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PubMed2002

Reduction of steps in the preparation of motile sperm for intrauterine insemination does not reduce efficacy of the procedure: simplified one-step swim-up method versus classic swim-up.

BACKGROUND: Intrauterine insemination (IUI) is a valid treatment for infertility with a cumulative pregnancy rate of >40-90% after 3-10 treatment cycles. We studied the efficacy of a simplified method for motile sperm preparation for IUI. METHODS: A prospective clinical trial was performed with 100 couples (male age 33-48 and female 28-37 years) with a 2-8 year history of primary infertility associated with slight oligozoospermia (16/100), oligomenorrhoea (32/100) or unknown (52/100). Motile sperm for IUI were prepared by: (A) the classic World Health Organization self-migration (swim-up) method which includes centrifugation, or (B) a simplified one-step swim-up procedure without centrifugation. Recombinant FSH was used for ovarian stimulation. Depending on the cause of infertility, patients were matched one-to-one at the time of IUI, so that when a total of 100 couples had been treated, 50/100 women received sperm prepared by method A and 50/100 by method B. RESULTS: A statistically significant correlation was found between the percentage motile sperm of the original semen sample and the percentage of motile sperm recovered by method A (r = 0.333, P < 0.01) and B (r = 0.400, P < 0.01). A highly significant correlation (r = 0.997, P < 0.001) was found between the two methods. CONCLUSIONS: The simplified one-step swim-up method was as effective as the classic swim-up method, but the former was easier and more economical.

Adult↗
PubMed1990

[Sperm survival test assessing the change of sperm motility after long-term incubation].

The purpose of this study is to establish a new screening test to assess the sperm fertilizing potential. This test was named sperm survival test (SST). At the beginning, following 6, 12, 24, 36 and 48 hour incubation, sperm motility was examined. It was defined that the SST result was positive when only motile swim-up sperm existed at the estimating point and negative when non-motile sperm existed. 1. The result of the SST was stable when there was a concentration of 10(5)-10(7) sperm/ml. 2. The SST could be used to assess sperm function that conventional semen analysis could not assess. 3. The result of the SST was positive at 36 hrs in 8 proven fertile men. In infertile men there was a significant correlation between a 36 hr positive SST result and the pregnancy rate (p less than 0.01). This result indicated that a 36 hr positive SST showed good sperm fertilizing potential and a 36 hr negative result poor fertilizing potential. 4. In IVF-ET, the fertilization rate was 92.9% at a 48 hr positive SST, 85.4% at a 36 hr positive SST, 68.1% at a 24 hr positive SST, 15.4% at a 12 hr positive SST and 0% at a 6 hr positive SST. Furthermore, the fertilization rate for a sperm concentration less than 10 x 10(6)/ml was 17.6% (0% at a 6 hr positive SST and 25% at a 12 hr positive SST) and at motility less than 30% was 28.9% (0% at a 6 hr positive SST, 18.2% at a 12 hr positive SST and 69.2%). In conclusion, SST is an accurate clinical test for predicting sperm fertilizing potential and the fertilization rate in IVF-ET.

Cells, Cultured↗
PubMed1981

Testosterone and gossypol effects on human sperm motility.

Testosterone concentration in seminal fluid has been found to be high in infertile males (75 +/- 11 pg/100 microliter). Fertile males have a testosterone concentration of 29 +/- 3 pg/100 microliter. The effects of adding 50, 150, and 300 pg of testosterone to 100 microliter of ejaculate have been studied by turbidimetric analysis (Sokoloski J, et al. Fertil Steril 28:1337, 1977). This method permits objective measurements of sperm velocity and percentage of rapidly moving sperm in a sample (%RM). A dose-dependent effect of testosterone on sperm motility was seen. Fifty pg/100 microliter had no effect on velocity or percentage of moving sperm; 150 pg of testosterone produced a decrease of 36% +/- 8; and 300 pg/100 microliter, a decrease of 62% +/- 8. Caffeine had a stimulatory effect on the percentage of motile sperm at doses of 400 microliter of semen. Likewise, dibutyryl cAMP (10 microgram/microliter) had a positive effect on sperm velocity. The stimulatory effect of these two drugs were negated when 300 pg of testosterone was added to the preparations. Other steroids (17 alpha-testosterone, 17 alpha-estradiol, and 17 beta-estradiol, DHT, and progesterone) tested under the same experimental conditions had no effect on sperm motility, but cottonseed oil (goosypol) had drastic effects. Doses a little as 100 pg/100 microliter produced a 90% decrease in sperm motility.

Bucladesine↗
PubMed2000

In vitro effects of sildenafil and phentolamine, drugs used for erectile dysfunction, on human sperm motility.

OBJECTIVE: The aim of this study was to determine the effects of sildenafil and phentolamine on sperm motility in vitro. STUDY DESIGN: Semen or washed sperm was mixed with various doses of sildenafil or phentolamine and analyzed for motility during a 30-minute period. The pH was measured for each of the samples tested. Statistical analyses were performed with analysis of variance. RESULTS: A 200-microg/mL dose of sildenafil had no effect on sperm motility. However, the highest dose (2000 microg/mL) significantly reduced motility by about 50%. The pH was reduced in this high-dose sample. The lowest dose of phentolamine (20 microg/mL) had no effect, whereas a dose of 200 microg/mL resulted in a significant reduction in sperm motility. The highest dose (2000 microg/mL) stopped virtually all sperm from moving. CONCLUSION: This study demonstrated a direct dose-related effect of phentolamine on reducing sperm motility. Only the highest dose of sildenafil had an effect, which may have been caused by a decline in pH.

Dose-Response Relationship, Drug↗
PubMedDate not supplied

The effect of reactive oxygen species on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation.

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for 30 minutes. Catalase (150 U/mL), superoxide dismutase (SOD, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the 30-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 73%+/-1%, respectively). Catalase, but not SOD, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs 30%+/-3%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the 30-minute incubation (53%+/-3%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

Acrosome↗
PubMed1999

Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility.

European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

Animals↗
PubMed1982

Stimulation of human sperm motility by relaxin.

Relaxin has recently been identified in human seminal plasma (SP). This investigation was undertaken to determine the effect of relaxin on sperm motility. Washed ejaculated human sperm were incubated with or without porcine relaxin. In a series of 37 normal specimens (motility greater than or equal to 60%) relaxin attenuated the decline in grade of forward progression and percentage of motile sperm. Relaxin had a similar effect on a series of 47 subfertile specimens (motility 30% to 40%). Endogenous relaxin may play a role in delaying declines of motility and grade of forward progress in ejaculated sperm.

Animals↗
PubMed2004

[Influence of the metabolite produced by Trichomonas vaginalis on human sperm motility in vitro].

OBJECTIVE: To investigate the effects of the metabolite produced by Trichomonas vaginalis on human sperm motility in vitro. METHODS: Trichomonas vaginalis having been cultured, the culture solution containing metabolite was obtained by removing the protozoa, then diluted into 3 kinds of concentration. Sperm was obtained from 10 healthy fertile men by masturbation and prepared by swim-up technique to produce a spermatozoon solution of high motility. Every sperm sample was divided into 4 groups (A, B, C, D). Unused culture solution was added to Group A as control, and the other 3 groups (B, C, D) were respectively incubated with the above used culture solution at 3 kinds of concentration (1.2 x 10(9)/L, 6 x 10(8)/L, 1.2 x 10(8)/L). Measurements were carried out at 30 s, 1 h, 2 h, 4 h, 6 h by CASA. RESULTS: Sperm motility decreased in both Group B and C markedly, and the effects displayed a concentration- and time-dependent manner. CONCLUSION: The metabolite of Trichomonas vaginalis can reduce human sperm motility in vitro, and may be one of the causes of infertility.

Animals↗
PubMedDate not supplied

Influence of the concentration of motile sperm inseminated on the ovum penetration assay results: toward a standardized method.

The influence of the concentration of motile sperm inseminated on the zona-free hamster egg penetration rate has been studied. In 3 fertile men, the higher the concentration of motile sperm inseminated, the higher the ovum penetration rate. This correlation emphasizes the usefulness of standardizing the technical conditions under which the ovum penetration assay is performed. It is also suggested to refer to a standard curve for results analysis. In the 22 patients studied, the outcome of the heterologous ovum assay was in most cases related to the initial sperm parameters. However, some disagreement was also observed. In the absence of a correlation between the initial sperm parameters and the ovum penetration rates, further investigation is needed in order to either more precisely determine the aetiology of unexplained couple infertility, or, in case of intrauterine insemination or in vitro fertilization, to better understand the conditions under which spermatozoa are capacitated in vitro.

Animals↗
PubMed1994

Effects of cholic acid and 'Protectaid' formulations on human sperm motility and ultrastructure.

Cholic acid (sodium cholate) and the other active ingredients of F-5 gel preparations in use for the impregnation of a new vaginal sponge (Protectaid) with contraceptive and anti-sexually transmitted disease properties, were assessed for their effects on human sperm motility and ultrastructure. Cholic acid (CA) produced an inhibition of motility which was both dose- and time-dependent. A complete suppression of motility was obtained at 30 s by a CA concentration of 1.25%. Nonoxynol-9 (NX9) compared with benzalkonium chloride (BZC) showed no significant difference at the concentration required (0.025%) to give a total inhibition of sperm motility after exposure for 30 s. The addition of F-5A gel containing 0.5% of each one of the spermicide ingredients (CA, NX9 and BZC) produced the total suppression of sperm motility within 30 s at a dilution of 1/50. Another preparation, F-5B gel, containing the spermicide ingredients at different concentrations (1.25% CA, 0.125% NX9 and 0.05% BZC) produced this same effect with a 1/10 dilution. Exposure of semen to a CA concentration of 1.25% or to 1/10 dilutions of F-5A gel for 30 s led to profound changes of sperm ultrastructure studied by scanning (SEM) and transmission (TEM) electron microscopy. SEM and TEM findings indicate that CA acts as a spermicide through its 'natural detergent' properties, damaging the outer plasma membrane of sperm cells. Protectaid formulations affect sperm motility and viability in a similar way.

Acrosome↗
PubMed2000

Effect of dietary selenium and vitamin E on the ultrastructure and ATP concentration of boar spermatozoa, and the efficacy of added sodium selenite in extended semen on sperm motility.

Three experiments evaluated the effects of dietary Se and vitamin E on the ultrastructure of spermatozoa, ATP concentration of spermatozoa, and the effects of adding sodium selenite to semen extenders on subsequent sperm motility. The experiment was a 2 x 2 arrangement of treatments in a randomized complete block design. A total of 10 mature boars were fed from weaning to 18 mo of age diets fortified with two levels of supplemental Se (0 or .5 ppm) or vitamin E (0 or 220 IU/kg diet). The nonfortified diets contained .06 ppm Se and 4.4 IU vitamin E/kg. In Exp. 1, the spermatozoa from all boars were examined by electron microscopy. Vitamin E had no effect on structural abnormalities in the spermatozoa. When the low-Se diet was fed the acrosome or nuclei of the spermatozoa was unaffected, but the mitochondria in the tail midpiece were more oval with wider gaps between organelles. The plasma membrane connection to the tail midpiece was not tightly bound as when boars were fed Se. Immature spermatozoa with cytoplasmic droplets were more numerous when boars were fed the low-Se diet, but the occurrence of midpiece abnormalities occurred in boars fed diets with or without Se or vitamin E. Our results suggest that Se may enhance spermatozoa maturation in the epididymis and may reduce the number of sperm with cytoplasmic droplets. In Exp. 2, the concentration of ATP in the spermatozoa was evaluated in the semen of all treatment boars. When the low-Se diet was fed, ATP concentration was lower (P < .01), whereas vitamin E had no effect on ATP concentration. Experiment 3 investigated the effect of diluting boar semen with a semen extender with sodium selenite added at 0, .3, .6, or .9 ppm Se. Three ejaculates from each boar were used to evaluate these effects on sperm motility to 48 h after dilution. Sperm motility declined (P < .01) when Se was added to the extender, and this decline was exacerbated as the concentration of added Se increased (P < .01). The added Se was demonstrated to be tightly adhered to the spermatozoa. Overall, these results suggest that low Se-diets fed to boars resulted in abnormal spermatozoal mitochondria, a lower ATP concentration in the spermatozoa, and a loose apposition of the plasma membrane to the helical coil of the tail midpiece, but no effect from inadequate vitamin E was demonstrated. Adding sodium selenite to the semen extender reduced sperm cell motility.

Adenosine Triphosphate↗
PubMed1997

Effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human sperm motility, viability and motion parameters.

Male genital tract infections and non-specific inflammatory conditions may be associated with unexplained infertility. Previous studies have shown the presence of cytokines such as tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the semen of infertile men. However, the mechanism of effect of these cytokines on human sperm function is still controversial. The present study was undertaken to investigate the in-vitro effects of TNF-alpha and IFN-gamma on human sperm motion, viability and the hypoosmotic swelling test (HOST). Washed spermatozoa from normal volunteers (n = 9) were incubated in the presence/absence of TNF-alpha (1 microgram/mL) plus IFN-gamma (0.1 microgram/mL). Sperm motility, viability, HOST, and video sequences were recorded at different time intervals (0, 30, 60 and 180 min). Sperm motion parameters were analysed using computer-assisted semen analysis. There was a time-dependent negative effect of TNF-alpha plus IFN-gamma on sperm motility, viability, HOST, and lateral-head displacement (ALH). The maximum decrease was observed between 60 and 180 min for sperm motility (50.8 +/- 5.6%), viability (52.8 +/- 4.0%), HOST (38 +/- 2%) and ALH (4.7 +/- 0.1 microns) compared to control samples (62.2 +/- 2.8, 62.4 +/- 2.9, 58 +/- 4, and 5.3 +/- 0.4, respectively; All p < 0.05). There was no significant effect on sperm straight-line velocity and mean linearity when compared to control. These data suggest that the common inflammatory cytokines TNF-alpha plus IFN-gamma have only partial detrimental effects on sperm motility, viability, membrane integrity and lateral head displacement, which may contribute to the poor fertilizing potential of human spermatozoa during inflammatory conditions.

Cell Membrane↗
PubMed2005

Effects of magnetic-activated cell sorting on sperm motility and cryosurvival rates.

OBJECTIVE: To evaluate the effect of magnetic-activated cell sorting in cryopreservation-thawing protocols on sperm motility and cryosurvival rate. DESIGN: Prospective-controlled study. SETTING: Andrology department at a university-based medical institution. PATIENT(S): Ten healthy volunteer sperm donors. INTERVENTION(S): Sperm populations were separated using annexin-V magnetic-activated cell sorting before and after the cryopreservation-thawing process. MAIN OUTCOME MEASURE(S): Sperm motility and cryosurvival rate. RESULT(S): Annexin-negative sperm separated by magnetic-activated cell sorting had statistically significantly higher motility following cryopreservation-thawing than sperm that were not separated. Similarly, annexin-negative spermatozoa also had higher cryosurvival rate than sperm cryopreserved without magnetic-activated cell sorting and sperm that were annexin-positive. CONCLUSION(S): Superparamagnetic annexin V-conjugated microbeads can separate spermatozoa with externalized phosphatidylserine, which is considered one of the early features of late apoptosis. The separation of a distinctive population of nonapoptotic spermatozoa with intact membranes may optimize the cryopreservation-thawing outcome. Magnetic-activated cell sorting using annexin-V microbeads enhances sperm motility and cryosurvival rates following cryopreservation.

Cell Separation↗
PubMed1995

Pentoxifylline-supplemented cryoprotectant improves human sperm motility after cryopreservation.

In this study, human spermatozoa obtained from donors (n = 15) with normal semen characteristics were cryopreserved in human sperm preservation medium, supplemented with the phosphodiesterase inhibitor pentoxifylline at concentrations of 0, 1, 3 and 10 mM. The effect of pentoxifylline on cryopreserved spermatozoa was determined by monitoring changes in sperm motility and acrosome morphology by labelling the spermatozoa with fluorescein-conjugated concanavalin A lectin. Cryoprotectant supplemented with 1 mM pentoxifylline was found to improve post-thaw progressive motility from 15.3 +/- 2.4 (control) to 23.1 +/- 3.8% (P < 0.01), and total motility from 27.4 +/- 3.3 (control) to 38.2 +/- 3.9% (P < 0.05) without reducing the percentage of spermatozoa with normal acrosomal regions, and so appears useful for cryopreservation purposes. The beneficial effects of 1 mM pentoxifylline on sperm motility were shown to be maintained post-thaw over a 6 h time course. Cryoprotectant supplemented with 3 mM pentoxifylline was found to improve only post-thaw progressive motility, from 15.3 +/- 2.4 (control) to 20.7 +/- 3.0% (P < 0.05). However, cryopreservation in the presence of 10 mM pentoxifylline was found to have a significantly (P < 0.01) detrimental effect on acrosome morphology post-thaw, reducing it from 29.0 +/- 2.0 (control) to 21.0 +/- 2.4% without affecting sperm motility. This suggests that assessment of the acrosomal region may indicate subtle deleterious effects of cryoprotectant supplements that cannot be determined from post-thaw motility assessments alone. These findings differ from previous studies in that a lower concentration of pentoxifylline (1 mM) was found to be optimal for cryopreservation purposes.

Acrosome↗
PubMed1994

The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration.

OBJECTIVE: To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. DESIGN AND METHODS: Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. RESULTS: After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. CONCLUSION: Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Calcium↗
PubMed1998

Use of a sperm analyzer for evaluating broiler breeder males. 1. Effects of altering sperm quality and quantity on the sperm motility index.

A new instrument for assessing mammalian semen attributes, the Sperm Quality Analyzer, was evaluated as a potential tool for determining rooster sperm quality. The Sperm Quality Analyzer measures the "activity" of sperm in a semen sample as the sperm motility index (SMI). The SMI is defined as the number and amplitude of deflections in a light path per second as a result of sperm movement within a capillary tube. In the present study, effects of sperm concentration, viability, and motility on the SMI were evaluated. Peterson broiler breeder males (n = 40) were used as semen donors. In the initial experiment, semen was diluted from 2- to 25-fold and SMI readings were obtained. The SMI was very low in neat semen samples but increased when semen was diluted up to threefold. However, at dilutions greater than fivefold, the SMI decreased. Apparently, sperm concentration in undiluted semen is so great that sperm are unable to move freely within the capillary tube. Maximum SMI values were obtained at sperm concentrations of approximately 1 billion sperm per milliliter. When thawed, dead sperm were mixed with incubated, live sperm, the SMI decreased with decreasing sperm viability even though sperm concentration was constant. Obviously, fewer sperm move across the light beam as sperm mortality increases. When motile, aerobically incubated sperm were mixed at different rates with immotile, anaerobically incubated sperm samples, the SMI increased with increasing concentrations of motile sperm, whereas total sperm concentration was static. In addition, the SMI was strongly correlated with motility scores obtained by microscopic analysis. The Sperm Quality Analyzer provides an estimate of the overall quality of sperm from broiler breeder males by reflecting sperm concentration, viability, and motility in a single value, the SMI.

Animals↗
PubMedDate not supplied

Effect of cyclosporine on human sperm motility in vitro.

Cyclosporine affects motility and viability of human sperm when incubated together in vitro. Sperm motility was almost reduced to nil following 10 min of incubation with cyclosporine at a concentration of 1 mg/mL. However, 200 micrograms/mL of the drug has no effect on motility and viability when tested for up to 60 min under standard laboratory conditions. Cyclosporine effect on sperm was both dose and time dependent. Sperm sensitivity and susceptibility to cyclosporine even to lower doses increased significantly following withdrawal of bovine serum albumin from the incubating medium. Compared to untreated controls, lactate dehydrogenase was estimated higher by more than 2 to 4 times in the sperm-free incubating media, suggesting an altered membrane porosity in the affected spermatozoa.

Cyclosporine↗
PubMed1991

Comparison of the hamster sperm motility assay to the mouse one-cell and two-cell embryo bioassays as quality control tests for in vitro fertilization.

The hamster sperm motility assay, mouse one-cell embryo, and mouse two-cell embryo bioassays were used to test modified Tyrode's solution and modified Ham's F-10 (Gibco, Grand Island, NY) medium prepared in tap water versus ultrapure water. Factors influencing the ability of each assay to discriminate water quality were evaluated to characterize these assays for quality control use in the in vitro fertilization laboratory. The hamster sperm motility assay reproducibly detected differences in treatment without significant interanimal, interanalyst, or interassay variation. Interanalyst and interanimal variation significantly affected the ability to detect treatment differences using the mouse bioassays. Sample sizes needed to predict clinically significant treatment effects were calculated using varying assay conditions. Ham's F-10 medium can be tested with the hamster sperm motility assay.

Analysis of Variance↗