluate the effect on pregnancy rates of the presence of collagenase and SP in semen samples prior to either cooling or freeze‐thawing.     

Omeprazole does not alter human sperm motility,viability or DNA integrity in vitro

The effect of omeprazole, a commonly used drug belongs to proton–‐pump inhibitor class, on human sperm function is still undetermined. Here, we hypothesised that addition of omeprazole to the ejaculated human semen may affect sperm parameters, and hence sperm function. Therefore, we assessed the in vitro effect of omeprazole on human sperm motility, viability and DNA integrity. Sixty‐six normozoospermic semen samples were collected randomly from men who attended the andrology laboratory at King Abdullah University Hospital. Sperm motility, viability and DNA breaks were assessed in the presence (1‐hr incubation at 37°C) of omeprazole at 5, 10, 20 and 50 µM compared to control (0 µM). None of the examined sperm parameters, at any tested omeprazole concentration, showed significant difference (p > 0.05) compared with the control. In conclusion, omeprazole at 5, 10, 20 and 50 µM does not alter human sperm motility, viability or DNA integrity in vitro.     

Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa

The aim of this study was to determine the effect of two equilibration temperatures (5 °C and room temperature) and two cryoprotectants (glycerol and dimethylformamide, both at 7%) on llama sperm cryopreservation. Llama ejaculates were divided into four aliquots. A lactose‐EDTA‐egg yolk (LEEY) extender with either 7% glycerol (LEEY‐G) or 7% dimethylformamide (LEEY‐DMF) was added to two of the aliquots, which were equilibrated for 20 min at room temperature and subsequently frozen. The other two aliquots were extended in LEEY, cooled to 5 °C, then LEEY‐G or LEEY‐DMF was added, equilibrated for 20 min at 5 °C and frozen. No significant differences (P > 0.05) were observed in membrane function and chromatin condensation between any of the freeze–thawing protocols. Post‐thaw motility was greater (P < 0.05) in LEEY‐DMF than LEEY‐G. DNA fragmentation was not different between raw and frozen semen with LEEY‐DMF but was high in all samples with glycerol. Our results indicate that 7% glycerol would be detrimental for llama spermatozoa, but further studies are needed to evaluate effectiveness if used at lower concentrations. Dimethylformamide preserved motility and DNA integrity of frozen–thawed llama spermatozoa and could be used to replace glycerol at the concentrations used in this study.     

Microencapsulation of human spermatozoa increases membrane stability and DNA longevity

The microencapsulation of spermatozoa offers potential benefits for maintaining sperm survival in vitro. The technique has also resulted in the production of offspring in several domestic animal species, but as yet, it has not been successfully applied in human reproductive medicine. This study examined the effect of alginic acid microencapsulation on human sperm membrane integrity (viability) and sperm DNA fragmentation (SDF) following storage for 24 hr at 37°C. The cumulative sperm viability (Log-rank, Mantel–Cox; Chi-square = 114.95, p = .000) and cumulative sperm DNA fragmentation (Log-rank, Mantel–Cox; Chi-square = 187.86, p = .000) of encapsulated spermatozoa were substantially improved when compared to control spermatozoa. Significant differences in the dynamic behaviour of different individuals were only apparent for sperm viability in microencapsulated samples (p = .021) while no significant differences were observed in control spermatozoa (p = .245); the equivalent comparison for SDF showed no differences (control p = .320; microencapsulated p = .432). We present potential scenarios for the use of microencapsulated human spermatozoa in reproductive medicine.     

DNA integrity and methylation changes of mouse spermatozoa following prolonged incubation

Sperm quality can be affected by different factors including the length of incubation time between sperm preparation and intracytoplasmic sperm injection. Here, we have evaluated the level of DNA methylation and expressions of related genes in mice spermatozoa. The spermatozoa were divided into three groups: fresh, spermatozoa incubated at room temperature (RT) and 37°C for 24 hr. The sperm chromatin structure assay was used to determine the DNA fragmentation index (DFI), and DNA methylation was analysed by flow cytometry. The expression levels of DNA methylation‐related genes were determined by quantitative real‐time PCR (qRT‐PCR). According to the results, we observed significantly higher sperm progressive motility and viability in the group incubated at RT compared to the spermatozoa incubated at 37°C (p < 0.05). Spermatozoa incubated at 37°C had a higher DFI compared to the other groups (p < 0.05), but the DNA methylation level significantly decreased (p < 0.05). qRT‐PCR analysis showed increased Dnmt‐1 expression in spermatozoa after 24‐hr incubation at 37°C. However, there were significantly higher expression levels of Dnmt‐3l, Dnmt‐3a and Dnmt‐3b after incubation at both RT and 37°C compared to the fresh group (p < 0.05). The 24‐hr incubation period affected both sperm DNA methylation and integrity. This study indicated that incubation at RT resulted in better sperm quality.     

Alpha‐lipoic acid minimises reactive oxygen species‐induced damages during sperm processing

Shearing forces during sperm preparation for assisted reproduction techniques may lead to excessive production of reactive oxygen species (ROS) which may have an unpleasant effect on embryonic development. In the current study, we assessed the effect of alpha‐lipoic acid (ALA) on ROS‐induced damages during sperm preparation process. Semen samples were collected from 15 normozoospermic men. Each semen sample was divided into two parts; one part was washed and centrifuged with sperm washing medium plus 0.02 mM ALA. Then, sperm pellet was diluted and incubated for 1 hr at 37°C in sperm washing media in the absence (ALA?) or presence of 0.02 mM ALA (ALA+). The second part was washed and centrifuged with sperm washing media in the absence of ALA, and then, sperm pellet was incubated for 1 hr at 37°C in sperm washing media in the absence (ALA?) or presence of 0.02 mM ALA (ALA+). Sperm viability, motility, intracellular oxidative stress and DNA fragmentation were assessed by eosin‐nigrosin, computer‐assisted sperm analysis system, H₂DCFDA staining and acridine orange staining respectively. Our results showed that addition of ALA as a fat‐ and water‐soluble antioxidant to sperm washing media maintains sperm viability and motility by reduction in ROS production and can also protect sperm DNA integrity.     

LED‐based red light photostimulation improves short‐term response of cooled boar semen exposed to thermal stress at 37°C

Pre‐treatment of boar semen with a red light photostimulation procedure increases its “in vivo” fertilising ability. However, “in vitro” conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short‐term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620–630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca²⁺ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca²⁺ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short‐term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.     

Gallic and carnosic acids improve quality of frozen‐thawed ram spermatozoa

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen‐thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris‐based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer‐aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreservation of ram semen in Tris‐based extender, supplementation with 2 mM GA increased post‐thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochondrial function.     

Incubation of frozen-thawed llama sperm with seminal plasma

Seminal plasma is intimately connected to sperm physiology and particularly in South American Camelids, has demonstrated to be involved in multiple physiological reproductive events. Different percentages of seminal plasma (0%, 10% and 50%) were added to thawed llama semen samples with the objective of evaluating the interaction with cryopreserved sperm over time (0, 1.5 and 3 hr at 37°C). A total of 20 ejaculates from five adult llama males (n = 5; r = 4) were evaluated. A significant decrease in sperm motility, membrane function and live sperm was observed in all thawed samples (0%, 10% and 50%) at 0 hr when compared to raw semen. Neither morphology nor chromatin condensation was altered in all thawed samples (p > .05), but a significant increase in the percentage of spermatozoa with fragmented DNA was observed after thawing all samples versus raw semen. When evaluating thawed samples over time, a significant decrease of motility and membrane function was observed, while the percentages of total live sperm were preserved over the 3 hr of incubation in all final concentrations evaluated. To conclude, the addition of 10% or 50% of seminal plasma was incapable of preserving motility or membrane function of frozen-thawed llama sperm during 3 hr of incubation.     

Famotidine does not affect human sperm fertility characteristics (motility,viability and DNA integrity) in vitro

Famotidine, a histamine‐2 receptor antagonist, is commonly used to relieve the acid‐related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty‐nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin–nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.     

Influence of post‐thawing thermal environment on bovine sperm characteristics and in vitro fertility

Our aim was to evaluate the effects of three thermal environments over time on kinetics, functionality and in vitro fertility of cryopreserved bovine spermatozoa. Four ejaculates from five bulls (n = 20) were cryopreserved. After thawing, semen was evaluated (0 hr), incubated for 4 hr in T36.0 (36.0°C), T38.0 (38.0°C) and T39.5 (39.5°C), and analysed every hour (1 hr, 2 hr, 3 hr, 4 hr). In vitro production of embryos was performed at 0 hr and 4 hr. Sperm motility and cell kinetics (Computer‐Assisted Sperm Analysis) were impaired after 2 hr at T38.0 and T39.5 (p < 0.05). Flow cytometry revealed an increase in the cells with injured plasma membrane to 39.5°C and a general reduction in the mitochondrial potential over time (p < 0.05). In vitro fertility was impaired in all temperatures after 4 hr, but there was no difference between 36.0°C and 38.0°C. Our results suggest that the ex situ resilience of semen at 36.0°C after thawing with no major damage to the quality is limited to 3 hr. In normothermia or in thermal stress, sperm cells present a gradual reduction of movement and functionality, which were more significant after 1 hr of incubation. The in vitro production of embryos is impaired when the semen is kept in a thermal environment ≥36.0°C for 4 hr.     

The presence of seminal plasma,especially derived from stallion semen,helps preserve chilled Asian elephant (Elephas maximus) sperm motility

The effects of seminal plasma (SP), derived from autologous, homologous and heterologous species (stallion, boar and dog) on chilled Asian elephant sperm quality, were determined. Semen was collected from eight males and samples with ≥30% motile spermatozoa were used in the study. Semen was diluted with Tris–glucose–egg yolk extender, supplemented with different SP types and preserved at 4°C for 48 hr. Experiment 1 (n = 31), showed that the presence of SP (autologous) helped to preserve sperm quality in terms of sperm motility and acrosome integrity (p < .05). Homologous SP did not result in better sperm quality than autologous SP. Heterologous SP from stallion provided higher sperm motility and velocities compared to autologous SP (p < .05). Experiment 2 (n = 14) determined the effect of different SP from four stallions. All stallion SP gave higher (p < .05) results for motile spermatozoa and sperm velocities than autologous SP. In conclusion, the presence of SP helps preserve Asian elephant sperm quality and stallion SP supports the motility of Asian elephant spermatozoa during cold storage.     

Chlorogenic acid improves the quality of boar semen subjected to cooled storage at 15°C

The aim of this study was to investigate the effect of chlorogenic acid (ChA) in boar semen stored at 15°C. Twelve ejaculates were processed into insemination doses at different concentrations of ChA (0.0, 1.5, 3.0, 4.0 and 6.0 mg/ml) or vitamin E (200 μl/ml) as positive control. Semen was analysed after 0, 24 and 72 hr of storage. ChA improved (p < .05) sperm motility, acrosome integrity and mitochondrial activity in all periods of storage. Furthermore, after 24 hr of storage, ChA above 1.5 mg/ml supported the sperm viability until 120 min after reheating (p < .05). Both ChA and vitamin E were similarly efficient in increasing the antioxidant capacity of semen, reducing the malondialdehyde levels before and after 72 hr of storage (p < .05). However, with 72 hr of storage, ChA at 3.0 mg/ml improved the mitochondrial activity over vitamin E (p < .05). In conclusion, results suggest that the concentration of 3.2 mg/ml of ChA is the best for semen stored for up to 24 hr. However, for semen stored for a longer period, 6.0 mg/ml or more should be used.