Motility Of Rabbit Spermatozoa Research Articles

Effect of platinum nanoparticles on rabbit spermatozoa motility and viability

Male reproductive health is becoming a frequently asked question nowadays. Spermatozoa can be highly sensitive to various substances found in the environment. With the accretive usage of nanoparticles in technology and medicine, nanoparticles may also pose as potential environmental contaminants. Platinum nanoparticles are increasingly used in medicine, mainly for cancer treatment. The aim of this study was to investigate the effect of platinum nanoparticles on the motility and viability of rabbit spermatozoa in-vitro. Rabbit semen (n = 5) was cultured with solutions with different concentrations of platinum nanoparticles (control group or CON = only 0.9% NaCl; experimental groups D = 62.5 µg/ml, E = 31.25 µg/ml, F = 15.63 µg/ml, G = 7.81 µg/ml, H = 3.91 µg/ml, I = 1.95 µg/ml, J = 0.98 µg/ml, K = 0.49 µg/ml, and L = 0.24 µg/ml platinum nanoparticles) at time intervals of 0, 2, 4, 6, 9 and 24 hours. Selected parameters were analysed by computer-assisted sperm analyzer (CASA) technology – total motility (MOT, %), progressive motility (PRO, %), curvilinear velocity (VCL, µm/s) and viability (metabolic activity) of spermatozoa using the MTT test. Platinum nanoparticles negatively affected spermatozoa motility, progressive motility, and curvilinear velocity at almost all assessed time intervals, mainly at the highest used concentrations. The initial time interval shows a slight but statistically significant increase in MOT and PRO. Spermatozoa viability was not significantly affected, according to the results of this experiment. It was shown that the effect of platinum nanoparticles is time and dose-dependent. However, further analyses are needed to draw complete conclusions.

High taurine concentrations negatively effect stallion spermatozoa parameters in vitro

Article Details: Received: 2020-09-29 | Accepted: 2020-11-27 | Available online: 2021-01-31 https://doi.org/10.15414/afz.2021.24.mi-prap.15-19 Over the past decades natural substances are widely used in the maintaining of spermatozoa viability. The target of present study was to evaluate the effect of various taurine concentrations on stallion spermatozoa during 37°C cultivation. Fresh semen was collected from 10 breeding stallions. The experimental groups were supplemented with six different concentration of taurine (in mg/ml): A – 2.5, B – 5, C – 7.5, D – 10, E – 15, F – 20 and compared to control (CON – 0). Spermatozoa motility was assessed using the Computer Assisted Semen Analyzer (CASA) system in 6 time periods (0, 1, 2, 3, 4 and 5 hours). The MTT test was used for detection of viability. For measuring antioxidant activity FRAP and TOS methods were used. Significantly negative effect was observed in the samples with the highest concentration of taurine (20 mg/ml). Spermatozoa viability was not significantly affected in analysed concentrations of taurine. Significant higher antioxidant activity was detected in the sample with the highest taurine concentration. Data clearly showed negative effects of high taurine concentrations on stallion spermatozoa. Keywords: taurine, CASA, antioxidant activity, spermatozoa, stallion References Benzie, I. F. and Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry , 239 (1), 70-76. https://doi.org/10.1006/abio.1996.0292 Bucak, M. N. et al. (2007). The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen: Microscopic and oxidative stress parameters after freeze–thawing process. Theriogenology , 67 (5), 1060-1067. https://doi.org/10.1016/j.theriogenology.2006.12.004 Erel, O. (2005). A new automated colorimetric method for measuring total oxidant status. Clinical Biochemistry , 38 (12), 1103-1111. https://doi.org/10.1016/j.clinbiochem.2005.08.008 Halo, M. and Tirpak, F. (2018) Stallion fertility - the basis of successful reproduction. Svet koni . 18. Halo Jr., M. et al. (2019) Time and dose-dependent effects of Viscum album quercus on rabbit spermatozoa motility and viability in vitro. Physiological Research, 68 (6), 955-972. https://doi.org/10.33549/physiolres.934223 Ijaz, A. and Ducharme, R. (1995). Effect of various extenders and taurine on survival of stallion sperm cooled to 5 C. Theriogenology , 44 (7), 1039-1050. https://doi.org/10.1016/0093-691x(95)00290-o Jambor, T. et al. (2017) In vitro effect of 4-nonylphenol on human chorionic gonadotropin (hCG) stimulated hormone secretion, cell viability and reactive oxygen species generation in mice Leydig cells. Environmental Pollution , 222, 219–225. https://doi.org/10.1016/j.envpol.2016.12.053 O'flaherty, L. et al. (1997) Intestinal taurine transport: a review. European Journal of Clinical Investigation , 27 (11), 873-880. https://doi.org/10.1046/j.1365-2362.1997.2000747.x Reddy, N. S. S. et al. (2010). Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation. Animal Reproduction Science , 119 (3-4), 183-190. https://doi.org/10.1016/j.anireprosci.2010.01.012 Slanina, T. et al. (2018) Effect of taurine on turkey (Meleagris gallopavo) spermatozoa viability and motility. Czech Journal of Animal Science , 63 (4),127-135. https://doi.org/10.17221/79/2017-CJAS Stephens, T. D. et al. (2013) Effects of pentoxifylline, caffeine, and taurine on post-thaw motility and longevity of equine frozen semen. Journal of Equine Veterinary Science , 33 (8), 615-621. https://doi.org/10.1016/j.jevs.2012.10.004 Tirpak, F. et al. (2017) Low taurine concentrations possitively affect rabbit spermatozoa properties in later time intervals. Journal of Microbiology, Biotechnology and Food Sciences , 7, 128-131. https://doi.org/10.15414/jmbfs.2017.7.2.128-131

Impact of Algerian date palm pollen aqueous extract on epididymal and ejaculated rabbit sperm motility during in vitro incubation

Date palm pollen (DPP) has been widely tested in vitro in preservation of spermatozoa of different farm animals except in rabbits. The objective of this study was to evaluate the effect of various concentrations (20, 40, 80 mg/mL) of DPP aqueous extract on epididymal and ejaculated rabbit spermatozoa during in vitro incubation at 37 °C during 120 min. NaCl Pollen Extender (NPE) and TRIS Base Extender (TPE) were respectively used in epididymal and ejaculated sperm preservation. Sperm motility parameters were objectively analysed by a Computer Aided Sperm Analyser (CASA). The results revealed that in epididymal sperm at 0 minute, sperm motility showed significant higher values (p ≤ 0.05) for all doses of DPP in comparison to the control group. After 30, 60 and 120 min, the motility rate was higher (p > .05) in all the experimental groups compared to the control. Concerning velocity traits (VCL and VAP), and trajectories parameters (LIN, ALH, BCF), the doses of 40 mg/mL and 80 mg/mL showed the highest values than Control. In ejaculated samples, the motility rate at 0 minute improved only with the dose of 80 mg/mL and DPP showed protective effect at all doses (20, 40, 80 mg/mL) up to 60 min of incubation. DPP enhanced spermatozoa velocity (VCL and VAP) and the trajectory of sperm (LIN, ALH, BCF) even from 20 mg/mL at any points of storage. In conclusion, aqueous extract of date palm pollen is a suitable supplement to the extender for rabbit sperm by protecting and improving sperm motility parameters. HIGHLIGHTS The addition of DPP extract improves the quality of epididymal and ejaculated rabbit sperm. The extender supplemented with 80 mg/mL of DPP in NPE has a beneficial effect on kinetic traits of epididymal sperm. Nevertheless, in TPE extender, 20 mg/mL of DPP was enough to improve sperm motility traits in ejaculated rabbit semen.

Time and Dose-Dependent Effects of Viscum Album Quercus on Rabbit Spermatozoa Motility and Viability in Vitro.

The target of this study was to evaluate the effect of extract of the European mistletoe - Viscum album quercus L. on spermatozoa motility and viability in vitro. The CASA system was used to determine the spermatozoa motility parameters at different time intervals (0, 1, 2 and 3 h) and spermatozoa viability was determined in five different doses of Viscum album quercus L [10 (QA), 6.6 (QB), 3.3 (QC), 2.5 (QD) and 2 (QE) mg/ml]. Results in experimental groups detected a significant deterioration on rabbit spermatozoa after 1, 2 and 3 hours, compared to the control. The initial total spermatozoa motility showed increased value for all doses of Viscum album quercus in comparison to control. After in vitro culture a dose-dependent decrease (QA: reduction of 69.7 %, QB: reduction of 40.9 %) was found. For the progressive spermatozoa most significant decrease (86.8 % for QA vs. 48.5 % for QB) was detected compared to the control after 3 hours of culture. Spermatozoa viability (MTT test) was decreased in all experiment groups at the end of experiment, but the differences were not significant. Significant alterations of membrane integrity were found in groups with the highest Viscum album quercus concentration (QA, QB), but acrosome integrity showed no significant changes. Results suggest negative dose- and time-dependent effect of Viscum album quercus at higher doses on spermatozoa motility and viability parameters in vitro.

VISCUM ALBUM PINI EFFECT ON RABBIT SPERMATOZOA MOTILITY, VIABILITY, MEMBRANE AND ACROSOME INTEGRITY IN VITRO

Mistletoe (Viscum album L.) was previously described as an effective additive in the therapy of patients with cancer after medical therapy of the primary neoplasia. The defence activity and also direct cytotoxic stimulation was found to be upstanding for the anti-carcinogenic action. The main target of this study was to detect the effect of Viscum album pini L. at various concentration 1.0 (PA); 0.66 (PB); 0.33 (PC); 0.1 (PD); 0.066 (PE) and 0.033 (PF) mg/mL on the fine rabbit spermatozoa motility parameters at various time periods (0; 1; 2 and 3 h) during the cultivation in vitro. The CASA methodology was applied to estimate the spermatozoa motility parameters. The spermatozoa motility at the beginning of the experiment (Time 0) found decreased rate for examined doses of Viscum album pini compared to the control, but the statistical significant reduction was observed (p

Assessment of rabbit spermatozoa characteristics after amygdalin and apricot seeds exposure in vivo.

This study evaluates rabbit spermatozoa motility parameters after in vivo administration of amygdalin and apricot seeds during a 28-day period. Apricot seeds are potentially useful in human nutrition and amygdalin is the major cyanogenic glycoside present therein. The rabbits were randomly divided into the five groups (Ctrl-Control, P1, P2, P3, P4) with 4 males in each group. Control group received no amygdalin/apricot seeds while the experimental groups P1 and P2 received a daily intramuscular injection of amygdalin at a dose 0.6 and 3.0 mg/kg b.w. respectively during 28 days. P3 and P4 received a daily dose 60 and 300 mg/kg b.w. of crushed apricot seeds mixed with feed during 28 days, respectively. CASA system was used to evaluate for motility, progressive motility, curvilinear velocity, amplitude of lateral head displacement and beat cross frequency. Intramuscular application of amygdalin resulted in a significant time- and dose-dependent decrease of spermatozoa motility as well as progressive motility. On the other hand, oral consumption of apricot seeds had no significant effect either on the rabbit spermatozoa motility or progressive motility over the entire course of the study. The analysis of the other motion characteristics revealed a similar trend depicting a continuous, time- and dose-dependent decrease of all parameters following intramuscular AMG administration, with significant differences particularly for the dose 3.0 mg AMG/kg b.w. On the other hand, oral administration of apricot seeds had no significant impact on spermatozoa motility parameters. The present study suggests that short-term intramuscular application of amygdalin decreased rabbit spermatozoa motility in vivo. Whereas, consumption of apricot seeds did not induce any change in rabbit spermatozoa in vivo. Our findings suggest dose-dependent negative effect of pure amygdalin, but not apricot seeds on the rabbit spermatozoa parameters.

In Vitro Effects of Enterococcus Faecalis and Selected Biomolecules on the Motility of Rabbit Spermatozoa

Summary This study assessed the potential efficiency of selected biologically active substances on the motility behavior of rabbit spermatozoa subjected to in vitro induced E. faecalis contamination. Semen samples were collected from 10 male rabbits and the presence of E. faecalis was confirmed using MALDI-TOF Mass Spectrometry. For the in vitro experiments rabbit spermatozoa were resuspended in the presence of 0,3 McF E. faecalis and different concentrations of selected biomolecules (resveratrol - RES, quercetin - QUE, curcumin - CUR, epicatechin - EPI, isoquercitrin - IZO). Sperm motility was assessed using the computer-aided sperm analysis at 0h, 2h, 4h, 6h and 8h. The presence of E. faecalis significantly decreased the motility (P<0.001) when compared to the untreated Control starting at 2h and maintaining this negative impact throughout the entire in vitro culture. Meanwhile, the motility was significantly higher in the experimental samples subjected to E. faecalis together 5 μmol/L RES (P<0.05), 10 μmol/L QUE (P<0.05) as well as 1 μmol/L (P<0.01) and 10 μmol/L CUR (P<0.05) when compared to the Positive Control (4h). No biomolecule was able to maintain the motion comparable to the Negative Control, and none was effective against the rapid decline of sperm motility caused by the presence of E. faecalis during later stages of the in vitro experiment (6h and 8h). We may conclude that RES, QUE and CUR may provide a selective advantage to spermatozoa in the presence of E. faecalis, particularly during short-term rabbit semen handling.

LOW TAURINE CONCENTRATIONS POSSITIVELY AFFECT RABBIT SPERMATOZOA PROPERTIES IN LATER TIME INTERVALS

The aim of this study was to determinate the effect of various taurine concentrations on rabbit spermatozoa in in vitro conditions with the potential use in routine artificial insemination. Taurine, amino acid distinguished by its strong antioxidant properties, has been widely tested on spermatozoa of all kinds of farm animals except for the rabbits. This study investigated differences in rabbit spermatozoa motility and viability between the control sample and samples enriched with taurine solutions: TAU3 (3.125 mM), TAU6 (6.250 mM) and TAU12 (12.5 mM). Ejaculates of nine adult rabbits were subjected to this study and observed at time intervals 0, 90, 180 and 270 minutes while cultivated at 39°C. For detection of viability the MTT test was used and motility parameters were carried out using CASA analysis. Experimental samples were compared against the control. Assessment of the mitochondrial toxicity revealed that the addition of taurine ensured the higher viability in supplemented samples however with no statistical significance. Monitored motility parameters (motility, progressive motility and velocity curved line of spermatozoa) showed that taurine treated spermatozoa have lower activity at the beginning of the study but along with increasing time the positive effect of taurine was induced. Significant (P

Factors affecting storage of Slovak native rabbit semen in the gene bank.

In this study, fresh and frozen-thawed semen of Nitra and Zobor rabbit breeds were evaluated for potential inter-breed or inter-male differences in sperm quality traits. Individual male semen from four rabbits of each breed were diluted (v:v; 1:1) in a freezing medium composed of a commercial diluent, 16% of dimethyl sulphoxide (DMSO), 4% of Ficoll 70 and 2% of sucrose and frozen in liquid nitrogen vapours before being plunged into liquid nitrogen. Different motility traits, viability and plasma membrane integrity of fresh and frozen-thawed semen were evaluated in vitro using computer-assisted sperm analysis and flow cytometry. To evaluate the sperm fertilization ability, artificial insemination of fresh and frozen-thawed sperm was performed. Our results showed the effect of breed (P ≤ 0.05) on frozen-thawed sperm viability and plasma membrane integrity. Moreover, individual variability in semen quality among the rabbits was revealed (0.31 to 0.71 among quality traits). Our results thereby confirmed that the cryopreservation procedure could not ensure comparable sperm post-thaw survival for different breeds or males. Nevertheless, correlations between numbers of fresh total motile and progressively moving sperm and several quality parameters measured post thawing were revealed. Therefore, we suggest that the objective assessment of fresh rabbit sperm motility may be an effective indicator of frozen-thawed semen quality. Consequently, regular semen assessment is required in order to preserve good-quality insemination doses from native breeds.

IN VITRO EFECTS OF SELECTED BIOLOGICALLY ACTIVE COMPOUNDS ON RABBIT SPERMATOZOA MOTILITY BEHAVIOUR

The aim of this study was to evaluate the in vitro effects of selected pure naturally occuring biologically active compounds (resveratrol-RES, quercetin-QUE, curcumin-CUR, epicatechin-EPI, isoquercitrin-ISO) on rabbit sperm motility using the IDENT staining method and the computer-aided sperm (CASA) analysis. Semen samples were collected regularly from 10 male rabbits, pooled, the seminal plasma was removed and the sperm fraction was exposed to different concentrations (1, 5, 10, 50 and 100 μmol/L) of chosen biomolecules. At culture times of 0h, 2h, 4h, 6h and 8h, the spermatozoa motility was assessed using the TOX IVOS II. CASA system and the IDENT fluorescent staining method. The motility assessment revealed different behavior patterns, specific and unique to each of the studied biomolecules. After 8h of in vitro culture, the highest sperm motility was detected in experimental groups subjected to 10 μmol/L RES (P

Lead induced alterations in rabbit spermatozoa motility and morphology in vitro

The aim of this in vitro study was to determine the effect of lead chloride (PbCl2) on rabbit spermatozoa motility and morphology. Lead concentrations in the medium ranged between 0.45 and 11.17 μg/ml; incubation time was 240 min (analyzed immediately after Pb addition followed by 30, 60, 120, 180, and 240 min), and temperatures of the culture environment were 22°C (laboratory), 4°C (refrigerator), and 37°C (incubator). Results were compared with a control group without Pb addition. After 30 min of culture at 22°C, a negative effect of Pb was noted as spermatozoa motility significantly decreased in groups with higher concentrations. After 120 and 240 min, a dose-dependent effect on spermatozoa motility was noted. At 4°C, spermatozoa motility analysis detected no significant differences between any of the experimental groups and control. At 37°C, a negative effect of Pb incubation on motility was detected at Times 30, 60, 120, 180, and 240 in groups with high concentrations. At Times 120, 180, and 240 a significant decrease in spermatozoa motility was also noted in all experimental groups in comparison to control. The analysis of pathological spermatozoa at Time 240 revealed an increasing trend of morphological abnormalities after incubation with Pb. Across three temperature regimes an increase of morphological changes was noted, particularly in the group with the highest Pb concentration. The predominant morphological abnormalities were knob twisted flagellum, flagellum ball, separated flagellum, and broken flagellum. Knob twisted flagella represented the most frequent pathological changes in the experimental group with the highest Pb concentration. Results suggest that the inhibitory effect of Pb on spermatozoa motility parameters depends on the concentration, incubation time, as well as environmental temperature during incubation. Furthermore, a negative effect of Pb in vitro on spermatozoa morphology indicates possible reproductive problems under in vivo conditions, too.

Effects of cholesterol-loaded cyclodextrins on the rate and the quality of motility in frozen and thawed rabbit sperm.

The motility of sperm after freezing and thawing is critical for effective cryopreservation. It is known that supplementation with cholesterol-loaded cyclodextrin (CLC) improves cryosurvival of sperm in various animals. To clarify the effects of supplementation with CLC on rabbit sperm motility after freezing and thawing, rabbit sperm motility was analyzed using a computer-assisted sperm analysis system. Sperm motility with CLC supplementation was 29.4 ± 9.6% (mean ± SD), which was significantly higher than that of controls (20.8 ± 7.1%, P<0.05). The curvilinear velocity of sperm with CLC exceeded that of controls, whereas the values for linearity and wobble were significantly lower in sperm with CLC compared with controls. After artificial insemination, 44.3% of recovered ova were fertilized in the CLC-supplemented group, which was higher than the percentage in the control group (36.4%). The results indicate that supplementation with CLC improves the rate and quality of motility in rabbit sperm after freezing and thawing, and would be advantageous for successful cryopreservation.

The time-dependent effects of prostate granules and seminal plasma on the capacitation, acrosome reaction, and motility of rabbit sperm

The present study investigated the role of rabbit seminal plasma and prostate granules on capacitation, the acrosomal reaction (AR), and sperm motility. Semen ejaculates obtained from five mature New Zealand White rabbit bucks in three collection series were used in the study. Raw semen, Percoll-selected sperm alone and in presence of either seminal plasma or prostate granules were incubated for 120min in capacitation medium. Chlortetracycline fluorescence (reflecting capacitation and the acrosome reaction) and sperm kinetic traits were analysed in each sample. All traits were observed from 0 to 120min of incubation at 37°C in 5% CO2. Both seminal plasma and prostatic granules markedly improved the sperm motility but seminal plasma mainly inhibited the capacitation progress whereas prostatic granules inhibited the AR. In conclusion, prostatic granules and seminal plasma have specific roles in synchronising sperm capacitation and the AR with egg availability.

Short communication: Influence of vitamins C and E on sperm motility of rabbit bucks

The objective of the study is to investigate the effect of vitamin C and E supplementation on rabbit sperm motility. Forty INAT breed bucks aged 8.5 mo were divided into 2 groups: control group (C) and treated group (T) receiving vitamins C (1 g/L) and E (1 g/L) incorporated in the drinking water. Semen was collected using an artificial vagina over 6 wk (C, total ejaculates=81; T, total ejaculates=76). The massal motility (MM) was evaluated in the fresh and raw semen. Then, the individual motility at 0 (IM0), 2 (IM2) and 4 h (IM4) after semen collection was determined in diluted semen and conserved at +4°C for the 2 groups. MM was higher in T than in C group (P=0.0012). However, the individual motilities IM0, IM2 and IM4 did not vary between the 2 groups. In conclusion, the supplementation of vitamins C and E in the drinking water for rabbit influenced only the MM in both fresh and raw semen.

Effect of the MACS technique on rabbit sperm motility

Abstract Magnetic-activated cell sorting (MACS) separates apoptotic spermatozoa by the use of annexin V-conjugated nanoparticles which bind to phosphatidylserine that is externalized on the outer leaflet of the sperm plasma membrane. This technique yields two fractions: annexin V-negative (AnV−) and annexin V-positive (AnV+). The aim of the study was to evaluate the effect of MACS application on the motility parameters of rabbit spermatozoa. Rabbit semen samples collected separately from 4 bucks (I, II, III, and IV) were filtered and separated in a MACS system. The semen samples from a control (untreated) group, AnV− and AnV+ fraction were evaluated using CASA system. The experiment was replicated 4 times for each buck. The AnV+ sperm had significantly lower concentration than the AnV− fractions and the control samples (P&lt;0.05 for bucks I, II, III, but not IV). We observed that the proportion of apoptotic spermatozoa in the semen of NZW bucks is about 20%. There was no significant difference in the percentage of motile and progressively motile spermatozoa between the AnVfractions and control samples. In conclusion, the MACS technique has no harmful effect on the rabbit sperm concentration and motility.

In vitro copper toxicity on rabbit spermatozoa motility, morphology and cell membrane integrity

In this in vitro study the effects of copper sulphate on the motility, morphology and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated by CASA method and Annexin analysis was used for detection of structural changes. For analysis of morphology samples of rabbit semen were fixed with Hancock's solution and stained with Giemsa, and for each sample at least 500 spermatozoa were evaluated. The concentration of copper in the medium varied from 3.57 to 4.85 μg CuSO4/mL. At Time 0 the highest motility was detected in the control group (57.78 ± 3.90%). Motility in groups with copper administration was lower in comparison to control. Significant differences were detected in groups with 3.70–4.85 μg CuSO4/mL (P<0.05) at Time 0. After 1 h of incubation with copper sulphate the motility significantly decreased almost in all experimental groups. However, at Time 2 h significant increase of total motility was observed in groups with lower concentrations of copper (3.57 and 3.63 μg CuSO4/mL). After 24 and 48 h of incubation almost all the spermatozoa were dead recording no motility at all concentrations. The concentration- dependent decrease of spermatozoa motility up to 50% of control was detected for the group receiving highest copper administration (4.85 μg CuSO4/mL) at Times 1 and 2 h. Progressive motility had an identical trend to that of motility in all experimental groups, at all culture times and for all concentrations. Evaluation of distance and velocity parameters indicated that a sort of stress tolerance developed in lower concentrations (3.57 and 3.63 μg CuSO4/mL). At lower concentrations, an increase was noted for distance parameter DCL and velocity parameter VCL, indirectly confirming the significant motility and progressive motility increase. Other motility parameters (straightness index, linearity index, wobble and amplitude of lateral head displacement) revealed decrease in the group with the highest copper concentration (4.85 μg CuSO4/mL) in comparison to the control group after 2 h of incubation, only. No significant alteration was noted for these parameters in comparison to control at Times 0 and 1 h. The total percentage of morphologically abnormal spermatozoa was significantly higher (P<0.05) in the group with the highest copper concentration (46.20 ± 5.54%) in comparison to control (30.60 ± 2.91). Predominant morphological abnormalities were acrosomal changes, knob-twisted flagellum and small heads. Detection of spermatozoa with disordered membrane was carried out for groups with higher copper concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the copper-exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In copper-exposed groups positive reaction proved alteration in anterior part of head (acrosome) and in connection segment (mid-piece) of spermatozoa. Detected data evidently confirm adverse effects of high copper sulphate concentrations in rabbit semen on parameters of spermatozoa motility, morphology and membrane integrity. This paper also indicates the lowest possible toxic concentration of copper (3.70 μg CuSO4/mL) to rabbit spermatozoa in relation to motility.

Cadmium toxicity at low concentration on rabbit spermatozoa motility, morphology and membrane integrity in vitro

In this study the effect of cadmium on various parameters of spermatozoa motility, morphology as well as on the spermatozoa membrane integrity in rabbits was analyzed in vitro, experimental concentrations ranging from 0.62 to 0.98 μ g CdCl2/mL. Pooled rabbit (n = 5) semen was cultured in vitro with cadmium and subsequently diluted to various experimental concentrations apart from control which received no cadmium exposure. Using computer assisted semen analysis method (CASA) we detected decrease of total motility with in the higher concentration range at Time 0. However, with increasing time (after 1 and 2 h of culture), cadmium exerted deleterious effect leading to significant motility reduction in comparison to control. A similar trend was exhibited in case of progressive motility, too. Most of the spermatozoa distance and velocity parameters detected no significant change in comparison to control at the beginning of culture (Time 0), although the toxic effect became significant (P < 0.05) with the passage of culture time (Times 1 and 2 h) in all concentrations. Analysis of spermatozoa morphology detected significant (P < 0.05) alterations at higher concentrations. At higher concentrations acrosomal changes, head without flagellum/separated flagellum, broken flagellum and other abnormalities were significantly higher (P < 0.05), while knob–twisted flagellum and small heads differed significantly (P < 0.05) in comparison to control at all concentrations. In regards to flagellum torso, flagellum ball and retention of cytoplasmic drop statistically higher values (P < 0.05) were noted at the maxium experimental concentration only. Annexin analysis for detection of spermatozoa with disordered membranes revealed higher occurrence of positive spermatozoa in cadmium exposed groups. Annexin–positive reactions suggested alterations in anterior part of head (acrosome) and in flagellum (mitochondrial segment) of spermatozoa. This paper underlines that cadmium is highly toxic for rabbit spermatozoa, as visualized by the toxic effects on parameters of spermatozoa motility, morphology and membrane integrity. The toxic effect is more drastic at higher concentrations. This study also indicates that cadmium requires a minimum one hour incubation time to exert its deletorious effects on various parameters of spermatozoa, particularly at low concentrations.

In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity

In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 μ g HgCl2/mL. At Time 0 the highest motility was detected in the control group (67.09 ± 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0–83.3 μ g HgCl2/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25–83.3 μ g HgCl2/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 – 83.3 μ g HgCl2/mL at Time 0, for groups receiving 12.5–83.3 μ g HgCl2/mL at Time 60 and 120, decreasing from 36.46 ± 18.73% to 1.03 ± 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In mercury-exposed groups positive reaction proved alteration in anterial part of head (acrosome), connection part (connection piece) and in mitochondrial segment. Detected data evidently confirm adverse effects of high mercuric chloride concentrations in rabbit semen on spermatozoa motility parameters.

In vitro gossypol induced spermatozoa motility alterations in rabbits

The objectives of the present study were to: (i) examine the in vitro dose response of rabbit spermatozoa motility to the antifertility agent gossypol (GOS) and (ii) determine whether filtered (FIL) and unfiltered (UNFIL) GOS differ in their magnitude of effect. Rabbit semen belonging to adult males (n = 5; 12–14 months) were cultured with UNFIL GOS and FIL GOS (5% solution) and subsequently diluted (1:1–7) for analysis using a Computer Assisted Semen Analyzer (CASA) system in 5 time periods (0, 60, 120, 180 and 360 minutes). At Time 0, no significant change in rabbit spermatozoa motility (MOT) and progressive motility (PROG) with GOS FIL was noted, while increases were observed with GOS UNFIL. At Time 60, weak changes were noted for MOT and PROG. After 120 minutes of culture with both GOS FIL and GOS UNFIL, MOT and PROG decreased significantly in some experimental groups. However, no differences were recorded for both the parameters at Times 180 and 360, with the exception of PROG in the GOS UNFIL category (groups A, B, E, F and G), where a significant decrease was noticed. Detailed evaluation of the distance and velocity parameters revealed reduction in all these studied markers after 60 and 120 minutes of in vitro culture with both GOS FIL and GOS UNFIL, indirectly confirming the PROG decrease. Straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) mostly remained unaltered at all time periods for GOS FIL, where as some minor alterations were noticed in GOS UNFIL category for STR, LIN, WOB, ALH and BCF parameters at Time 0, 60 and 120. The present study confirms the dose and time dependent alterations of rabbit spermatozoa motility parameters by GOS. The GOS dynamics in our experiment shows that rabbit spermatozoa as a biological material can indicate a GOS inhibition of motility. Obtained data for the first time indicates a higher immobilizing potential of unfiltered GOS in comparison to filtered GOS in its inhibitory action of spermatozoa motility parameters in rabbits.

Influence of a 50 Hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rates in rabbits

Effects of a 50 Hz extra-low frequency electromagnetic field (ELF EMF) on in vitro rabbit spermatozoa motility were analyzed, as well as the effect on fertilization rates after insemination. Pooled semen samples and a control were exposed to 50 Hz ELF EMF. The difference of the samples of the test groups G1 and G2 with the control group CG (75.56%) for spermatozoa motility were found to be significant (P < 0.01). Differences were significant (P < 0.01) for curvilinear velocity (VCL) between the test group G3 (122.38 μ m/s) and the control group CG (112.02 μ m/s). Hormonally stimulated adult (9–12 months) females (n = 140) were inseminated with semen samples from G1, G2, G3 and CG (0.88 × 109 spermatozoa/0.5 mL average insemination portion) immediately after ELF EMF exposure and fertilization (kindling) rates were calculated. For the G2 it was 54.28% data indicate 50 Hz ELF EMF induced alterations of spermatozoa motility and kindling rate in rabbits, therefore influencing fertility.