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.     

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.     

Supplemental effect of varying L-cysteine concentrations on the quality of cryopreserved boar semen

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk （LEY） extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L^-1 （group Ⅰ, control）, 5 mmol L^-1 （group Ⅱ）, 10 mmol L^-1 （group Ⅲ） and 15 mmol L^-1 （group Ⅳ）. Semen suspensions were loaded in straws （0.5 mL） and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher （P 〈 0.01） percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups （group Ⅱ and group Ⅲ） compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group Ⅲ. In conclusion, 5 or 10 mmol L^-1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.     

Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality

The resazurin reduction assay depends on the ability of metabolically active cells to reduce the resazurin redox dye to resorufin. In the present study we applied and made a diagnostic evaluation of a spectrophotometric application of the resazurin reduction assay to assess the colour change of resazurin reduction in butanol extracted colour to evaluate boar semen quality. Forty-one samples of boar semen from various breeds were included in the study. The absorption peaks for resazurin and resorufin were found to be 610 and 575 nm, respectively. Absorbance at 610 nm, where the minimum overlap of the two peaks was observed, was used in further analysis. Spearman rank correlation analysis was used to determine the correlation between the resazurin reduction assay and various semen parameters. The highest correlations were observed with the concentration of motile spermatozoa (r = -0.841; p < 0.001), sperm concentration (r = -0.833; p < 0.001), sperm index (-0.826; p < 0.001) and concentration of viable spermatozoa (r = -0.763; p < 0.001). Sensitivity and specificity, at 94.1 and 91.7%, respectively, indicate that the present test is highly accurate in discriminating between the samples according to the sperm index. When motile sperm concentration was used to distinguish between good and poor samples, high sensitivity (93.6%) was also found, whereas the test was only moderately, 80%, specific. The stability of butanol extracts in terms of A610 at different times of measurement confirmed that the resazurin reduction could be spectrophotometrically measured within 7 days from the time of assay performance, making the assay much more useful. Based on these results, the assay could be used as an additional tool for evaluating the quality of boar semen.     

Supplementing oregano essential oil to boar diet with strengthened fish oil: Effects on semen antioxidant status and semen quality parameters

Previous research has shown benefits of dietary fish oil supplementation on semen quality of boars. However, little is known about how antioxidant protects lipid peroxidation on spermatozoa from n‐3 polyunsaturated fatty acid (PUFA) addition. This study evaluated the effect of oregano essential oil (OEO) supplementation on semen antioxidant status and semen quality in boars fed a diet enriched with fish oil. Thirty‐four mature boars of proven fertility, received daily 2.5 kg basal diet top‐dressed with 45 g soybean oil and 15 g fish oil to meet the n‐3 PUFA requirement of spermatozoa, randomly allocated to one of four groups supplemented with 100 mg α‐tocopheryl acetate kg^?1 (control), or 250 or 500 or 750 mg OEO kg^?1 for 16 weeks. Semen was collected at weeks 0, 8, 12 and 16 for measurements of sperm production, motion characteristics, sperm α‐tocopherol content, antioxidant enzyme activities, reactive oxygen species (ROS), DNA damage (8‐hydroxydeoxyguanosine, 8‐OHdG), lipoperoxidation (malondialdehyde, MDA) and seminal total antioxidant capacity (TAC). Sperm production and motion characteristics were similar (p > .05) among groups throughout the experimental week 16, but increased (p < .01) with experimental week. Although higher α‐tocopherol content and superoxide dismutase (SOD) activities were in OEO group spermatozoa, feeding diet with 500 mg/kg OEO resulted in elevation in seminal TAC, decrease in sperm ROS, MDA and 8‐OHdG than control group (p < .05). Overall, these results support the view that oregano essential oil has a positive effect on antioxidant capacity in boar when used fish oil.     

Effect of storage in short--and long-term commercial semen extenders on the motility, plasma membrane and chromatin integrity of boar spermatozoa

For artificial insemination (AI) in pigs, preservation of liquid boar semen at 16-20 degrees C is still common practice as sperm cryopreservation remains suboptimal in this species. To meet the different needs of the swine industry, several extenders have been developed to preserve semen in liquid form for short--and long-term storage. In the present study, three different commercial extenders devised for short-term (BTS+) or long-term preservation (MR-A and X-Cell), were used to test whether storage of semen from four mature, fertile boars at 17 degrees C for 96 h would affect sperm characteristics relevant for fertility, such as motility, membrane integrity and chromatin stability. Computer-assisted sperm analysis, and stainings with the acylated membrane dye SYBR-14/propidium iodide, and acridine orange in connection with flow cytometry were used to evaluate these variables. Percentages of total motile spermatozoa decreased slightly, but significantly, after 72-96 h. While membrane integrity values varied during the period of study, no significant changes in either membrane integrity or chromatin stability were, however, registered. This suggests a customary 96-day storage at 17 degrees C in these extenders was too short an interval to cause losses of integrity in nuclear DNA in the boar population studied.     

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.     

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.     

Tests to measure the quality of spermatozoa at spermiation

This commentary is to critique the revised World Health Organization （WHO） semen analysis manual as it pertains to characteristics of a spermatozoon at spermiation. The aims of the revised WHO manual include improving the ＇quality of semen analysis＇ without any restriction to clinical use. Furthermore, the manual states that semen analysis may be useful for （a） ＇investigating male fertility status＇ and （b） ＇monitoring spermatogenesis during and following male fertility regula- tion.＇ However, if the analysis of ejaculated spermatozoa is intended for the purposes described in （b）, then cells that are abnormal at spermiation must be identified. This paper takes the position that the manual does not identify methods to estimate the quality of spermatozoa at spermiation. Instead, it uses a ＇gold standard＇ of sperm passing through the cervical mucus or arriving near the site of fertilization. Although this standard is appropriate for drawing conclusions regarding the probability that an individual could impregnate his partner, it is not appropriate for studying illness of the testes per se. Herein, the measures of sperm quality presented in the WHO manual are critiqued with respect to the detection of spermatozoa that were abnormal at spermiation vs. those that became abnormal subsequently. Quality assessments based on the percentage of motile or ＇viable＇ spermatozoa are meaningless. Alternative quality attributes defining spermatozoa at spermiation are presented in this paper. In conclusion, assessment of spermatozoal quality at spermiation, on the basis of quality attributes of individual ejaculated spermatozoa, is best achieved through application of （a） a new paradigm for the morphological evaluation of sperm quality and （b） modern analytical techniques to evaluate, in an adequate sample, several appropriate independent attributes in each spermatozoon in order to more accurately identify the proportion of abnormal spermatozoa.     

Selenium and vitamin E supplementation enhances the antioxidant status of spermatozoa and improves semen quality in male dogs with lowered fertility

Studies showed a beneficial effect of supplementation with selenium (Se) and vitamin E on semen quality. The aim of the study was to investigate the effect of Se and vitamin E supplementation on the antioxidant status of spermatozoa and semen quality in dogs with lowered fertility. Ten dogs were supplemented daily with Se (6 μg/kg organic Se yeast) and vitamin E (5 mg/kg) per os for 60 days. Control group consisted of 10 males without the supplementation. Semen was collected on day 0, 30, 60 and 90. Sperm quality parameters were evaluated using CASA and a microscope. Concentrations of Se and vitamin E in blood as well as glutathione peroxidase (GSH‐Px) activity and total antioxidant capacity (TAC) in the spermatozoa were determined. After 60 days of supplementation the concentration of spermatozoa, the majority of motility indicators and the percentage of normal morphology and live spermatozoa increased significantly (p < .05). An increase (p < .05) in concentration of Se and vitamin E in blood and GSH‐Px‐activity and TAC in the spermatozoa was detected. The study results indicate that Se and vitamin E supplementation for 60 days enhances the antioxidant status of spermatozoa and improves the quality of the semen in dogs with lowered fertility.     

Vitrified sperm banks: the new aseptic technique for human spermatozoa allows cryopreservation at −86 °C

The vitrification technique is simple, quick, cost‐effective and has showed a significantly stronger cryoprotective effect in contrast to conventional freezing. The method is based on the rapid cooling of the cell by direct immersion in liquid nitrogen (LN ₂), thereby avoiding the formation of ice crystals, due to the lower risk of water thawing, which impairs cell function. The aim of this study was to evaluate the effect of storage at ?86 °C compared to the conventional ?196 °C (under LN ₂) on essential parameters of the functioning of aseptically vitrified human sperm. Sperm motility, integrity of mitochondrial membrane potential and the rate of DNA fragmentation were determined. The comparison of ?86 °C and ?196 °C demonstrated no statistical difference in sperm progressive motility (73% vs. 77%), integrity of mitochondrial membrane potential (71% vs. 74%) or DNA fragmentation (3.1% vs. 2.9%). In conclusion, aseptically vitrified sperm can be preserved at ?86 °C; eliminating the use of LN ₂ simplifies and significantly reduces the costs associated with storage in sperm banks by decreasing the time and space needed for storage, the effort in finding stored samples, and by improving safety for the operator. However, for prolonged storage further studies are needed.