Sperm Motility Analysis System Research Articles

P-022 Relationship between sperm movement characteristics assessed by a sperm motility analysis system (SMAS) and normal fertilization rates in vitro

Abstract Study question Is there a relationship between movement characteristics of human sperm assessed by SMAS and normal fertilization rates in vitro? Summary answer The curvilinear path wobble of motile sperm before insemination is predictive of normal fertilization rates after in vitro fertilization (IVF). What is known already Assessment of semen parameters is very important in both the diagnosis of male infertility and the selection of methods for treatment with conventional-IVF (C-IVF) or intracytoplasmic sperm injection (ICSI). Several studies have reported that objectively movement characteristics of motile sperm assessed by SMAS are related to the functional capacity of human sperm. However, there are no established the objective indicators and criteria for sperm motility characteristics assessed by SMAS. In this study, we investigated the utility of the movement characteristics of motile sperm measured by SMAS as an indicator of normal fertilization rates. Study design, size, duration Retrospective experimental trial conducted in a single IVF center between January 2018 and February 2020. Sperm movement characteristic analysis was performed using the SMAS in 300 IVF cycles using sperm from 270 men. At least three metaphase II oocytes were inseminated in C-IVF. The SMAS estimates were measured in semen and before insemination. This study was approved by the Ethics Committee of the Institutional Review Board (IRB) of Kinutani Women’s Clinic, Hiroshima, Japan. Participants/materials, setting, methods Motile sperm from at least 3 different fields were observed in each sample and their parameters were examined. First, we investigated the correlation between the normal fertilization rates and sperm motility characteristics. Next, the group comprising 300 cycles was classified into a group of a ≥ 60% normal fertilization (group 1: 229 cycles) and a group with <60% normal fertilization (group 2: 71 cycles) to assess the sperm characteristics described below. Main results and the role of chance The following indicators were evaluated: amplitude lateral head displacement (ALH), best cross frequency (BCF), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN=VSL/VCL), straightness (STR=VSL/VAP), and curvilinear path wobble (WOB=VAP/VCL).There were significant correlations between normal fertilization rates and semen parameters, including VCL (r = 0.014; P < 0.05) and ALH (r = 0.177; P < 0.05). However, there were no significant correlations for other parameters. Moreover, there were no significant correlations between normal fertilization rates and sperm parameters before insemination. The SMAS estimates in semen and sperm before insemination were compared between group 1 and group 2. There were no significant differences in most baseline characteristics across groups, except for the number of normally fertilized oocytes. However, there were significant differences in sperm WOB before insemination between group 1 and group 2 (0.589 vs. 0.572; P < 0.05). Sperm motility before insemination was more curvilinear in group 1 than in group 2. The optimal cut-off value for WOB of motile sperm before insemination for predicting≥60% normal fertilization was 0.587 (AUC was 0.60; P < 0.05). These results were suggested that the WOB of sperm before insemination is useful as an indicator in determining the selection of methods for fertilization in vitro. Limitations, reasons for caution The main limitations of this study are its retrospective design and small sample size. A prospective study is necessary to further clarify the prediction criteria. Wider implications of the findings This study suggests that the WOB of motile sperm before insemination may be used as one of the indicators to predict good fertilization rates after C-IVF. Trial registration number N.A.

Protective effect of Scrophularia striata combined with trehalose and cysteine added to diluents on cryopreservd goat epididymal sperm

Objective: To evaluate antioxidant effects of Scrophularia (S.) striata ethanol extract, trehalose and cysteine added to diluents on cryopreserved goat epididymal sperms. Methods: Motility and standard motion parameters of sperm were assessed by using computer assisted sperm motility analysis system. Sperm viability was evaluated by eosin-nigrosin staining method. Hypo-osmotic swelling test was used to evaluate membrane health. Thiobarbituric acid testing was used to measure malondialdehyde (MDA) concentrations. To assess DNA fragmentation, sperm chromatin dispersion test was used. In Experiment 1, treatments consisting of basal Tris diluent supplemented with 25, 50 or 100 μg/mL of S. striata ethanol extract gave the best concentration to the freezing diluents. Experiment 2 was carried out to compare the best concentration of S. striata ethanol extract (50 μg/mL) resulting from the first experiment with 150 mM trehalose and/or 5 mM cysteine alone or in combination. Results: S. striata ethanol extract (50 μg/mL) significantly increased sperm viability, motility and progressive motility and at the same time decreased MDA concentration and DNA fragmentation compared to other treatments (P<0.05). In addition, all treatment groups resulted in viability, membrane health, total motility, progressive motility, curvilinear velocity, straight-line velocity higher and MDA lower compared to the control group (P<0.05). Acrosome integrity was significantly higher in 50 μg/mL of S. striata ethanol extract combined with cysteine, trehalose, or cysteine+trehalose groups than those in the control, trehalose, cysteine, and 50 μg/mL of S. striata ethanol extract groups (P<0.05). Regarding DNA, extenders supplemented with 50 μg/mL of S. striata ethanol extract, 50 μg/mL of S. striata ethanol extract+trehalose, and 50 μg/mL of S. striata ethanol extract+trehalose+cysteine were superior to other treatments. Conclusions: Adding 50 μg/mL of S. striata ethanol extract alone or in combination with trehalose and cysteine can improve the quality of cryopreserved epididymal sperms of goats.

SPERM DNA FRAGMENTATION AND FUNCTIONAL FEATURES OF HUMAN SPERM SELECTED BY MICROFLUIDIC SORTING FOR CLINICAL USE

High sperm DNA damage is associated with decreased normal fertilization, embryo development and pregnancy rates, and an increased miscarriage rate. Thus, selecting sperm with minimal DNA damage is important for assisted reproductive technology. A microfluidic sperm selection chamber (MSS, ZyMōt™; DxNow) is a device designed to collect sperm with high motility and chromatin integrity, which has recently been clinically used for ICSI/IVF. Here we aimed to comprehensively evaluate MSS compared to density-gradient centrifugation (DGC) in terms of (1) sperm DNA fragmentation by sperm chromatin dispersion (SCD) test and comet assay, (2) sperm motility and morphological features of motile sperm, and (3) blastocyst development after ICSI and clinical outcomes after vitrified-thawed single blastocyst transfers (vSBT). Sperm analysis was performed in 23 cases between October 2020 and February 2021. SCD test was optimized as a rapid procedure (≤30 min), with sperm showing a halo deemed normal, and those without a halo abnormal. Comet assay results were analyzed using CometScore 2.0, with comparison of %Tail DNA, Tail Length (TL), and Tail Moment (TM). Sperm motility parameters were investigated using the Sperm Motility Analysis System (SMAS, Dietect). To assess motile spermatozoa characteristics, middle piece morphology and active mitochondrial distribution were labeled by MitoTracker-Red. The reactive oxygen species (ROS) level was labeled using CM-H2DCFDA. ICSI outcomes (normal fertilization, blastocyst development, and pregnancy rates) were compared between DGC and MSS for 620 cycles between September 2019 and December 2020. In SCD test, MSS yielded a significantly lower abnormality rate than for raw sperm and DGC in all cases. The comet assay showed that %Tail DNA, TL, and TM were significantly lower with MSS than for raw sperm or DGC. Sperm motility rates were significantly higher with DGC and MSS than for raw sperm. Sperm motility analysis demonstrated significantly lower straightness, linearity, and beat-cross frequency (Hz) with MSS compared to DGC. The morphological abnormality rate of the sperm midpiece was significantly higher with MSS than DGC. Abnormal sperm midpieces exhibited significantly high ROS activity around mitochondria. Fertilization after ICSI and blastocyst rates were similar between MSS and DGC, while the vitrified blastocyst rate was significantly higher with MSS than DGC. Clinical pregnancy and miscarriage rates after vSBT did not significantly different between MSS and DGC. MSS-selected sperm had high DNA integrity and were homogeneous. The sperm had a higher motility rate, but the characteristic sperm motility pattern was meandering motion. Additionally, MSS did not exclude sperm that were morphologically abnormal in the middle piece with high ROS levels. MSS-ICSI yielded an improved vitrified blastocyst rate, and MSS is an effective device for sperm collection for ICSI.

Ability of Ureaplasma parvum to invade mouse sperm, fertilize eggs through infected sperm, and impair mouse sperm function and embryo development

To examine the effect of Ureaplasma parvum (U. parvum) infection on mouse sperm motility, structure, and fertilizing ability and on embryo development. Invitro model of the effects of U. parvum serovar 3 infection on mouse sperm. Basic research laboratory. None. Mice. Mouse sperm motility was examined using the swim-up method, and their motility parameters were analyzed using the sperm motility analysis system. Localization and invasion of U. parvum were observed with fluorescence, confocal, and scanning electron microscopy. After invitro fertilization with U. parvum-infected sperm, the quality of the fertilized egg and embryo development were assessed. U. parvum was attached and internalized into mouse sperms and localized mainly at the sperm head and midpiece. U. parvum-infected mouse sperms exhibited decreased motility in a dose- and duration-dependent manner. Electron micrographs revealed that U. parvum infection induced the aggregation and morphological destruction of mouse sperm. Infected mouse sperm transported U. parvum into the fertilized egg with reduced fertilization rates, and infected embryo development was impaired. U. parvum infection caused deterioration of the mouse sperm quality and its functions, which affected the fertilization rate and embryo development.

Comparison between semen parameters in specimens collected early in the morning and in the evening

ABSTRACTThe majority of facilities in Japan that offer artificial insemination as part of assisted reproduction programs currently perform semen collection in the early morning. The total motile sperm count of the semen used in intrauterine insemination is an important factor in achieving successful fertilization and subsequent childbirth. The present study was initiated to determine whether semen parameters varied with the time of day at which the semen sample was collected. The study subjects were 20 fertile males and 20 infertile males with abnormal seminograms who attended our Reproduction Center. Semen was collected early in the morning (morning collection group) and in the evening (evening collection group) from the same subjects, and total motile sperm count was assessed as the primary outcome measure. As secondary outcome measures, semen volume, sperm concentration, sperm motility and total sperm count were assessed. A sexual abstinence period of 3 days was set for all participants. The semen samples were analyzed using CASA CEROS, a sperm motility analysis system, and the data from the morning and evening collection groups were compared using a Wilcoxon signed rank test. We found that the fertile males had a significantly higher total motile sperm count and total sperm count in the evening collection group than in the morning collection group. In contrast, the male infertility patients showed no significant difference in total sperm count between the two collection times; however, the total motile sperm count was significantly higher in the evening collection group than the morning collection group. Our analyses indicate that total motile sperm count in ejaculated semen is significantly higher after evening collection than after morning collection. From a male side perspective, we suggest that successful intrauterine insemination might be easier to achieve using semen collected in the evening than in the early morning.Abbreviations: IUI: intrauterine insemination; OAT: oligoasthenoteratozoospermia; TSC: total sperm count; TMSC: total motile sperm count

Sperm motility analysis system implemented on a hybrid architecture to produce an intelligent analyzer

Much research and analysis in biomedicine involve image and video inspection using microscopes. Presently, scientists are dissatisfied with manual observations and assessments, when objective and enhanced data can be obtained by applying new technologies (such as image and video inspection) to biomedical fields, such as sperm analysis. Computer Assisted Sperm Analysis (CASA) systems, developed in the late 1980s, constitute third-generation methods of sperm analysis. This study aimed to develop a standalone medical image and video analysis system that is reconfigurable, flexible, reliable, deterministic, and robust. It proposed a new sperm motility analysis system running on a dual core Central Processing Unit (CPU) + field programmable gate arrays (FPGA) platform, under a real-time operating system (RTOS), which is a step ahead of the third-generation CASA systems. The system hardware and related sperm detection and tracking algorithms were the novelty of this work. The image processing functions mainly run on FPGA, image acquisition, and calculations run on CPU, parallel with FPGA. The result is a much faster, reliable, reconfigurable, and compact intelligent analyzer system. Our prototype system was applied to sperm motility analysis; however, other image processing systems can be applied to this architecture. Additionally, the proposed tracking method for sperm track determination is simple, effective, and does not exert a load on the system.

113 Effect of antioxidants on motility and fertility of liquid-stored bovine sperm

It is difficult to maintain sperm in liquid storage for a long time, compared with permanent frozen storage in liquid nitrogen. Antioxidants have been reported to improve the quality and fertility of liquid-stored semen. In this study, we investigated whether antioxidants can extend the motility and fertility of frozen-thawed sperm in liquid storage. Frozen-thawed semen from one Japanese black bull (one ejaculate) was diluted in Tris-citrate-fructose (TCF) diluent with 10% (v/v) egg yolk to a sperm concentration of 1×107 spermmL−1. The antioxidants β-mercaptoethanol (βMe) and glutathione (GSH) were added independently, at various concentrations (0.1, 0.5, 1, and 5mM) to sperm suspensions, and these preparations were compared with Control (no added antioxidant). Sperm suspensions were packaged in centrifuge tubes and placed at 17°C in air and monitored daily until sperm motility had stopped (up to 14 days). Sperm motility was analysed by the Sperm Motility Analysis System (SMAS; Ditect Co. Ltd), and the percentage of progressively motile sperm (straight-line velocity (VSL) of >25μm s−1; Grade A classified by WHO manual), compared with that recorded on Day 0 (100%), was determined each day. For evaluation of fertilizing ability, after incubation in liquid storage for 0, 3, 5, and 7 days, sperm were used for IVF with invitro-matured oocytes (30 oocytes per treatment, three replicates). Embryo development was recorded as the proportion of embryos that reached blastocyst by 8 days after IVF. Data for motility were analysed using one-way ANOVA with Tukey test, and embryo development using chi-squared test. A P-value<0.05 was considered statistically significant. At 7 days, the percentage of progressively motile sperm was significantly higher for 0.5, 1, and 5mM βMe than for Control (30.8%, 48.1%, and 50.3%, vs. 0%, respectively). Treatments with 1 and 5mM βMe maintained some sperm progressive motility for 14 days (9.5% and 14.5%). Treatment with GSH showed the same trend at 7 days (32.2%, 36.3%, and 13.7% for 0.5, 1, and 5mM, vs. 0% for Control); 1 and 5mM GSH maintained sperm progressive motility over 10 days (24.8% and 4.4%). In both antioxidant treatments, embryo development was achieved with sperm stored for up to 5 days (Day 0 vs. Day 5 for 0.1mM βMe: 17.6% vs. 13.8%; for 1.0mM GSH: 26.0% vs. 6.7%; for Control: 17.6% vs. 0%). In this study, antioxidants extended both motility and fertility of frozen-thawed bovine sperm in liquid storage. This result suggests the possibility of application to AI using liquid-stored bovine semen.

Effect of Lepidium meyenii on in vitro fertilization via improvement in acrosome reaction and motility of mouse and human sperm.

PurposeThe direct effects of Lepidium meyenii (Maca) on sperm remain unclear. Herein, we examined the direct effect of Maca on in vitro fertilization.MethodsWe examined the fertilization rate in a mouse model and the rate of acrosome reaction in sperm from transgenic mice expressing enhanced green fluorescent protein (EGFP) in a Maca extract‐containing human tubal fluid (HTF) medium. Using human sperm, we assessed acrosome status via fluorescein isothiocyanate‐conjugated peanut agglutinin (FITC‐PNA) staining and performed detailed analysis using a sperm motility analysis system (SMAS).ResultsIn the mouse model, the fertilization rate in the Maca extract‐containing HTF was significantly higher than that in the control medium. The acrosome reaction rate in sperm from transgenic mice expressing EGFP was also significantly higher in the Maca extract‐containing HTF than that in the control medium. Similarly, a high acrosome reaction rate, identified via FITC‐PNA staining of human sperm samples, was found in the Maca extract‐containing HTF compared with that in the control medium. Human sperm motility in the Maca extract‐containing HTF was also increased compared with that in the control medium as measured using an SMAS.ConclusionsMaca improved in vitro fertilization rates by inducing an acrosome reaction and increasing sperm motility.

43 Cryopreservation of Bovine Sperm Using Antifreeze Polyamino-Acid

Carboxylated poly-l-lysine (CPLL), an ampholytic polymer compound, is reported to have a cryoprotective property similar to that of antifreeze proteins. We previously reported the effectiveness of CPLL as cryoprotective material for bovine sperm (43rd Annual Conference of International Embryo Technology Society, Austin, TX, USA; http://www.iets.org/2017/IETS_2017_Program_Book_FINAL.pdf). In this research, we investigated additional aspects of CPLL for bovine sperm. The conventional cryopreservation medium used for Control group consisted of 6.5% (v/v) glycerin, and the cryopreservation medium used for the CPLL group consisted of 3.25% (v/v) glycerin and 0.5% CPLL (w/v). In experiment 1, sperm motility was measured 1, 3, and 6 h after thawing. The post-thaw motility was assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). The CPLL treatment yielded better motility rate at 6 h (Control v. CPLL; 23.7% v. 38.5%; P < 0.01), average path velocity (μm s−1) at 1 and 3 h (Control v. CPLL; 49.8 v. 57.7, 35.8 v. 42.8; P < 0.01), straight-line velocity (μm s−1) at 1 h (Control v. CPLL; 35.2 v. 45.7; P < 0.01), and curvilinear velocity (μm/s) at 1 and 3 h (Control v. CPLL; 93.7 v. 106.2, 59.9 v. 68.4; P < 0.01) than the Control. In experiment 2, sperm membrane integrity was assessed by using the LIVE/DEAD Sperm Viability Kit (Thermo Fisher Scientific K.K., Kanagawa, Japan). The CPLL group yielded greater sperm membrane integrity rate than control (Control v. CPLL; 49.6% v. 60.6%; P < 0.01). In experiment 3, AI was carried out on 111 cows (Control v. CPLL; 49 v. 62) and the conception rate of the CPLL group was significantly higher than that of the control group (53.1% v. 79.0%; P < 0.01). Previously, we reported the effectiveness of CPLL for bovine sperm. In this study, we clarified how CPLL works to improve the conception rate of AI: CPLL maintains post-thaw motility and protects the sperm membrane. These results suggest that CPLL has potential as a new cryoprotective material for bovine sperm.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa.

The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500μgNPml(-1) for 1, 2, 3 or 4h. Computer-assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500μgNPml(-1) was detrimental to progressive motility (P<0.05), and its adverse effect was significant at lower (100μgNPml(-1) ) concentration (P<0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250μgml(-1) NP (P<0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose-dependent NP effects (P<0.05) on acrosomal integrity of both species at as low as 1μgml(-1) NP for boar spermatozoa and 10μgml(-1) NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.

Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility

This study compared the sperm nuclear vacuoles and semen quality in the evaluation of male infertility. One hundred and forty-two semen samples were obtained from patients who visited the Male Infertility Clinic at Toyama University Hospital. Semen samples were evaluated by conventional semen analyses and the Sperm Motility Analysis System (SMAS). In addition, spermatozoa were analyzed at 3,700-6,150x magnification on an inverted microscope equipped with DIC/Nomarski differential interference contrast optics. A large nuclear vacuole (LNV) was defined as one or more vacuoles with the maximum diameter showing > 50% width of the sperm head. The percentage of spermatozoa with LNV (% LNV) was calculated for each sample. Correlations between the % LNV and parameters in SMAS and conventional semen analyses were analyzed. Processed motile spermatozoa from each sample were evaluated. The mean age of patients was 35 years old. Semen volume was 2.9 ± 1.6mL (0.1-11.0; mean ± standard deviation, minimum-maximum), sperm count was 39.3 ± 54.9 (x106/mL, 0.01-262.0), sperm motility was 25.1 ± 17.8% (0-76.0), and normal sperm morphology was 10.3 ± 10.1% (0-49.0). After motile spermatozoa selection, we could evaluate % LNV in 125 ejaculates (88.0%) and at least one spermatozoon with LNV was observed in 118 ejaculates (94.4%). The percentage of spermatozoa with LNV was 28.0 ± 22.4% (0-100) and % LNV increased significantly when semen quality decreased. The correlation between the % LNV and the semen parameters was weak to moderate; correlation coefficients were -0.3577 in sperm count (p < 0.0001), -0.2368 in sperm motility (p = 0.0084), -0.2769 in motile sperm count (p = 0.019), -0.2419 in total motile sperm count (p = 0.0070), and -0.1676 in normal sperm morphology (p = 0.0639). The % LNV did not show a significant correlation with the SMAS parameters except for weak correlation to beat/cross frequency (r = -0.2414, p = 0.0071). The percentage of spermatozoa with LNV did not have a strong correlation with parameters in conventional semen analysis and SMAS in the patients with male infertility; however, a certain level of negative influence of LNV to sperm quality cannot be excluded.

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Ile de France rams

The aims of this study were to identify different motile sperm subpopulations in fresh ejaculates from six Ile de France rams, by using a computer-assisted sperm motility analysis (CASA) system, and to evaluate the effects of individual ram and season on population distribution. Overall sperm motility and individual kinematic parameters of motile spermatozoa were evaluated for 125,312 spermatozoa, defined by curvilinear velocity (VCL), linear velocity (VSL), average path velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR), wobble coefficient (WOB), mean amplitude of lateral head displacement (ALH) and frequency of head displacement (BCF). A multivariate cluster analysis was carried out to classify these spermatozoa into a reduced number of subpopulations according to their movement patterns. The statistical analysis clustered the whole motile sperm population into five separate groups: subpopulation 1, constituted by rapid, progressive and non sinuous spermatozoa (VCL = 126.41 μm/s, STR = 92.87% and LIN = 86.47%); subpopulation 2, characterized by progressive spermatozoa with moderate velocity (VCL = 74.74 μm/s and STR = 84.03%); subpopulation 3, represented by rapid, progressive and sinuous spermatozoa (VCL = 130.45 μm/s, STR = 76.02% and LIN = 47.68%); subpopulation 4 represents rapid nonprogressive spermatozoa (VCL = 128.69 μm/s and STR = 44.09%); subpopulation 5 includes poorly motile, nonprogressive spermatozoa with a very irregular trajectory (VCL = 36.81 μm/s and STR = 47.04%). Our results show the existence of five subpopulations of motile spermatozoa in ram ejaculates. The frequency distribution of spermatozoa within subpopulations was quite similar for the six rams, and the five subpopulations turned out to be very stable along seasons.

A comparison of conventional and computer-assisted semen analysis (CRISMAS software) using samples from 166 young Danish men

The aim of the present study was to compare assessments of sperm concentration and sperm motility analysed by conventional semen analysis with those obtained by computer-assisted semen analysis (CASA) (Copenhagen Rigshospitalet Image House Sperm Motility Analysis System (CRISMAS) 4.6 software) using semen samples from 166 young Danish men. The CRISMAS software identifies sperm concentration and classifies spermatozoa into three motility categories. To enable comparison of the two methods, the four motility stages obtained by conventional semen analysis were, based on their velocity classifications, divided into three stages, comparable to the three CRISMAS motility categories: rapidly progressive (A), slowly progressive (B) and non-progressive (C+D). Differences between the two methods were large for all investigated parameters (P < 0.001). CRISMAS overestimated sperm concentration and the proportion of rapidly progressive spermatozoa and, consequently, underestimated the percentages of slowly progressive and non-progressive spermatozoa, compared to the conventional method. To investigate whether results drifted according to time of semen analysis, results were pooled into quarters according to date of semen analysis. CRISMAS motility results appeared more stable over time compared to the conventional analysis; however, neither method showed any trends. Apparently, CRISMAS CASA results and results from the conventional method were not comparable with respect to sperm concentration and motility analysis. This needs to be accounted for in clinics using this software and in studies of determinants of these semen characteristics.

Carp transferrin can protect spermatozoa against toxic effects of cadmium ions

Cadmium is a widespread heavy metal that enters the aquatic environment and affects many processes involved in fish reproduction such as sperm motility. Fish seminal plasma proteins can protect spermatozoa against toxic effects of heavy metals. The objective of this study was to demonstrate the ability of a major carp seminal plasma protein-transferrin (TF) to bind cadmium ions and to neutralize the toxic effect of cadmium on carp sperm motility. To obtain a high quantity of carp seminal plasma TF necessary for the experiment, immunoaffinity chromatography as a one-step isolation procedure was established. The titration of TF with cadmium ions spectrophotometrically at 247nm revealed that TF binds cadmium ions at only one spectrophotometrically-sensitive binding site, which suggests that TF is capable of neutralizing the cadmium toxic effect. Indeed, the addition of carp TF to carp semen incubated with 50ppm cadmium for 48h led to about a four-times higher percentage of sperm motility (30.3±1.1%) in comparison to samples incubated with only 50ppm cadmium (8.2±5.2%). Similarly, higher values of other parameters of sperm movement measured by a computer-assisted sperm motility analysis system (VSL, VCL and ALH) were observed at the presence of transferrin. In conclusion, our study provides the first evidence that transferrin from carp seminal plasma can protect sperm motility from cadmium toxicity.

Analysis of sperm motility and fertilization rates after the separation by microfluidic sperm sorter made of quartz

Analysis of sperm motility and fertilization rates after the separation by microfluidic sperm sorter made of quartz

Comparative study of Sperm Motility Analysis System and conventional microscopic semen analysis.

Background and Aim: Conventional manual sperm analysis still shows variations in structure, process and outcome although World Health Organization (WHO) guidelines present an appropriate method for sperm analysis. In the present study a new system for sperm analysis, Sperm Motility Analysis System (SMAS), was compared with manual semen analysis based on WHO guidelines. Materials and methods: Samples from 30 infertility patients and 21 healthy volunteers were subjected to manual microscopic analysis and SMAS analysis, simultaneously. We compared these two methods with respect to sperm concentration and percent motility. Results: Sperm concentrations obtained by SMAS (Csmas) and manual microscopic analyses on WHO guidelines (Cwho) were strongly correlated (Cwho=1.325×Csmas; r=0.95, P<0.001). If we excluded subjects with Csmas values >30×106sperm/mL, the results were more similar (Cwho=1.022×Csmas; r=0.81, P<0.001). Percent motility obtained by SMAS (Msmas) and manual analysis on WHO guidelines (Mwho) were strongly correlated (Mwho=1.214×Msmas; r=0.89, P<0.001). Conclusions: The data indicate that the results of SMAS and those of manual microscopic sperm analyses based on WHO guidelines are strongly correlated. SMAS is therefore a promising system for sperm analysis. (Reprod Med Biol 2006; 5: 195-200).

Effects of Diethylstilbestrol Administration on Sperm Motility and Reproductive Function in Male Japanese Quail (Coturnix japonica)

The present study was undertaken to evaluating the effects of diethylstilbestrol (DES) on sperm motility and reproductive function in male Japanese quail. To do so, we have identified the method that ensures maximal quail sperm motility, i.e. the semen from the ductus deferens was diluted with Lake’s solution supplemented with 0.005% (w/v) caffeine and 10% (v/v) quail cloaca fluid. Sperm motility was analyzed by the computer-assisted sperm motility analysis system (CASA). Compared with control birds, administration of DES (0.1 or 1 mg DES on a daily basis for a week) caused a significant reduction in testicular weight, a marked disintegration of the seminiferous epithelium and a significant decrease in the sperm population of the seminiferous lumen. Sperm recovery and motility also showed a significant decrease in treated individuals. These results suggested that enhanced estrogenic effect caused by DES injection could cause a disruption in reproductive function by affecting spermatogenesis and influencing sperm motility.

Survival of boar spermatozoa frozen in diluents of varying osmolality

We investigated the effects of freezing diluents of differing levels of osmolality on boar sperm cryosurvival. The spermatozoa were frozen using a pellet technique. Cryosurvival was evaluated in terms of motility, intact acrosomes and membrane integrity. The motility parameters were assessed using a computer-assisted sperm motility analysis (CASA) system. Acrosomal status was monitored by means of FITC-labeled peanut agglutinin, and membrane integrity was evaluated after double staining with SYBR-14 and propidium iodide. At 3 h of incubation after thawing, the highest motility was found in the 420 mOsm/kg group, and progressive motility in the 420 to 580 mOsm/kg groups was higher than that in the hypo- (225 mOsm/kg) and iso-osmotic (290 mOsm/kg) groups (P < 0.05). The intact acrosomes of the spermatozoa frozen in the 510 and 580 mOsm/kg BF5 diluents were more numerous than in other groups (P < 0.05). The 420 and 510 mOsm/kg groups yielded maximal values of post-thaw membrane integrity. These observations obtained in the present study indicate that moderately hypertonic BF5 diluents are favorable for the cryopreservation of boar spermatozoa.

Effects of methyl-beta-cyclodextrin on cryosurvival of boar spermatozoa.

Methyl-beta-cyclodextrin (MBCD), which leads to the stimulation of cholesterol efflux from the cell membrane, was examined for its ability to increase the cryosurvival of spermatozoa from Göttingen miniature pigs. The intactness of the acrosome and various motion parameters of spermatozoa after freezing and thawing were used to monitor cryosurvival. Spermatozoa were exposed to MBCD or a combination of MBCD and cholesterol-3-sulfate over a period of 3 hours while being cooled slowly from 25 degrees C to 5 degrees C, and were subsequently cryopreserved by the pellet method. After freezing and thawing, the acrosomal status was monitored by fluorescein isothiocyanate-labeled peanut agglutinin, and the motion parameters were assessed with a computer-assisted sperm motility analysis system. In post-thaw spermatozoa the values of the intact acrosome, motility, progressive motility, progressive velocity, straightness, and linearity of the cell path increased greatly with the concentration of MBCD (P < .05). In contrast, the lateral head displacement amplitude and the beat cross-frequency of post-thaw spermatozoa were not different among all treatments. On the contrary, the addition of cholesterol-3-sulfate to the diluent containing MBCD abolished the protective effect against freeze-thaw injury that MBCD provides to spermatozoa. These results indicate that cryosurvival of boar spermatozoa is enhanced by exposure to MBCD before freezing. This protective effect of MBCD may be due to active depletion of sperm membrane cholesterol.