Computer-aided Sperm Analysis Research Articles

Busulfan induces apoptotic and cytotoxic effects on testis and epididymal sperm of adult male mouse following low dose treatment

At adequate concentrations busulfan selectively attacks the dividing spermatogonia and spermatocytes. But, there is not enough information about side effects of low dose busulfan. The aim of this study was to assess changes that occur on seminiferous tubules morphology and epididymal sperm of adult male mouse following treatment with low dose busulfan. So, adult male NMRI mice (25-35 g) were assigned in three groups including; Group 1: Control, Group 2; treated with busulfan for 21 days (0.06 mg/kg/day) and Group 3: treated with busulfan for 21 days (0.8 mg/kg/day). The effects of busulfan on seminiferous tubules morphology and epididymal sperm were evaluated by light microscopy, Computer–Aided Sperm Analysis (CASA), MTT assay and flowcytometry. The results showed that busulfan caused significant germinal epithelium destruction in treated mice versus control. Also, busulfan had significant cytotoxic and apoptotic effects in epididymal sperm of treated mice. The percentages of early apoptotic, late apoptotic and necrotic sperms in groups 2 and 3 demonstrated significant increase versus control. Also, analysis of sperm parameters and sperm viability using CASA and MTT assay demonstrated significant differences between control and busulfan treated mice. In conclusion, chemotherapy with low dose busulfan causes significant changes on sperm viability, sperm parameters and morphology of seminiferous tubules. All side effects of busulfan are dose dependent and they are considerable in mice treated with 0.8 mg/kg busulfan. Hence, the treatment with low dose busulfan may reduce toxicity of chemotherapy.

Ability of Escherichia coli to produce hemolysis leads to a greater pathogenic effect on human sperm

To determine the effect on human sperm of Escherichia coli strains separated on the basis of their ability to produce hemolysis. Experimental study. University-based laboratory. Semen samples from healthy donors. Five million sperm, selected via the swim-up method, were incubated with 3E. coli concentrations to obtain ratios of sperm to E. coli of 1:2, 1:16, and 1:128. The E. coli strains were: a hemolytic isolated strain (H), a nonhemolytic American Type Culture Collection strain (NH-ATCC), and a nonhemolytic isolated strain (NH-I). Aliquots of human sperm were used to measure progressive motility using computer-aided sperm analysis, mitochondrial membrane potential (ΔΨm) with a JC-1 (5,5',6,6' tetrachloro-1,1',3,3'-tetraethylbenzamidazolocarbocyanin iodide) and propidium iodide stain, and intracellular reactive oxygen species (iROS) with a dihydroethidium (DHE) stain. Sperm ΔΨm and iROS were measured by flow cytometry. Sperm vitality was considered the mean of propidium iodide-negative and DHE-negative cells. Sperm incubated with the H strain in a 1:2 sperm to bacteria ratio demonstrated a significant decrease in motility and ΔΨm, and an increase of iROS. The NH-ATCC strain decreased sperm motility and ΔΨm, but in a ratio of sperm to bacteria of 1:128; it increased iROS at a ratio of 1:16. The NH-I strain did not affect the analyzed sperm functions, even at a 1:128 sperm to bacteria ratio. Results show a greater pathogenic effect on human sperm of E. coli strains with, versus without, hemolytic capacity.

The future of computer-aided sperm analysis.

Computer-aided sperm analysis (CASA) technology was developed in the late 1980s for analyzing sperm movement characteristics or kinematics and has been highly successful in enabling this field of research. CASA has also been used with great success for measuring semen characteristics such as sperm concentration and proportions of progressive motility in many animal species, including wide application in domesticated animal production laboratories and reproductive toxicology. However, attempts to use CASA for human clinical semen analysis have largely met with poor success due to the inherent difficulties presented by many human semen samples caused by sperm clumping and heavy background debris that, until now, have precluded accurate digital image analysis. The authors review the improved capabilities of two modern CASA platforms (Hamilton Thorne CASA-II and Microptic SCA6) and consider their current and future applications with particular reference to directing our focus towards using this technology to assess functional rather than simple descriptive characteristics of spermatozoa. Specific requirements for validating CASA technology as a semi-automated system for human semen analysis are also provided, with particular reference to the accuracy and uncertainty of measurement expected of a robust medical laboratory test for implementation in clinical laboratories operating according to modern accreditation standards.

Comparison of Electro Ejaculator and Artificial Vagina on Semen Collection from South African Indigenous Goat Following Assessment by Computer Aided Sperm Analysis

South African indigenous bucks are raised under extensive conditions and are more likely to reject artificial vagina (AV) due to their limited contact with human beings, as they are less handled. The purpose of the study was to compare goat semen collection techniques used for goats based on semen traits of South African indigenous bucks during the natural breeding season. A total of eight South African indigenous bucks were used, four bucks per semen collection technique due to their scarcity. Before semen collection with AV group, ten bucks were trained and exposed to AV. Semen was then collected from both groups over a period of six weeks per individual buck. Semen volume, pH and sperm concentration were evaluated immediately. The Computer Aided Sperm Analysis (CASA) assessed the sperm motility rates. Sperm morphology was evaluated using a fluorescence microscope with the aid of eosin-nigrosin staining. Analysis of variance was used to test the differences among the semen collection groups following assessment by CASA. Semen collected with EE resulted in higher semen volume (1.1 mL) compared with AV (0.5 mL) technique. However, AV technique resulted in higher total sperm motility and rapid movement (91.9% and 48.7%), live sperm (72%) and sperm concentration (635.6 × 106 sperm/mL) compared with EE (86.3% and 30.4%; 69% and 463.7 × 106 sperm/mL, respectively). There were no significant differences observed in pH and sperm morphology characteristics between two semen collection techniques. Moreover, EE had higher sperm moving in a progressive (53.6%) and medium rate compared with AV (41.1% and 21.9%) technique. It was concluded that the AV yielded higher total sperm motility rate, sperm concentration and rapid sperm movement compared with EE. Therefore, AV was more suitable for semen collection of South African indigenous bucks, even though training was problematic to such indigenous bucks.

Effects of Inclusion of Polyunsaturated Fatty Acids and Antioxidants on Spermatozoa Morphology of Potchefstroom Koekoek Cockerels

This study was conducted to determine the effects of increasing dietary polyunsaturated fatty acids (PUFA) and antioxidants (Se and vitamin E) on spermatozoa morphology. At 24 weeks of age, Potchefstroom Koekoek cockerels (n = 60) were selected from the base population. Cockerels were randomly allocated into five dietary treatments with 12 cockerels per dietary treatment. The diets consisted of commercial layer diet (CL), commercial cockerel diet (CC), modified layer diet (ML), modified layer diet +PUFA (MLP), and modified layer +PUFA+ antioxidants (MLPA) diet. At 36 weeks of age, six cockerels with ≥ 75% sperm motility rate following analysis by Computer Aided Sperm Analysis (CASA) were selected from the base population in each dietary treatment group. Semen volume, sperm cells concentration and dead cells did not differ significantly among any of the groups. However, live and normal spermatozoa were significantly higher in the MLPA group. The bent mid-piece, bent mid-piece plus droplets, proximal droplets and distal droplets were significantly lower in the MLPA group. From this study it can be concluded that supplementation of polyunsaturated fatty acids and antioxidants may be of vital importance for normal morphology characteristics of spermatozoa in Potchefstroom Koekoek cockerels.

Visual Tracking for Abrupt Motions of Human Sperm Using Smoothing Stochastic Approximate Monte Carlo

Characteristic of sperm movement or sperm motility is one of important quality of sperm. Computer-aided Sperm Analysis (CASA) systems have attempted to give more accurate information about sperm motility. For collecting a single good sperm in Intracytoplasmic Sperm Injection (ICSI) procedures, achieving correct sperm trajectory is necessary. On the other hand, visual tracking of a sperm is a challenging issue because: (i) sperms have similar size and shape, (ii) a good sperm moves fast and has unpredictable motions. Furthermore, our sperm videos were taken by regular cameras which have low frame rate (about 20 fps), which cause the sperm-motion more abrupt. To address this problem, we propose searching driven stochastic sampling framework for visual tracking of abrupt motions. In here, searching algorithm will give promising regions of the target, and thus can replace the role of transition model in tracking system. For searching, we employ Coherency Sensitive Hashing (CSH) in a window to constrain search space in each frame, called as Search Window. And then for stochastic sampling framework, we adopt Smoothing Stochastic Approximate Monte Carlo (SSAMC) to specifically finding the target. The experimental results on human-sperm sequences show that: (i) comparing to our previous tracker which use geometric transition dynamic model, the proposed tracker can handle abrupt motions robustly, (ii) by using appropriate Search Window, the tracker can localize the sperm target and restrict observation of other similar sperms.

349 SUPEROVULATORY RESPONSE AND EMBRYO QUALITY RECOVERED FOLLOWING FLUSHING NGUNI HEIFERS AND COWS

Most of the South African indigenous cattle breeds are facing genetic degradation due to unselective crossbreeding and irregular matings especially in small scale farming. The aims of the study were to compare superovulatory (SO) response, fertilization rate and to evaluate embryo quality recovered from superovulated Nguni stud cows and heifers. Nguni stud cows (n = 15) and heifers (n = 10) aged 4–6 and 2 years, respectively, were used as embryo donors for ex situ conservation. Nine days following heat observation for the first oestrus synchronisation protocol, superstimulation of donor cows and heifers were administered with a total of 350 mg of FSH (Follotropin-V®, porcine pituitary glands extract) divided into 2 injections daily 12 h apart (6 a.m. and 6 p.m.) for 4.5 days on a decreasing dosage. Two injections of prostaglandin F2α were administered on Day 3 and 4 of FSH injections. Semen was collected from 2 Nguni bulls (bull 1 and 2) and assessed by computer aided sperm analysis before artificial insemination (AI). The AI was conducted with fresh semen 3 times, 12 h apart, and the first AI was performed at the onset of oestrus. Semen from bull 1 was used to inseminate the cows, and bull 2 was used for heifers. Embryos were flushed 7 days after AI using a nonsurgical technique. Embryos were evaluated under stereo microscope and classified according to IETS standard codes (code 1, good and excellent; code 2, fair). Recovered embryos were then vitrified and stored for future use. Superovulatory response was measured by counting number of corpus lutea in each ovary. Data were analysed using 1-way ANOVA (SAS, 2003, SAS Institute Inc., Cary, NC, USA). There was no statistical difference between bull 1 (93.7%) and bull 2 (83.5%) on total sperm motility rate. Furthermore, no significant differences were recorded on fertilization rate between cows (67.5%) and heifers (53.5%). There was also no significant difference on the proportion of Nguni cows (40%) and heifers (40%) that responded to superovulation treatment. There was a significant difference on the ovary reaction (number of corpus lutea) of cows (11.3 ± 1.41) and heifers (4.0 ± 0.57). There were no significant differences recorded on the embryo quality recovered between Nguni cows (code 1, 2.5 ± 1.00; code 2, 1.3 ± 0.59) and heifers (code 1, 0.8 ± 0.41; code 2, 1.0 ± 0.36). However, cows had higher numbers of unfertilized ova than heifers (5.5 ± 1.05 and 1.8 ± 0.47, respectively) and degenerate embryos (3.7 ± 1.00 and 1.3 ± 0.39, respectively). Although superovulatory response of both Nguni cows and heifers was low, Nguni cows had higher ovarian response than heifers. Moreover, the quality of embryos recovered was similar for both Nguni cows and heifers.

Association of DNA methylation and mitochondrial DNA copy number with human semen quality.

Whether there is a relationship between quality, DNA methylation, and mitochondrial DNA (mtDNA) copy number in human-derived sperm specimens is unknown. A cohort (n = 118) of male partners of couples who were undergoing fertility assessment because of an idiopathic inability to conceive were recruited. Sperm motility parameters were determined by computer-aided sperm analysis (CASA), while sperm quality was assessed using World Health Organization criteria, mtDNA copy number was measured by real-time PCR, and DNA methylation patterns were analyzed employing high-melting resolution PCR and bisulfite sequencing PCR. The mtDNA copy number negatively correlated with semen parameters, including sperm motility, concentration, morphology, progression, and motion characteristics (r for -0.19 to -0.54; P < 0.05 for all). As a surrogate marker for global DNA methylation, LINE-1 negatively correlated with sperm motility (r = -0.25; P = 0.009). Meanwhile, after adjustment for age, length of abstinence, smoking, and alcohol intake, there was a suggested association for increased LINE-1 methylation and mtDNA copy number tertiles versus sperm motility (odd ratios were 1.0, 2.6, and 4.7, and 1.0, 2.5, and 4.9, respectively). Altered mtDNA copy number and DNA methylation may serve as genetic and epigenetic markers to assess human sperm quality together with CASA parameters.

Use of computer-assisted semen analysis for evaluation of Rosy-faced lovebird (Agapornis roseicollis) semen collected in different periods of the year

The seminal characteristics of the Rosy-faced lovebird (Agapornis roseicollis) were analyzed, both in and out of season, using a computer-aided sperm analyzer. Avian semen collection and artificial insemination techniques have great potential for captive breeding programs, and computer-aided sperm analyzer allows an objective and quantitative assessment of sperm motility and kinetics. Although Agapornis roseicollis is a largely diffuse species, its seminal parameters have never been fully investigated. Using the massage technique, 38 ejaculates were collected in the breeding season, and 6 ejaculates were collected outside of the breeding season. Semen color, volume, degree of contamination, spermatozoa concentration, total and progressive motility, and kinetics parameters were recorded. Seasonal significant differences were found in the ejaculate volume (1.6 ± 0.6 and 1.1 ± 0.2 μL in and out season, respectively, P < 0.01) and spermatozoa concentration (7194.0 ± 6735.1 × 106 and 327.5 ± 314.0 × 106 spermatozoa/mL, P < 0.01); among the motility parameters, only beat cross frequency, indicating the frequency of flagellar beats, was significantly higher out of the reproductive season (29.8 ± 2.6 vs. 24.5 ± 3.8 Hz, P < 0.01). There was very large individual variation in semen characteristics that could qualify a male as a potentially good or bad semen donor for future assisted reproduction in captivity.

Potential importance of transition metals in the induction of DNA damage by sperm preparation media.

What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen samples employed for this analysis involved a cohort of >50 unselected donors and at least three independent samples were used for each component of the analysis. The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®); analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.

Effect of mitochondrial calcium uniporter blocking on human spermatozoa.

Calcium (Ca(2+) ) regulates a number of essential processes in spermatozoa. Ca(2+) is taken up by mitochondria via the mitochondrial calcium uniporter (mCU). Oxygen-bridged dinuclear ruthenium amine complex (Ru360) has been used to study mCU because it is a potent and specific inhibitor of this channel. In bovine spermatozoa, it has been demonstrated that mitochondrial calcium uptake inhibition adversely affects the capacitation process. It has been demonstrated in human spermatozoa that mCU blocking, through Ru360, prevents apoptosis; however, the contribution of the mCU to normal human sperm function has not been studied. Therefore, the aim of this study was to evaluate the effect of mCU blocking on human sperm function. Spermatozoa obtained from apparently healthy donors were incubated with 5 and 10μm Ru360 for 4h at 37°C. Viability was assessed using propidium iodide staining; motility was determined by computer-aided sperm analysis, adenosine triphosphate (ATP) levels using a luminescence-based method, mitochondrial membrane potential (ΔΨm) using JC-1 staining and reactive oxygen species (ROS) production using dihydroethidium dye. Our results show that mCU blocking significantly reduced total sperm motility and ATP levels without affecting sperm viability, ΔΨm and ROS production. In conclusion, mCU contributes to the maintenance of sperm motility and ATP levels in human spermatozoa.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.

A study on aconitase-2 expression change and possible cryo-injury mechanisms after human sperm cryo-thawed

Objective To investigate the change of aconitase-2 (ACO2) between pre-freeze and post-thaw and possible mechanism of cryo-injury.Methods The parameters of sperm from 15 donaters were analyzed by computer-aided sperm analysis (CASA) and revealed a loss in motility; the Western blotting revealed a reduction of ACO2 content after thawing and ACO2 was located in mitochondrial shealth by immunofluorescence test.The change of adenosine-triphosphate (ATP) contents was determined by Chemiluminescence test.The sperm mitochondrial membrane potential was determined by JC-1 test and indirectly revealed mitochondrial function loss by red/green fluorescence,and quantification of fluorescence was determined by Flow Cytometry.T-test was used to compare these two groups.Results Compared with prefreezing group,the motility decreased 18％,ATP contents decreased 32％ and R1 proportion reduced by 27％ (P ＜ 0.05).Conclusion ACO2,degraded after thawing and resulted in mitochandrial functional iniury and reduction in ATP contents,was responsible for sperm motility loss. Key words: Sperm, human; Cryopreservation; Thawing; Aconitase-2 ; Injury

53 COMPARISON OF 4 DIFFERENT CRYOPROTECTANTS ON FREEZING NGUNI BULL SPERMATOZOA EVALUATED BY COMPUTER-AIDED SPERM ANALYSIS

Cryopreservation has been reported to damage approximately 40 to 50% of viable spermatozoa in bulls. It is critical to evaluate frozen-thawed spermatozoa with computer-aided sperm analysis (CASA) and find a suitable cryoprotectant for Nguni semen. The study was conducted to compare cryo-effectiveness of glycerol (GLY), dimethyl sulfoxide (DMSO), propanediol (PND), and ethylene glycol (EG) cryoprotectants at 12% concentrations during freezing of Nguni bull semen. Semen was collected from 18 stud Nguni bulls of proven fertility with the use of an electro-ejaculator. Collected semen samples were kept in a thermo flask (37°C) and transported to the laboratories. Semen samples were pooled and sperm motility rate was evaluated using CASA. Semen was then diluted (1 : 2; vol : vol) with egg-yolk citrate extender supplemented with either 12% GLY, DMSO, PND, or EG. Semen samples were equilibrated for 4 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled rate programmable freezer using a customized freezing curve (from 5 to –5°C at 0.008°C min–1 and from –3 to – 130°C at 6°C min–1). Following thawing of semen, artificial insemination was conducted on 104 oestrus-synchronised Nguni cows. The IVF was also conducted on 120 oocytes to check the cleavage rate. Data were analysed using ANOVA. There was a significant difference (P &lt; 0.05) between raw total sperm motility (94.70 ± 2.63) and frozen-thawed sperm total motility with GLY (77.80 ± 11.03), EG (20.35 ± 11.86), DMSO (15.68 ± 10.14), and PND (11.19 ± 11.27) groups. The pregnancy rate following artificial insemination was 75.9% and a total of 86.6% oocytes had cleaved after fertilization with frozen (12% GLY)/thawed semen. In conclusion, cryopreservation process reduced sperm motility and velocity rates, regardless of cryoprotectant. Egg-yolk citrate extender supplemented with 12% glycerol had recorded the highest post-thaw sperm motility rate.

Evaluation of the spermicidal and contraceptive activity of Platycodin D, a Saponin from Platycodon grandiflorum.

BackgroundThe extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity, as well as the safety, of Platycodin D (PD), a major saponin in Platycodon grandiflorum.MethodsUsing the computer-aided sperm analysis (CASA) test criteria, the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation.ResultsA dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD, indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats, was non-DNA damaging and was not harmful to the vaginal tissue in the rats.ConclusionPD has significant spermicidal activity that should be explored in further studies.

In vitroeffects 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.

Successful use of aromatase inhibitor letrozole in NOA with an elevated FSH level: a case report

Aromatase inhibitors inhibit the conversion of testosterone to oestrogens and could reduce serum oestradiol concentrations. Letrozole is one of aromatase inhibitors frequently used in treatment of men with oligospermia. We present the case of an infertile man with small testes and an elevated FSH level, which was diagnosed as NOA, hypospermatogenesis proven by testicular biopsy. After taking letrozole for 3months, semen analyses by computer-aided sperm analysis present that this man had normal spermatogenesis. This is the first case report of the activation of spermatogenesis, in man who was NOA with elevated FSH level, resulting from the use of the one of aromatase inhibitors.

Cluster analysis reveals a binary effect of storage on boar sperm motility function.

Storage of liquid-preserved boar spermatozoa is associated with a loss of fertilising ability of the preserved spermatozoa, which standard semen parameters barely reflect. Monitoring responses to molecular effectors of sperm function (e.g. bicarbonate) has proven to be a more sensitive approach to investigating storage effects. Bicarbonate not only initiates capacitation in spermatozoa, but also induces motility activation. This occurs at ejaculation, but also happens throughout passage through the oviduct. In the present study we tested whether the specific response of boar sperm subpopulations to bicarbonate, as assessed by motility activation, is altered with the duration of storage in vitro. Three ejaculates from each of seven boars were diluted in Beltsville thawing solution and stored at 17°C. Only minor changes in the parameters of diluted semen were revealed over a period of 72h storage. For assessment of bicarbonate responses, subsamples of diluted spermatozoa were centrifuged through a discontinuous Percoll gradient after 12, 24 and 72h storage. Subsequently, spermatozoa were incubated in two Ca2+-free variants of Tyrode's medium either without (TyrControl) or with (TyrBic) 15mM bicarbonate, and computer-aided sperm analysis motility measurements were made. Cluster analysis of imaging data from motile spermatozoa revealed the presence of five major sperm subpopulations with distinct motility characteristics, differing between TyrBic and TyrControl at any given time (P<0.001). Although there was an increasing loss of motility function in both media, bicarbonate induced an increase in a 'fast linear' cohort of spermatozoa in TyrBic regardless of storage (66.4% at 12h and 63.9% at 72h). These results imply a binary pattern in response of sperm motility function descriptors to storage: although the quantitative descriptor (percentage of motile spermatozoa) declines in washed semen samples, the qualitative descriptor (percentage of spermatozoa stimulated into fast linear motion by bicarbonate) is sustained independent of the duration of storage.

Computer-aided sperm analysis: past, present and future

Computer-aided sperm analysis (CASA) system has been accepted and used commonly as a routine semen analysis instrument in hospital clinical laboratories worldwide. However, technicians in clinical laboratories have little informed knowledge about the principles of CASA system and the sources of analysis errors. In this review, we focus on the concept of CASA, the development course of CASA technology, the clinical application of CASA systems and the factors influencing the accuracies of results, such as frame rate, sperm counting chambers affiliated to the CASA system, algorithms and sperm concentration. These factors and lack of internal quality control may result in huge errors of the CASA between systems and laboratories. It is therefore necessary to perform the standardisation and quality control for CASA.

Effect of 830-nm diode laser irradiation on human sperm motility

Sperm motility is known as an effective parameter in male fertility, and it depends on energy consumption. Low-level laser irradiation could increase energy supply to the cell by producing adenosine triphosphate. The purpose of this study is to evaluate how the low-level laser irradiation affects the human sperm motility. Fresh human semen specimens of asthenospermic patients were divided into four equal portions and irradiated by 830-nm GaAlAs laser irradiation with varying doses as: 0 (control), 4, 6 and 10 J/cm(2). At the times of 0, 30, 45 and 60 min following irradiation, sperm motilities are assessed by means of computer-aided sperm analysis in all samples. Two additional tests [HOS and sperm chromatin dispersion (SCD) tests] were also performed on the control and high irradiated groups as well. Sperm motility of the control groups significantly decreased after 30, 45 and 60 min of irradiation, while those of irradiated groups remained constant or slightly increased by passing of time. Significant increases have been observed in doses of 4 and 6 J/cm(2) at the times of 60 and 45 min, respectively. SCD test also revealed a non-significant difference. Our results showed that irradiating human sperms with low-level 830-nm diode laser can improve their progressive motility depending on both laser density and post-exposure time.