Fluorescein Isothiocyanate-conjugated Pisum Sativum Agglutinin Research Articles

Evaluation of Cryopreserved Ram Sperm with Nano-Ozone Solution and Post-Thaw Life Span by Flow Cytometric Analysis.

Ozone has been used as a therapy tool in medical science for conditions such as ulcers, peritonitis, wounds, and mostly joint problems. Ozone therapy strengthens the resistance to infections by kick-starting antioxidant, anti-inflammatory, and immune modulation systems. Ozone creates a defensive response against oxidative stress in membranes and protects metabolism against reactive oxygen species (ROS). Sperm membranes are one of ROS's main targets; therefore, the cells' cryopreservation process requires more defensive elements for better results. This study aimed to investigate the protective effect of nano-ozone solution (NOS) on ram sperm cryopreservation and the influence of the process on various sperm parameters for post-thaw (0 hour) and postincubation (6 hours) time points. Samples were collected from six Merino rams in the breeding season by electroejaculation five times at 3-day intervals. The study was conducted by cryopreservation of the samples using a tris citric acid-egg yolk-based extender. The samples were subjected to freezing in control and NOS (0.5, 1, and 2 μg/mL nano-ozone supplemented). Post-thaw motility, hypo-osmotic swelling test, acrosome (fluorescein isothiocyanate-conjugated Pisum sativum agglutinin [PSA-FITC]), and DNA integrities (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) were evaluated with a phase-contrast microscope. Mitochondrial membrane potential (MMP) assessments were conducted by JC1-PI dual staining with a flow cytometer. Malondialdehyde and glutathione (GSH) levels were measured by a spectrophotometer. Sperm kinematics were investigated by a computer-assisted sperm analyzer (CASA) at the post-thaw time point. Compared with the control, relatively low doses of NOS (0.5 and 1 μg/mL) yielded better results in many parameters (motility, membrane and acrosomal integrities, MMP, various sperm kinematics, and GSH levels) (p < 0.05). The addition of low ozone doses to cryopreservation extenders improved the results compared with the control group at post-thaw and postincubation time points. Despite the valuable potential of nano-ozone supplementation in ram sperm cryopreservation, this subject requires further investigations with fertility trials soon.

Iberiotoxin and clofilium regulate hyperactivation, acrosome reaction, and ion homeostasis synergistically during human sperm capacitation.

Potassium channels play essential roles in the regulation of male fertility. However, potassium channels mediating K+ currents in human sperm (IKSper ) remain controversial. Besides SLO3, the SLO1 potassium channel is a potential candidate for human sperm KSper. This study intends to elucidate the function of SLO1 potassium channel during human sperm capacitation. Human sperm were treated with iberiotoxin (IbTX, a SLO1 specific inhibitor) and clofilium (SLO3 inhibitor) separately or simultaneously during in vitro capacitation. A computer-assisted sperm analyzer was used to assess sperm motility. The sperm acrosome reaction (AR) was analyzed using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Sperm protein tyrosine phosphorylation was studied using western blotting. Intracellular Ca2+ , K+ , Cl- , and pH were analyzed using ion fluorescence probes. Independent inhibition with IbTX or clofilium decreased the sperm hyperactivation, AR, and protein tyrosine phosphorylation, and was accompanied by an increase in [K+ ]i , [Cl- ]i , and pHi , but a decrease in [Ca2+ ]i . Simultaneously inhibition with IbTX and clofilium lower sperm hyperactivation and AR more than independent inhibition. The increase in [K+ ]i , [Cl- ]i , and pHi , and the decrease in [Ca2+ ]i were more pronounced. This study suggested that the SLO1 potassium channel may have synergic roles with SLO3 during human sperm capacitation.

KCNQ1 Potassium Channel Expressed in Human Sperm Is Involved in Sperm Motility, Acrosome Reaction, Protein Tyrosine Phosphorylation, and Ion Homeostasis During Capacitation.

Potassium channels are involved in membrane hyperpolarization and ion homeostasis regulation during human sperm capacitation. However, the types of potassium channels in human sperm remain controversial. The voltage-gated ion channel KCNQ1 is ubiquitously expressed and regulates key physiological processes in the human body. In the present study, we investigated whether KCNQ1 is expressed in human sperm and what role it might have in sperm function. The expression and localization of KCNQ1 in human sperm were evaluated using Western blotting and indirect immunofluorescence. During capacitation incubation, human sperm were treated with KCNQ1- specific inhibitor chromanol 293B. Sperm motility was analyzed using a computer-assisted sperm analyzer. The acrosome reaction was studied using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Protein tyrosine phosphorylation levels and localization after capacitation were determined using Western blotting and immunofluorescence. Intracellular K+, Ca2+, Cl−, pH, and membrane potential were analyzed using fluorescent probes. The results demonstrate that KCNQ1 is expressed and localized in the head and tail regions of human sperm. KCNQ1 inhibition reduced sperm motility, acrosome reaction rates, and protein tyrosine phosphorylation but had no effect on hyperactivation. KCNQ1 inhibition also increased intracellular K+, membrane potential, and intracellular Cl−, while decreasing intracellular Ca2+ and pH. In conclusion, the KCNQ1 channel plays a crucial role during human sperm capacitation.

Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways

BackgroundHeat shock protein 90 (Hsp90) is a highly abundant eukaryotic molecular chaperone that plays important roles in client protein maturation, protein folding and degradation, and signal transduction. Previously, we found that both Hsp90 and its co-chaperone cell division cycle protein 37 (Cdc37) were expressed in human sperm. Hsp90 is known to be involved in human sperm capacitation via unknown underlying mechanism(s). As Cdc37 was a kinase-specific co-chaperone of Hsp90, Hsp90 may regulate human sperm capacitation via other kinases. It has been reported that two major mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38, are expressed in human sperm in the same locations as Hsp90 and Cdc37. Phosphorylated Erk1/2 has been shown to promote sperm hyperactivated motility and acrosome reaction, while phosphorylated p38 inhibits sperm motility. Therefore, in this study we explored whether Hsp90 modulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.MethodsHuman sperm was treated with the Hsp90-specific inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) during capacitation. Computer-assisted sperm analyzer (CASA) was used to detect sperm motility and hyperactivation. The sperm acrosome reaction was analyzed by using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC) staining. The interactions between Hsp90, Cdc37, Erk1/2 and p38 were assessed using co-immunoprecipitation (Co-IP) experiments. Western blotting analysis was used to evaluate the levels of protein expression and phosphorylation.ResultsHuman sperm hyperactivation and acrosome reaction were inhibited by 17-AAG, suggesting that Hsp90 is involved in human sperm capacitation. In addition, Co-IP experiments revealed that 17-AAG reduced the interaction between Hsp90 and Cdc37, leading to the dissociation of Erk1/2 from the Hsp90-Cdc37 protein complex. Western blotting analysis revealed that levels of Erk1/2 and its phosphorylated form were subsequently decreased. Decreasing of Hsp90-Cdc37 complex also affected the interaction between Hsp90 and p38. Nevertheless, p38 dissociated from the Hsp90 protein complex and was activated by autophosphorylation.ConclusionsTaken together, our findings indicate that Hsp90 is involved in human sperm hyperactivation and acrosome reaction. In particular, Hsp90 and its co-chaperone Cdc37 form a protein complex with Erk1/2 and p38 to regulate their kinase activity. These results suggest that Hsp90 regulates human sperm capacitation via the Erk1/2 and p38 MAPK signaling pathways.

150 α6 Integrin for evaluating sperm quality in bulls with different capacities of invitro embryo production

The α6 integrin, an adhesion molecule, is expressed on bovine sperm, but major questions about the role of integrins in sperm-oocyte fusion remain unsolved. In this work, we show the results of characterisation of sperm α6 integrin from 4 bulls with different capacities of invitro embryo production using flow cytometric analysis. The bull capacities judged by the rate of blastocyst formation after invitro embryo production with semen from 5 ejaculates per animal were 44.3, 17.1, 13.2, and 15.0% for bulls 1, 2, 3, and 4, respectively (P&amp;lt;0.05). For flow cytometric analysis, surface expression of α6 integrin was evaluated using a fluorescence-activated cell sorter (FACScan, Coulter Electronics) using a 520-nm excitation from an argon laser at 150 mW for excitation. Frozen-thawed sperm were centrifuged at 700×g for 10min and washed once in warm phosphate buffered saline (PBS). Briefly, the spermatozoa (5×105 cells per sample) were resuspended in 100μL of PBS containing 1% bovine serum albumin. After washing 3 times, the live cells were incubated at room temperature for 1h with 100μL (1:500) of α6 monoclonal antibody (Chemicon) in PBS (0.1M, pH 7.4) with 1% bovine serum albumin. After washing three times, the cells were incubated at 4°C for 1h in the dark with the fluorescein isothiocyanate-conjugated F(ab)2 fragment of affinity isolated goat anti-mouse antibody (Invitrogen). The cells were washed three times, resuspended in 100μL of PBS, and analysed. For each sample, 10 000 cells were recorded at a flow rate of 200-300 cells s−1 using forward scatter (cell size) and side angle of light scatter (cell density), the first using a logarithmic amplifier and the second using a linear amplifier. The fluorescence data were collected using the logarithmic amplifier. The percentage of positive cells and the mean fluorescence channel on a 1023-channel scale were calculated using Epics Profile II software (Epics II Software). To define the forward and side-scatter regions corresponding to sperm, binding of fluorescein isothiocyanate-conjugated Pisum sativum agglutinin to the acrosome was used for setting the bitmap on the dot plot. Initially, the percentage of spermatozoa stained with α6 antibody was calculated on a per-individual basis. Subsequently, the mean±standard deviation was calculated for each group. The Mann-Whitney U test was used to compare the differences in the expression of α6 between groups. Expression of α6 integrin was higher in bull 1 (control) than in bulls with low capacities of invitro embryo production. The spermatozoa from bull 1 was distributed in a single broad peak with a mean fluorescence intensity of 36.16±4.17%. The increased distance of the fluorescence intensity in bull 1 compared with the weak fluorescence peaks in the others bulls reflects the increased width and distance of the population distribution. In conclusion, α6 integrin may be used as a biomarker to evaluate sperm quality. This study was supported by FAPESP grants 2010/01077-9, 2011/18085-7, and 2016/00976-6.

Effect of the herbicide atrazine and its major metabolite, DACT, on bovine sperm cryotolerance

During freezing and thawing procedures, sperm are exposed to chemical and/or physical stressors that may cause adverse and harmful changes to sperm membranes. Accurate evaluation of the structural and functional integrity of fresh as well as cryopreserved sperm is highly important in predicting sperm fertilization capacity and success of artificial insemination (AI). The herbicide atrazine (ATZ) and its major metabolite, diaminochlorotriazine (DACT) are considered a ubiquitous environmental contaminants and endocrine disruptors, which deleteriously effect sperm function. Taking into consideration possible damage caused by environmental contaminants to sperm membranes, additive effects during cryopreservation cannot be ruled out. The aim of the current study was to evaluate the effect of ATZ (0.1 or 1 μM) and DACT (1 or 10 μM) exposure during or after cryopreservation on bovine sperm cryotolerance. Sperm membrane integrity and functionality were evaluated using fluorimetric probes: (1) double-stranded DNA was examined by 4′,6-diamidino-2-phenylindole; (2) plasma membrane integrity was examined by propidium iodide; (3) acrosome reaction (AR) was examined by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin; mitochondrial membrane potential (ΔΨm) was examined by 5,5′,6,6′-tetra-chloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide fluorescent probe. The findings demonstrate, that exposure of sperm to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during cryopreservation increased the proportion of dead sperm relative to the control (P < 0.09); exposure to DACT (1 or 10 μM) increased ΔΨm (P < 0.03). Neither ATZ nor DACT affected spontaneous AR. In contrast, the proportion of sperm with Ca++ ionophore-induced AR was lower after exposure to 1 μM DACT (P < 0.05). Following freezing and thawing procedures, exposing sperm to 1 μM ATZ increased the proportion of dead sperm relative to the control (P < 0.05), but had no significant effect on sperm ΔΨm or AR. In conclusion, exposing sperm to endocrine-disrupting chemicals such as ATZ or DACT during cryopreservation reduces sperm cryotolerance and resistance post-thawing.

Effects of egg yolk and soybean lecithin on sperm quality determined by computer-assisted sperm analysis and confocal laser scanning microscope in chilled canine sperm.

The reduction of spermatozoa survival time is a major problem of canine chilled sperm for artificial insemination. The aim of the study was to improve the quality of canine chilled sperm during storage time. We therefore, evaluated the effects of eight treatments with different levels of soybean lecithin concentration (1, 3 and 5%) and egg yolk (20%) in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extender on chilled canine sperm quality during 10 days of storage. The sperm motility was analysed by computer‐assisted sperm analysis (CASA), whereas plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential parameters were determined using a fluorescent staining combination of propidium iodide (PI), Hoechst 33342 (H342), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) by confocal laser scanning microscope. The results showed that egg yolk was found to be better than soybean lecithin in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extender for maintaining the quality of chilled canine sperm within 10 days of storage (P < 0.05). Although egg yolk in Tris‐citric‐fructose extender could maintain the motility better than other extenders, egg yolk in Tris‐citric‐fructose‐mineral salts extender was the highest in intact plasma membrane, intact acrosome membrane and high mitochondrial membrane potential (P < 0.05). In contrast, the sperm quality of soybean lecithin in Tris‐citric‐fructose‐mineral salts extender was lower than that of soybean lecithin in Tris‐citric‐fructose extender, and soybean lecithin 1% was greater than soybean lecithin 3% and 5% in plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential (P < 0.05). In conclusion, soybean lecithin cannot replace egg yolk in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extenders, and egg yolk in Tris‐citric‐fructose‐mineral salts extender is superior to other extenders in chilling canine sperm.

Fluorimetric Techniques for the Assessment of Sperm Membranes.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes

Fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were used to simultaneously evaluate the integrity of plasma and acrosomal membranes as well as mitochondrial membrane function in cryopreserved bovine semen and to verify its influence on fertility and postinsemination uterine vascularization. One hundred eighty-two Nellore cows were distributed for artificial insemination (AI) using semen batches separated according to the cell percentage presenting intact plasma membrane, intact acrosome, and high mitochondrial function (IPIAH): group G (44.5% IPIAH, n = 68), group M (23.0% IPIAH, n = 56), and group R (8.5% IPIAH, n = 58). The uterine hemodynamic was evaluated by Doppler sonogram in three periods: 30 hours before AI, 4 and 24 hours after AI were considered the resistance index and the uterine vascularization score. The pregnancy rate of group G (64.7%) was greater (P > 0.05) compared with group R (36.2%), but both did not differ from group M (50.0%). There was no effect (P > 0.05) of semen quality on uterine vascularization. Greater vascularization was noticed 4 hours after AI than 30 hours before and 24 hours after AI. Semen evaluation using fluorescent probes contributes to predicting fertilizing potential of semen. The use of semen with less percentage of IPIAH sperm does not alter uterine hemodynamic in cows.

Morphofunctional Characterization of Cooled Sperm With Different Extenders to Use in Equine-Assisted Reproduction

The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide [JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen.

Estudio de los daños estructurales tras la inmovilización espermática previa a la microinyección intracitoplasmática de espermatozoides en sujetos teratozoospérmicos

ObjectiveTo describe and compare the ultrastructural alterations, DNA fragmentation and acrosome status that could cause sperm immobilisation prior to intracytoplasmic sperm injection, in samples from normozoospermic and teratozoospermic males. Material and methodsFor this study we used 15 sperm samples from consenting normozoospermic donors and 20 samples from teratozoospermic males, submitted for assisted reproduction at Instituto Bernabeu of Alicante. To assess the ultrastructural alterations induced by immobilisation, human oocytes were used as containers for spermatozoa and then observed by transmission electron microscopy. DNA fragmentation was assessed using TUNEL and acrosome status using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. The control group consisted of sperm without manipulation. Statistical analysis between groups was performed by ANOVA. ResultsThe results showed that immobilised spermatozoa presented the same damage in plasma and acrosomal membranes in the samples from both normozoospermic and teratozoospermic subjects. After assessing DNA fragmentation in the cells from normozoospermic and teratozoospermic patients by the TUNEL technique, we observed that the percentage of spermatozoa with DNA fragmentation did not increase in the immobilised group compared with the control group. However, the percentage of acrosome-reacted sperm was significantly greater in immobilised spermatozoa than in the control group in both normozoospermic and teratozoospermic males. ConclusionThe results of this study show that the structural damage caused by immobilisation in spermatozoa from teratozoospermic and normozoospermic males is independent of sperm morphology. In addition, the immobilisation does not cause DNA fragmentation. However, the percentage of acrosome-reacted sperm was greater in the immobilised group than in the control group, in both normozoospermic and teratozoospermic subjects.

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.

20 LIPID PEROXIDATION AND GENERATION OF HYDROGEN PEROXIDE FROM SUBFERTILE STALLION SPERMATOZOA DURING STORAGE AT REFRIGERATION TEMPERATURE

The aim of this study was to evaluate the generation of hydrogen peroxide (H2O2) and membrane lipid peroxidation of subfertile spermatozoa stored at 5°C for 24 h. Semen samples, collected from 5 subfertile stallions (≤40% of conception rate), were diluted in a skim-based extender (BotuSemen; Botupharma) and stored in a passive transport container at 5°C over a period of 24 h. Sperm motility was determined by computer-assisted semen analysis (CASA; IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA) for total motility (TM), progressive motility (PM), and rapid sperm (RAP). Plasma membrane integrity, generation of H2O2, and membrane lipid peroxidation were determined by flow cytometry (LSRFortessa cell analyzer, BD Biosciences, Franklin Lakes, NJ, USA). For evaluation of acrosome and plasma membrane integrity, samples were stained with Hoechst 33342 dye (H33342; Molecular Probes, Eugene, OR, USA), iodide propidium (IP; Sigma, St. Louis, MO, USA), and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA; Sigma). For the assessment of generation of H2O2, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma), H33342, and IP were added to the sperm suspension. For the assessment of sperm lipid peroxidation, samples were stained with C11-BODIPY581/591 (Molecular Probes), H33342, and IP. Samples were evaluated after semen collection (0 h) and after 24 h of cooled storage. A total of 10 000 gated events were analyzed per sample by flow cytometry. The green fluorescence (FL1) was collected through a 580-nm band-pass filter and the red fluorescence (FL3) through a 635-nm band-pass filter. Statistical analysis was performed using GraphPad Prism version 4.03 (2005; GraphPad Software Inc., La Jolla, CA, USA), through paired t-test to identify the significant differences (P ≤ 0.05). In general, sperm motility parameters of TM and RAP, and acrosome and plasma membrane integrity significantly decreased after 24 h of refrigeration at 5°C (P ≤ 0.05). Interestingly, no differences were found in PM at 0 and 24 h (P ≥ 0.05). The percentage of sperm cells with high generation of H2O2 did not differ at 0 and 24 h (P ≥ 0.05), whereas the percentage of sperm cells with membrane peroxidation increased (0.56 ± 0.3 v. 2.24 ± 1.3; P ≤ 0.05) at these periods. The percentage of viable sperm cells with low generation of H2O2 had a significant decrease from 22.6 ± 11.2 at 0 h to 0.08 ± 0.1 after 24 h of storage (P ≤ 0.05), although no differences were found in the percentage of viable sperm cells without membrane peroxidation (P ≥ 0.05). In conclusion, the cooled storage of subfertile spermatozoa for 24 h drastically decreased the number of viable spermatozoa with low generation of H2O2 and increased the percentage of membrane lipid peroxidation, which is related to the decrease in sperm motility and increase in dead sperm. These results make it difficult to use refrigerated semen of subfertile stallions with poor semen quality in commercial breeding programs. São Paulo Research Foundation (FAPESP) is acknowledged for supporting this research.

Season-Induced Changes in Bovine Sperm Motility Following a Freeze-Thaw Procedure

Decreased conception rate of dairy cows in the summer is mainly associated with the deleterious effects of environmental thermal stress on the female reproductive tract. Here, we suggest that decreased reproductive performance might be partially due to inferior-quality semen. Semen from five representative bulls was collected in summer (August to September) and winter (December to January) and evaluated with a computerized sperm-quality analyzer for bulls (SQA-Vb). No seasonal effect was found in fresh ejaculate, but sperm examined post-thawing showed lower velocity, motility and progressive motility (P<0.04) in summer vs. winter samples. Element concentrations in the seminal plasma, determined by inductively coupled plasma-atomic emission spectrometry, differed between seasons, with higher (P<0.01) concentration values of K, Mg, Na and S elements in winter vs. summer samples. Therefore, season-induced alterations in seminal plasma element concentration should be taken into account when using an extender for cryopreservation. Acrosome integrity was assessed by a triple-fluorescence test using Hoechst 33342, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Acrosome reaction was examined by a one-step staining method using FITC-PSA. The proportion of sperm cells with a damaged acrosome post-thawing tended to be higher (P<0.07) in semen collected during the summer vs. winter. Such alterations suggest that seasonal reductions in sperm function might also be involved in the decreased conception rate of dairy cows in summer.

Exogenous glucose improves long-standing human sperm motility, viability, and mitochondrial function

A relevant fraction of human sperm can be maintained in simple phosphate-buffered saline (PBS) for more than 1 week at room temperature, a time that could be doubled if glucose was available. This does not seem to be directly due to sperm mitochondrial activity.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Simultaneous assessment of plasmatic, acrosomal, and mitochondrial membranes in ram sperm by fluorescent probes

In this experiment, it was defined a protocol of fluorescent probes combination: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and JC-1. For this purpose, four ejaculates from three different rams (n=12), all showing motility &gt;80% and abnormal morphology &lt;10%, were diluted in TALP medium and split into two aliquots. One of the aliquots was flash frozen and thawed in three continuous cycles, to induce damage in cellular membranes and to disturb mitochondrial function. Three treatments were prepared with the following fixed ratios of fresh semen:flash frozen semen: 0:100 (T0), 50:50 (T50), and 100:0 (T100). Samples were stained in the proposal protocol and evaluated by epifluorescence microscopy. For plasmatic membrane integrity, detected by PI probe, it was obtained the equation: v=1.09+0.86X (R²=0.98). The intact acrosome, verified by the FITC-PSA probe, produced the equation: v=2.76+0.92X (R²=0.98). The high mitochondrial membrane potential, marked in red-orange by JC-1, was estimated by the equation: v=1.90+0.90X (R²=0.98). The resulting linear equations demonstrate that this technique is efficient and practical for the simultaneous evaluations of the plasmatic, acrosomal, and mitochondrial membranes in ram spermatozoa.

13 SEASONAL CHANGES IN ION CONCENTRATION, ION-CHANNEL EXPRESSION, ACROSOME INTEGRITY, AND BOVINE SPERM QUALITY

The decrease in dairy cow fertility during the summer is mainly associated with the deleterious effects of heat stress on the female reproductive tract. The present study suggests that the decreased reproductive performance is, in part, a result of using semen of inferior quality. Evaluated parameters included (1) ionic concentrations in the seminal fluid of bull semen, including [Ca2+], [K+], and [Na+]; (2) expression of ion channels CNGB1, CNGA3, and IP3R; (3) parameters of semen quality such as volume, concentration, motility, and progressive motility; and (4) acrosome integrity. Semen was collected from 5 representative bulls throughout the summer (August and September) and winter (December and January), and was evaluated according to a computerized sperm quality analyzer for bulls (SQA-Vb, Medical Electronic Systems, Caesarea, Israel). [Ca2+], [K+], and [Na+] in the seminal fluid were determined by inductively coupled plasma-atomic emission spectrometry. A semiquantitative PCR and the computer program Scion Image (Scion Corporation, Frederick, MD) were used to determine RNA expression of ion channels CNGB1, CNGA3, and IP3R. Acrosome integrity was assessed by a triple-fluorescence test, which included Hoechst 33342 (h33342), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), and propidium iodide (PI) staining. Data were presented as mean ± SD and analyzed by t-test (JMP-in 5.1). Findings revealed seasonal differences in [K+], [Na+] (P &lt; 0.01), and [Ca2+] (P &lt; 0.08) in the seminal fluid. These were associated with differences in RNA expression for ion channels CNGB1, CNGA3 (P &lt; 0.01), and IP3R (P &lt; 0.09), which are known to be involved in acrosome reactions. Although no differences were found in fresh semen, a progressive decrease in motility was noted for post-thaw semen collected in the summer (P &lt; 0.05). Furthermore, the proportion of sperm cells with damaged acrosomes was higher in post-thaw semen collected in the summer than in its counterpart collected in winter (54.2 ± 3.5% v. 51.4 ± 1.9%, respectively; P &lt; 0.08). The results suggest that semen collected during the summer is less able to survive cryopreservation, as reflected by its inferior vitality post-thawing. Further examination is required to determine whether such alterations are involved in the low summer fertility of dairy cows. The authors thank the Israeli Artificial Insemination Center, Sion, for semen and Medical Electronic Systems (Caesarea, Israel) for providing the SQA-Vb sperm quality analyzer.

Physiological action of oestradiol on the acrosome reaction in human spermatozoa

The acrosome is a secretory vesicle located in the sperm head. The acrosome reaction consists in the fusion of the sperm plasma membrane with the external acrosomal membrane. It has been observed that this reaction does not take place in spermatozoa incubated in cervical mucus, hydrogel that contains high concentrations of oestradiol in the peri-ovulatory period. The objective of the present study was to analyse the influence of oestradiol on the acrosome reaction in human spermatozoa to evaluate the possible inhibitory effect of this hormone. Spermatozoa were incubated in progesterone (10.1 nmol l(-1)); oestradiol plus progesterone (oestradiol at 840 pmol l(-1) and progesterone at 10.1 nmol l(-1)), oestradiol (840 pmol l(-1)) and control (without steroidal hormones) for 30 min, 60 min, 240 min and 24 h. The acrosome reaction was evaluated by stain with Hoechst 33258 and fluorescein isothiocyanate-conjugated Pisum sativum agglutinin lectin. Progesterone-incubated spermatozoa showed the highest percentage of acrosome reaction (P < 0.05). Spermatozoa incubated with oestradiol and oestradiol plus progesterone showed the lowest percentage of acrosome reaction. The present study demonstrates the inhibitory role of oestradiol on the acrosome reaction, stimulated by progesterone in human spermatozoa under physiological conditions.

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology </=10%. The semen was diluted in Modified Tyrode's medium (TALP) (25 x 10(6) spermatozoa/ml) and split into two aliquots, one sample was flash-frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash-frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.