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.     

Investigations on the influence of bradykinin upon the motility of ram spermatozoa

The influence of bradykinin, a component of the kallikrein-kinin system, on the motility of ram spermatozoa was examined in vitro (photometric motility evaluation, penetration test in cervical mucus of sheep, estimation of the percentage of forward-moving spermatozoa in the thermal resistance test). Whereas the motility was found to be influenced by bradykinin the acrosomal status remained undisturbed by it. With fresh as well as with frozen semen, the drug mainly increased the motility of samples with a low initial motility. With frozen semen, the penetration test revealed greater motility rises at 22 degrees C than at 38 degrees C. The importance of the results in relation to the angiotensin-converting enzyme (kininase) as well as the motility evaluation capabilities of the methods used are discussed. There was, however, no positive drug effect after adding the drug in the course of cryopreservation prior to freezing semen in straws although bradykinin solutions frozen in liquid nitrogen maintained their effectiveness.     

精子染色质完整性对IVF/ICSI临床结局的预测价值

目的:通过分析精子染色质完整性与体外受精胚胎移植(IVF-ET)、卵细胞胞质内单精子注射(ICSI)结局之间的关系,探讨精子染色质完整性检测在辅助生殖技术(ART)中的预测价值。方法:采用精子染色质结构分析试验(SCSA)方法检测187个ART周期中精子的染色质完整性,以精子DNA损伤指数(DFI)为参数,分为高DFI组(DFI≥30%)和低DFI组(DFI<30%),两组中根据采用不同的体外受精方式分为IVF亚组和ICSI亚组。分别比较高DFI组、低DFI组中IVF亚组和ICSI亚组间受精率、卵裂率、D2天胚胎质量、D3天胚胎质量、临床妊娠率差异性。结果:在高DFI组中,行ICSI治疗的夫妇临床妊娠率显著高于行IVF治疗的夫妇,具有统计学意义;在行IVF治疗的夫妇中,比较高DFI组与低DFI组的各临床结局,未见统计学差异;在行ICSI夫妇中,比较高DFI组与低DFI组的各临床结局,未见统计学差异。结论:精子染色质完整性影响辅助生育技术的结局,在选择辅助生殖技术方案时,可作为常规精液检查的一个补充。     

In vitro initiated sperm forward motility in caput spermatozoa: weak and transient

Testicular spermatozoa during journey through epididymis acquire forward motility, which is essential for fertility. To understand the biochemistry of sperm motility initiation, various initiation media have been developed that permitted high level of motility induction (55-60%) in the immature caput-spermatozoa in presence of activating principles: theophylline, bicarbonate and epididymal plasma (EP) when analysed microscopically. Here, we show for the first time using caprine model that stability and quality of in vitro-induced motility in the caput spermatozoa is insignificant in contrast to naturally induced motility in mature cauda spermatozoa. In vitro-induced motility of the immature spermatozoa was lost completely upon the removal of these activators by centrifugation. Selective withdrawal of either EP or HCO(3) by dilution retains 50-60% of the in vitro-induced motility. Spectrophotometric analysis revealed that in vitro-induced vertical motility in immature spermatozoa is too little when compared to mature spermatozoa. In in vitro-initiated caput spermatozoa, cyclic adenosine monophosphate level becomes doubled but lesser than cauda spermatozoa. This revelation concludes that scientific knowledge generated over the years on the basis of in vitro initiation method is insignificant and needs improvisation to delineate biochemical regulation of sperm motility which in turn has remarkable potential in wide biological fields, especially in infertility treatment.     

In vitro effect of gold and silver nanoparticles on human spermatozoa

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60′–120′) with 30, 60, 125, 250 and 500 μM Au/Ag‐NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au‐NPs were characterised and localised with field emission gun‐based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose‐dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag‐NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au‐NPs were localised in spermatozoa, whereas Ag‐NPs were undetectable. In conclusion, Au‐NPs and Ag‐NPs do not appear to be harmful for human spermatozoa up to high concentrations (250–500 μM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.     

Effects of temperature and storage time on the motility,viability, DNA integrity and apoptosis of processed human spermatozoa

The aim of this study was to evaluate motility, viability, DNA integrity and apoptosis of spermatozoa when washed semen samples were kept for up to 12 days at 4–6°C and 25°C. In this experimental study, 26 normozoospermic semen samples were washed twice in Modified Ham's F10 and resuspended in IVF fertilisation medium. Half of the specimens were stored at 4–6°C, and the other half was kept at 25°C for 12 days. The proportions of viable, motile, spermatozoa with double-stranded DNA and apoptotic spermatozoa were examined during storage time. Apoptosis was measured using annexin V-PI staining followed by flow cytometry. Results showed that sperm motility and viability decreased during 12 days of sample storage (p < .001). There was no significant difference between the two temperatures in terms of motility and viability for up to 2 days (p < .05). The percentage of spermatozoa with double-stranded DNA remained unchanged during the 12 days of storage at both temperatures (p > .05). Although there was no difference between the two temperatures in terms of motility, viability and apoptosis during the first two days of storage, storage of spermatozoa at 4–6°C is better than storage for a longer period than storage at 25°C. Sperm DNA resisted against denaturation during storage.     

Preparation and incubation conditions affect the DNA integrity of ejaculated human spermatozoa

Appropriate semen processing and assessment are critical for successful infertility treatment. We investigated whether laboratory procedures including semen preparation and incubation affect sperm DNA integrity. A total of 153 infertile men were involved. Conventional semen parameters and sperm chromatin structure assay （SCSA） parameters, that is, DNA fragmentation index （%DFI） and high DNA stainability （%HDS）, were assessed on the flesh ejaculated semen samples, which were treated and incubated under different conditions. Negative correlations were identified between the %DFI and sperm concentration, motility, progressive motility and morphology. A lower percentage of DFI was detected in spermatozoa when density gradient centrifugation （DGC） was followed by swimup treatment in comparison with DGC alone （P 〈 0.01）. Although the %DFI increased in a time-dependent manner with incubation both at room temperature （RT） and at 37℃ in air, the %DFI after 24 h at RT was significantly lower than that at 37℃ （P 〈 0.05）. Incubation with 5% CO2 was effective in maintaining sperm motility （P 〈 0.01）; however, it induced further elevation of %DFI （P 〈 0.001）. Thus, sperm DNA damage was associated with longer incubation periods. Interestingly, common culture conditions, such as maintaining pH and temperature, compromised the sperm DNA integrity.     

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.     

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.     

In vitro effects of nicotine on human spermatozoa

Washed human spermatozoa from 12 normozoospermic donors were treated with different concentrations of nicotine 0.1, 1.0, 5.0 and 10.0 mm and were compared to spermatozoa suspended in nutrient medium only (control). Computer‐aided sperm analysis was used to assess sperm kinematic properties after 30, 60, 120 and 180 min of incubation. Viability was assessed by means of a dye exclusion staining technique (eosin/nigrosin), while acrosome‐reacted cells were identified under a fluorescent microscope using fluorescein isothiocyanate–Pisum sativum agglutinin as a probe. Nicotine significantly reduced total motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement, beat cross‐frequency, viability and caused spontaneous acrosome reaction at concentrations of ≥5.0 mm after 2 and 3 h of exposure. Nicotine concentrations of 0.1 and 1.0 mm had no significant effect (P < 0.05) on spermatozoa except that 1.0 mm significantly decreased (P < 0.05) sperm progressive motility at 2 and 3 h of incubation as well as viability after 3 h of incubation. This study concludes that the occurrence of high levels of nicotine in the body and seminal fluid might adversely affect fertilisation capacity of human spermatozoa through a mechanism that involves decreased motility, viability and premature induction of the acrosome reaction.     

In vitro effects of peroxynitrite on human spermatozoa

In the present study, the in vitro effects of peroxynitrite on sperm motility, lipid peroxidation and sulfhydryl content were examined. Sperm percentage motility and movement characteristics were assessed by a computer-assisted system. Lipid peroxidation was measured by determining malondialdehyde levels using the thiobarbituric acid (TBA) method. Sperm sulfhydryl content was measured by a spectrophotometric method based on reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) by sulfhydryl groups. Percentage motility, movement characteristics and sulfhydryl content decreased significantly in peroxynitrite-treated samples compared to decomposed peroxynitrite-treated samples. Lipid peroxidation in peroxynitrite-treated samples was significantly higher than in decomposed peroxynitrite-treated samples. These results indicate that peroxynitrite anion may cause sperm dysfunction through lipid peroxidation stimulation and total SH depletion.     

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.     

Sperm chromatin structure assay （SCSA）： a tool in diagnosis and treatment of infertility

Diagnosis of male infertility has mainly been based on the World Health Organization (WHO) manual-based semen parameter's concentration, motility and morphology. It has, however, become apparent that none of these parameters are reliable markers for evaluation of the fertility potential of a couple. A search for better markers has led to an increased focus on sperm chromatin integrity testing in fertility work-up and assisted reproductive techniques. During the last couple of decades, numerous sperm DNA integrity tests have been developed. These are claimed to be characterized by a lower intraindividual variation, less intralaboratory and interlaboratory variation and thus less subjective than the conventional sperm analysis. However, not all the sperm chromatin integrity tests have yet been shown to be of clinical value. So far, the test that has been found to have the most stable clinical threshold values in relation to fertility is the sperm chromatin structure assay (SCSA), a flow cytometric test that measures the susceptibility of sperm DNA to acid-induced DNA denaturation in situ. Sperm DNA fragmentation as measured by SCSA has shown to be an independent predictor of successful pregnancy in first pregnancy planners as well as in couples undergoing intrauterine insemination, and can be used as a tool in investigation, counseling and treatment of involuntary childlessness. More conflicting data exist regarding the role of sperm DNA fragmentation in relation to fertilization, pre-embryo development and pregnancy outcome in in vitro fertilization and intracytoplasmic sperm injection (ICSI).     

Capacitation status and in vitro fertility of boar spermatozoa: effects of seminal plasma, cumulus-oocyte-complexes-conditioned medium and hyaluronan

In the present study, the effects of seminal plasma (SP), cumulus-oocyte-complexes (COCs) conditioned medium (CCM) and hyaluronan (HA) on functional changes and in vitro fertilizing ability of porcine spermatozoa were examined. In in vitro fertilization (IVF) experiments, 10% (v/v) of exogenous SP in the fertilization medium prevented sperm penetration (using fresh-extended and frozen-thawed ejaculated spermatozoa). Analysis of frozen-thawed CCM revealed a HA content to levels of 30 ng/mL per incubated COC. Presence of frozen-thawed CCM did not, however, prove effective to increase (furthermore decreasing) oocyte penetration in vitro, and neither did supplementation with exogenous HA at the same concentration as that present in the CCM (secreted by COCs). Analysis of sperm capacitation using the chlortetracycline (CTC) assay showed that frozen-thawed CCM had no elevating effect on 'B-pattern' spermatozoa (implying capacitation-like changes) and that addition of 10% (v/v) SP held spermatozoa in the 'F-pattern' (intact) status. Dose of 500 microg/mL HA and freshly prepared CCM increased, however, the frequency of capacitated spermatozoa (B-pattern) without resulting in increased rates of 'AR-pattern' (acrosome-reacted) spermatozoa, compared with controls. The present results confirm the decapacitating effect of SP and suggest capacitating actions of HA (dose-related) and CCM (freshly prepared) on boar spermatozoa in vitro. The unclear effects of frozen-thawed CCM and a low dose of HA on penetration rates of boar spermatozoa call for further researches of their function in vivo.     

The effects of acidic epididymal glycoprotein (AEG) and some other proteins on the motility of rat epididymal spermatozoa

Acidic epididymal glycoprotein (AEG) had a slight stimulatory effect on the motility of spermatozoa showing low initial motility which had been removed by micropuncture from the caput and corpus epididymidis of rats. However, similar effects were seen when bovine serum albumin (BSA) or purified gammaglobulin against AEG was used instead of AEG. Furthermore, BSA, normal rabbit serum and serum from a rabbit immunized against AEG reduced the motility of spermatozoa which showed high initial motility after removal from the caput, while AEG had no effect. These studies emphasize the importance of the effects of proteins on the motility of spermatozoa but do not provide any clear evidence for a specific effect of AEG.     

Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa

The accuracy of computer-assisted sperm morphometry analysis (CASMA) depends on the careful preparation, fixation and staining of spermatozoa. The efficiency of CASMA may be enhanced by developing optimized protocols. The aim of the present study was to evaluate the influence of sperm washing and the use of three staining techniques [rapid Panoptic, Hemacolor and Harris's Haematoxylin (HH)] on image-processing accuracy and boar sperm head morphometry. Sperm washing had a significant effect on samples stained with rapid Panoptic, increasing the percentage of correctly binarized sperm heads and the contrast between cells and background. However, rapid Panoptic yielded the lowest percentage of properly digitized sperm heads. HH provided the highest cell/background contrast, and also greater sperm head staining intensity, but discrimination of sperm midpieces was considered insufficient. Hemacolor occupied an intermediate position, providing acceptable colour intensity and satisfactory cell/background contrast. Use of different staining procedures prompted dimensional differences in sperm head morphometry. Significant differences between animals were observed for all morphometric parameters. Low within-animal variation coefficients reflected a homogeneous sperm head population. Between-animal variation coefficients were relatively high for Hemacolor and HH, and significantly high for the rapid Panoptic stain. Using Panoptic and HH, stable morphometric measurements required at least 100 properly digitized sperm heads rather than 200, while Hemacolor required only 50 spermatozoa. These results indicate that both washing of semen and staining procedures significantly affect the accuracy of image processing and sperm head dimensions. Hemacolor and HH proved to be the best staining techniques for evaluating sperm head dimensions in boar.     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

The influence of pentoxifylline on motility and viability of spermatozoa from normozoospermic semen samples

In order to evaluate the effects of pentoxifylline on sperm motility and longevity, a controlled in-vitro study was conducted on normozoospermic donor semen samples using the Cellsoft automated system for sperm motility analysis. After incubation and selection, pentoxifylline was found to improve the recovery of spermatozoa and to increase their velocity. In the subgroup of progressively motile spermatozoa, curvilinear velocity was also enhanced. It is concluded that pentoxifylline has an effect on the vigour, but not on the pattern, of sperm motion. Pentoxifylline did not improve the motility characteristics of senescent spermatozoa in normozoospermic sperm samples. Sperm survival, as shown by supra-vital staining, and motility longevity both decreased with time after pentoxifylline treatment.