Showing papers on "Sperm plasma membrane published in 2015"

New insights into the regulation of cholesterol efflux from the sperm membrane

Abstract: Cholesterol is an essential component of the mammalian plasma membrane because it promotes membrane stability without comprising membrane fluidity. Given this important cellular role, cholesterol levels are tightly controlled at multiple levels. It has been clearly shown that cholesterol redistribution and depletion from the sperm membrane is a key part of the spermatozoon's preparation for fertilization. Some factors that regulate these events are described (e.g., bicarbonate, calcium) but the mechanisms underlying cholesterol export are poorly understood. How does a hydrophobic cholesterol molecule inserted in the sperm plasma membrane enter the energetically unfavorable aqueous surroundings? This review will provide an overview of knowledge in this area and highlight our gaps in understanding. The overall aim is to better understand cholesterol redistribution in the sperm plasma membrane, its relation to the possible activation of a cholesterol transporter and the role of cholesterol acceptors. Armed with such knowledge, sperm handling techniques can be adapted to better prepare spermatozoa for in vitro and in vivo fertilization.

Influence of ejaculation frequency on seminal parameters.

Abstract: Several factors have been shown to influence semen parameters, one of which is sexual abstinence; a clinical criteria included in the semen evaluation to provide maximum sperm quality. The aim of the present study was to assess the effect of a daily ejaculation frequency on conventional and functional semen parameters. Semen samples were collected daily over a period of two weeks of which every second sample per person was processed and analyzed according to the World Health Organization guidelines. Furthermore, mitochondrial function, intracellular reactive oxygen species production and sperm DNA fragmentation were evaluated by flow cytometry. Total sperm count and seminal volume per ejaculation declined and remained decreased for the duration of the daily ejaculation period. However, conventional parameters such as sperm concentration, motility, progressive motility, morphology, vitality and functional parameters such as sperm plasma membrane integrity, mitochondrial membrane potential and DNA fragmentation was not significantly affected and remained similar to the initial measurement throughout the daily ejaculation period. Despite intra- and inter individual variations, the average values of the basic semen parameters remained above the WHO (2010) reference values throughout the daily ejaculation period. Interestingly, a decreasing trend in intracellular ROS production was observed, although statistically not significant. The study shows that an extended 2 week period of daily ejaculation does not have major clinical effects on conventional and functional seminal parameters.

Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation

Abstract: To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 ...

AMPK up-activation reduces motility and regulates other functions of boar spermatozoa

Abstract: We recently demonstrated that AMPK inhibition in spermatozoa regulates motility, plasma membrane organization, acrosome integrity and mitochondrial membrane potential. As AMPK activity varies in different energy conditions induced by sperm environment, this work investigates the functional effects of AMPK activation in boar spermatozoa. Spermatozoa were incubated under non-stimulating (TBM) or Ca 2+ and HCO3 − -stimulating (TCM) media in the presence/absence of AMPK activator, A769662, for different times. AMPK activity, evaluated as Thr 172 phosphorylation by western blot, is effectively increased by A769662 in spermatozoa. AMPK activation significantly reduces the percent- age of motile spermatozoa under Ca 2+ and/or HCO3 − -stimulating conditions. Moreover, AMPK activation in spermatozoa incubated in TBM or TCM significantly reduces curvilinear VCL, straight-line VSL and average VAP velocities, which subsequently lead to a significant decrease in the percentage of rapid spermatozoa (VAP . 80 mm/s). The effect of AMPK activation on motility is intensified by the absence of BSA in the incu- bation medium. AMPK activation for a short time prevents the decline in cell viability and in the sperm population displaying high mitochondrial membrane potential which is induced by Ca 2+ and HCO3 − . Sustained (24 h) AMPK activation under TBM or TCM significantly increases both lipid disorganization and phosphatidylserine externalization in the sperm plasma membrane, and diminishes the acrosome membrane integrity. In summary, AMPK activation modifies essential sperm processes such as motility, viability, mitochondrial membrane potential, acrosome mem- brane integrity, and organization and fluidity of plasma membrane. As these spermatozoa processes are required under different environmental conditions when transiting through the female reproductive tract to achieve fertilization, we conclude that balanced levels of AMPK activity are essential for regulating sperm function.

Seminal Vesicle Secretion 2 Acts as a Protectant of Sperm Sterols and Prevents Ectopic Sperm Capacitation in Mice

Abstract: Seminal vesicle secretion 2 (SVS2) is a protein secreted by the mouse seminal vesicle. We previously demonstrated that SVS2 regulates fertilization in mice; SVS2 is attached to a ganglioside GM1 on the plasma membrane of the sperm head and inhibits sperm capacitation in in vitro fertilization as a decapacitation factor. Furthermore, male mice lacking SVS2 display prominently reduced fertility in vivo, which indicates that SVS2 protects spermatozoa from some spermicidal attack in the uterus. In this study, we tried to investigate the mechanisms by which SVS2 controls in vivo sperm capacitation. SVS2-deficient males that mated with wild-type partners resulted in decreased cholesterol levels on ejaculated sperm in the uterine cavity. SVS2 prevented cholesterol efflux from the sperm plasma membrane and incorporated liberated cholesterol in the sperm plasma membrane, thereby reversibly preventing the induction of sperm capacitation by bovine serum albumin and methyl-beta-cyclodextrin in vitro. SVS2 enters the uterus and the uterotubal junction, arresting sperm capacitation in this area. Therefore, our results show that SVS2 keeps sterols on the sperm plasma membrane and plays a key role in unlocking sperm capacitation in vivo.

Storage temperature of boar semen and its relationship to changes in sperm plasma membrane integrity, mitochondrial membrane potential, and oxidoreductive capability

Abstract: The aim of this study was to analyze changes in sperm plasma membrane integrity, mitochondrial activity, and extracellular environment during storage of boar semen at 5 °C, 16 °C, and 25 °C for 10 days. Progressive sperm motility, plasma membrane integrity (SYBR-14/PI-test and HOS test), aspartate aminotransferase (AAT), and mitochondrial activity (JC-1-test and NADH-dependent NBT assay), as well as pH and osmolality were assessed in nine ejaculates of Polish Landrace boars. The plasma membrane integrity of the semen stored at 5 °C was similar to that of the semen stored at 16 °C and 25 °C; however, an increase in AAT activity of the semen stored at 5 °C revealed sperm membrane disorders as early as day 2. Significant differences in the progressive motility of the semen stored at 5 °C and 16 °C were observed at each of the evaluation times. This reduced motility was consistent with decreased sperm mitochondrial transmembrane potential and oxidoreductive capability. We inferred that the cooling of boar semen to 5 °C increases the permeability of the sperm plasma membrane, which may not be revealed by SYBR-14/PI staining or HOS testing. The loss of boar sperm motility that occurs during hypothermic liquid preservation may be connected with alterations in the plasma membrane stability and disorders of mitochondrial transmembrane potential and oxidoreductive capability.

Chapter 3 – The Spermatozoon

Abstract: This chapter examines the novel structural features of the mammalian spermatozoon, its composition and assembly, and the mechanisms regulating its function. The spermatozoon is a highly specialized cell with unique structural features and functional characteristics required to deliver the male genome to the egg. The head of a spermatozoon contains a highly condensed nucleus, an enzyme-filled acrosome, and a small amount of cytoplasm. The flagellum is a highly novel feature that provides the spermatozoon with the ability to propel itself through liquid medium. New findings about genes that are expressed only in spermatids and encode novel proteins unique to spermatozoa are reviewed in this chapter. The gene knockout approach has identified many proteins required for normal sperm functions in mice that are likely to be required in humans as well. However, many critical questions remain to be answered about the composition, organization, and function of spermatozoa.

Semen characteristics and selected biochemical markers of canine seminal plasma in various seasons of the year

Abstract: The aim of this study was to evaluate the influence of season on selected qualitative semen characteristics and biochemical markers of canine seminal plasma. Whole ejaculates were collected from 5 crossbred dogs aged 2-8 years. The study covered a period of one year divided into four seasons: spring (March, April, May), summer (June, July, August), autumn (September, October, November) and winter (December, January, February). Semen samples were subjected to macroscopic and microscopic analyses to determine semen volume, total sperm counts and sperm morphology parameters. The study also involved the determination of sperm motility parameters (CASA system), sperm plasma membrane integrity (SPMI, fluorescent staining SYBR-14/PI), sperm mitochondrial membrane potential (MMP, fluorescent staining JC-1/PI) and the ATP content of sperm cells. Total protein content (TPC) and the activity of alkaline phosphatase (AP) and acid phosphatase (AcP) were determined in biochemical analyses of seminal plasma. No significant differences in ejaculate volume, SMPI or ATP content of sperm cells were observed between seasons. The highest total sperm counts were reported in ejaculates acquired in summer and autumn. The lowest MMP values were determined in summer ejaculates. No significant differences in sperm motility (MOT) were observed throughout the experiment, but ejaculates collected in autumn and winter were characterized by the highest progressive motility (PMOT). AP activity and TPC were not significantly affected by season. However, AcP activity levels were significantly lower in autumn than in the remaining seasons. Seasonal variations in the analyzed macroscopic and microscopic parameters of ejaculates and biochemical markers of seminal plasma did not exert a clear negative effect on the quality of canine semen.

Protective effect of lycopene on human spermatozoa during cryopreservation and its mechanism

Abstract: Objective To investigate the protective effect of lycopene against cryopreservation injury of post-thawing human sperm and its mechanism. Methods Semen samples were collected from 25 volunteers, each sample equally divided into four parts to be cryopreserved with cryoprotectant only (Ly0 control) or cryoprotectant + lycopene at the concentrations of 2 (Ly2), 5 (Ly5), and 10 µmol/L (Ly10), respectively. Before and after thawing, the semen samples were subjected to computer-assisted semen analysis ( CASA) for sperm kinematics, flow cytometry for sperm apoptosis, thiobarbituric acid assay for malondialdehyde (MDA) concentration, and JC-1 fluorescent staining for the sperm mitochondrial membrane potential (MMP). Results After cryopreservation, sperm motility was markedly decreased in all the groups (P 0.05). Conclusion Addition of lycopene at a proper concentration to cryoprotectant may reduce oxidative damage to sperm mitochondria in the freezing-thawing process, attenuate oxidative stress injury induced by reactive oxygen species to sperm plasma membrane, and improve the anti-apoptosis ability of sperm.

Sperm Selection Based on Surface Electrical Charge

Abstract: Sperm selection is an important part of the ICSI and is based solely on sperm morphology and motility. The latter parameters may not be sufficient to select sperm with intact chromatin. Therefore, sperm selected based on sperm functional characteristics may result in selection of the most appropriate sperm for the ICSI procedure. In this chapter, we introduce two advanced sperm selection procedures based on electric charge on the sperm plasma membrane, Zeta method and electrophoretic method, and also discuss the importance of these methods for ART.

The effect of two packaging systems on the post-thaw characteristics of canine sperm

Abstract: The aim of this study was to compare the effect of different packaging systems on some parameters of cryopreserved canine spermatozoa. The experimental material consisted of the sperm-rich fractions of ejaculates collected from four Beagle dogs. Semen samples for cryopreservation were stored in 0.25 ml plastic straws and two aluminum tubes with a total volume of 5.0 ml. Semen was frozen in static nitrogen vapor for 10 minutes (0.25 ml straws) or 15 and 20 minutes (aluminum tubes). Post-thaw assessments involved the determination of sperm motility parameters using a computer assisted sperm analyzer (CASA), sperm plasma membrane integrity (SPMI), mitochondrial membrane potential (MMP) and acrosome integrity (normal apical ridge, NAR). Regardless of the packaging system applied, no significant differences in total sperm motility (TMOT) or selected kinematic parameters were observed after freezing-thawing. However, spermatozoa frozen in 0.25 mL straws were characterized by improved functionality, in particular mitochondrial function, after thawing. The results indicate that large quantities of canine semen can be frozen in aluminum tubes. Further studies are required, however, to evaluate different freezing and thawing rates of aluminum tubes.

Effect of Conjugated Linoleic Acid on Boar Semen Quality After Long-term Refrigeration at 17°C

Abstract: Contents In this study, the effect of conjugated linoleic acid (10 trans, 12 cis) (CLA) on refrigerated boar sperm quality parameters up to 14 days at 17°C was assessed. Semen was extended in Androhep and divided into four treatments supplemented with CLA (25, 50, 100 and 200 μm) and control group, then kept for 2 h at 22°C. Afterwards an aliquot of each treatment was removed, and mitochondrial activity, viability, lipid membrane peroxidation (LPO) and stability of the sperm plasma membrane were assessed by flow cytometry. The remaining extended semen was maintained at 17°C until 336 h, repeating the same analysis every 48 h. Regarding percentage of live spermatozoa, no statistical differences were observed among treatments up to 96 h. After this time, viability decreased significantly (p < 0.05) for CLA concentrations of 100 and 200 μm. Despite these results, there was an individual response to CLA. Although in the control group, the boar A presented better results when compared with the other boars, especially at concentrations of 50 and 100 μm boar B showed significantly higher results (p < 0.05). Supplementation with CLA improved (p < 0.05) LPO, but not the mitochondrial membrane potential of sperm. The highest two CLA concentrations showed to be toxic for sperm as all results were lower than the observed for the control. In conclusion, CLA at 50 μm seems to be an efficient concentration for reducing the oxidative stress, decreasing LPO, maintaining viability, membrane stability and mitochondrial potential on refrigerated boar spermatozoa.

Effect of Silymarin on Plasma Membrane and Acrosome of Sperm Treated with Aluminum Chloride

Abstract: Background: Aluminum, as an environmental pollutant, has destructive effects by inducing oxidative stress on male reproductive system and sperm. Silymarin, an effective substance extracted from Silybum marianum, is a potent antioxidant which inhibits oxidative stress. Because of toxic effects of aluminum and the antioxidant role of silymarin, this study was performed to investigate if silymarin can prevent the adverse effects of aluminum chloride on plasma membrane integrity and acrosome integrity in ram sperm. Materials and Methods: In this experimental study, epididymal spermatozoa from Farahani's ram are divided into five groups: sperm at 0 hour, sperm at 180 minutes (control), sperm treated with aluminum chloride (0.5mM) for 180 minutes, sperm treated with silymarin (0.5μM) + aluminum chloride (0.5μM) for 180 minutes and sperm treated with silymarin (0.5μM) for 180 minutes. To evaluate sperm plasma membrane integrity and sperm acrosome integrity, propidium iodide-Hoechst and comassie brilliant blue staining were used, respectively. The results were analyzed using one-way ANOVA and p<0.05 was considered as significant level. Results: The percentage of sperm plasma membrane integrity and acrosome integrity were significantly decreased in aluminum chloride group compared to the control. The simultaneous use of silymarin+aluminum chloride could significantly compensate the adverse effects of aluminum chloride on the sperm plasma membrane integrity and acrosome integrity compared to aluminum chloride. Conclusion: Aluminum chloride induces toxic effect on ram sperm plasma membrane integrity and acrosome integrity and silymarin is able to compensate the adverse effect this pollutant on these parameters.

A Comprehensive and Systematic Study of Semen Quality and SpermFunctional Status in Normozoospermic Controls and Infertile Males fromSouth India

Abstract: Objective: Despite, several global studies indicate the variations in semen characteristics that accounts for male infertility, the association of specific changes in semen quality and fertility status among different Indian communities are poorly investigated. With wide range of geographical locations, diverse lifestyle patterns, seasonal variations combined with heterogeneous population, India offers an excellent system to study genotype-to-phenotype correlation. Hence, the current study has been initiated in South Karnataka region of India, in order to examine the variations in semen quality and sperm functional status in infertile individuals compared with normozoospermic controls. Methods: WHO strict guidelines are followed for systematic semen analysis of 239 infertile and 244 normozoospermic control subjects. Results: Interestingly, compared to normozoospermic controls, higher percentage of physical abnormalities such as, low semen volume and reduced sperm count are observed in infertile men. Additionally, semen characteristics namely, vitality and motility values are significantly reduced in infertile than controls. Further, in sperm function test the lower scores are documented for hypo-osmotic swelling assay, but not for sperm chromatin decondensation and acrosome intactness examination, suggesting loss of sperm plasma membrane integrity in infertile men. Moreover, the observed changes in semen parameters and sperm function are also evident in different infertile sub-conditions with varied responses. Surprisingly, age wise analysis revealed reduction in sperm morphology scores, whereas, vitality, count, motility and volume remain unchanged with increasing age of infertile males. However, we recorded inverse relationship between age and sperm vitality as well as motility in normozoospermic control men. Together, though the scores for different semen parameters in normozoospermic control group are in accordance with WHO reference range, the infertile men displayed poor semen quality. Conclusion: Thus, our data establishes basic differences between infertile and normozoospermic control group in terms of semen characteristics and sperm functional status, but the cause may be attributable to genetic or environmental factors or interaction of the two, which necessitates further detailed examination in larger cohort among heterogeneous population.

Effect of oleic-linoleic acid and β-sitosterol to freezing extender of bulls and stallions semen.

Abstract: Addition of polyunsaturated fatty acids and/or cholesterol to a freezing diluent can modify the sperm plasma membrane composition, influencing its behavior during cryopreservation, thus, favoring seminal cryoresistance. The present study aimed to evaluate the effects of the addition of oleic-linoleic acid, (OLA); ?-sitosterol (?-sit), a plant analog of cholesterol; and OLA + ?-sit in combination to a freezing diluent, on the cryopreservation bull and stallion semen. The following variables were analyzed: motility/vigor, plasma and acrosomal membrane integrity (by Trypan Blue/Giemsa staining), mitochondrial activity (by DAB staining), and lipid peroxidation (by a TBARS assays). The lipids were added according to experimental treatments: C – control group, A1 and A2 – OLA at concentrations of 37 ?M and 74 ?M, B1 and B2 – ?-sit at concentrations of 1 ?g mL-1 and 2 ?g mL-1; AB1 and AB2 – OLA 37 ?M + ?-sit 1 ?g mL-1 and OLA 74 ?M + ?-sit 2 ?g mL-1, respectively. The study was divided into three experiments; in Experiment 1, the concentrations of the groups A1, B1, and AB1 were evaluated, whereas in Experiment 2 the concentrations of the groups A2, B2, and AB2 were analyzed, both experiments were performed with bull semen. We conducted Experiment 3 using equine semen with the addition of lipids at all of the concentrations described. Data were subjected to analysis of variance, using the GLM procedure of SAS, with treatment means compared by Duncan test considering 5% significance. These variables differed significantly after thawing the semen post-collection. However, there was no significant difference between treatments when variables were compared within the same time point, except for Experiment 2, where there was a decrease in motility and vigor decrease post-thaw in the groups following ?-sit addition (C – 51.0 ± 13.7%/2.9 ± 0.4; B2 – 35.8 ± 15.8%/2.3 ± 0.6; AB2 – 38.5 ± 16.6%/2.5 ± 0.5, respectively; p < 0.05). In conclusion, the tested concentrations of these lipids did not confer greater cryoresistance to the spermatozoa, and were not effective in preserving the structural integrity of plasma and acrosomal membranes after thawing. Furthermore, there was no change in the mitochondrial activity and lipid peroxidation due to lipids addition.

Effect of oleic-linoleic acid and β-sitosterol to freezing extender of bulls and stallions semen Efeito da adição de ácido oleico-linoleico e β-sitosterol ao diluente de congelação do sêmen de touros e garanhões

Abstract: Addition of polyunsaturated fatty acids and/or cholesterol to a freezing diluent can modify the sperm plasma membrane composition, inAuencing its behavior during cryopreservation, thus, favoring seminal cryoresistance. The present study aimed to evaluate the effects of the addition of oleiclinoleic acid, (OLA); β-sitosterol (β-sit), a plant analog of cholesterol; and OLA + β-sit in combination to a freezing diluent, on the cryopreservation bull and stallion semen. The following variables were analyzed: motility/vigor, plasma and acrosomal membrane integrity (by Trypan Blue/Giemsa staining), mitochondrial activity (by DAB staining), and lipid peroxidation (by a TBARS assays). The lipids were added according to experimental treatments: C – control group, A1 and A2 – OLA at concentrations of 37 μM and 74 μM, B1 and B2 – β-sit at concentrations of 1 µg mL -1 and 2 µg mL -1 ; AB1 and AB2 – OLA 37 μM + β-sit 1 µg mL -1 and OLA 74 μM + β-sit 2 µg mL -1 , respectively. The study was divided into three experiments; in Experiment 1, the concentrations of the groups A1, B1, and AB1 were evaluated, whereas in Experiment 2 the concentrations of the groups A2, B2, and AB2 were analyzed, both experiments were performed with bull semen. We conducted Experiment 3 using equine semen with the addition of lipids at all of the concentrations described. Data were subjected to analysis of variance, using the GLM procedure of SAS, with treatment means compared by Duncan test considering 5% signi?cance. These variables differed signi?cantly after thawing the semen post-collection. However, there was no signi?cant difference between treatments when variables were compared within the same time point, except for Experiment 2, where there was a decrease in motility and vigor decrease postthaw in the groups following β-sit addition (C – 51.0 ± 13.7%/2.9 ± 0.4; B2 – 35.8 ± 15.8%/2.3 ± 0.6; AB2 – 38.5 ± 16.6%/2.5 ± 0.5, respectively; p < 0.05). In conclusion, the tested concentrations of these lipids did not confer greater cryoresistance to the spermatozoa, and were not effective in preserving the structural integrity of plasma and acrosomal membranes after thawing. Furthermore, there was no change in the mitochondrial activity and lipid peroxidation due to lipids addition.

Efeito de diferentes diluidores sobre a membrana plasmática do espermatozóide eqüino e fertilidade do sêmen resfriado Effect of different extenders on sperm plasma membrane and fertility of equine cooled semen

Abstract: Stallions semen shows individual differences regarding to its preservation suitability. Although the causes for that are not clear, phenomena that occur during semen manipulation anel storage in +5°C exerce considerable influence on functional anel morphological integrity of equine sperm plasma membrana. Besides fertility rate, the present work evaluated the efficiency of different semen extenders on spermatozoal motility, plasma membrana functional anel morphological integrity of equine liquid preserved semen at +5°C for (2h. lt could be concluded that sperm functional anel morphological integrity evaluation is usefull for equine semen rotine analysis anel could aid to preview the fertility suitability of cooled semen.

Homeopathic Medicine Improves the Motility and Vigor of Semen in Rams

Abstract: The semen of rams suffers during cryopreservation, and the sperm plasma membrane breaks down during the process, creating the deleterious effect of reducing their fertilization capacity.

Comparison of different osmolarities and sugarsalt solutions for hypo-osmotic swelling test of frozen-thawed water buffalo spermatozoa

Abstract: Hypo-osmotic swelling test (HOST) is a method of assessing the functional integrity of sperm plasma membrane which in turn was claimed as indicator of sperm fertility. Standard protocol of HOST has been established in different species but not in buffaloes. Thus, the current study was conducted to establish the HOST assay for buffalo frozen-thawed sperm cells. Frozen-thawed sperm cells were incubated in different osmolalities (0, 50, 100, 150, 200, 250, and 300 mOsm) of sodium citrate-fructose solution to assess sperm reaction. Then, the effectiveness of sucrose vs. fructose was compared as sugar component of the HOST solution. Results showed higher (P<0.05) number of HOS positive (+) spermatozoa in 150 mOsm compared to 0, 250 and 300 mOsm/L though no significant difference was observed with 50, 100, and 200 mOsm/L. Sucrose- and fructose-containing solutions are both effective in enhancing swelling among treated spermatozoa. The results demonstrated that 150 mOsm is the efficient osmolality to effect optimum reaction of frozen-thawed buffalo spermatozoa and that either sucrose or fructose could be used for HOST solution to assess the functional membrane integrity of buffalo sperm cells.

Treating boar sperm with cholesterol-loaded cyclodextrins or cyclodextrins prior to cryopreservation: effects on post-thaw in vitro sperm quality of sperm cryopreserved in different freezing extenders.

Abstract: [EN] Cryopreserved boar sperm is not used extensively for artificial insemination due to poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged when cooled from body temperature to 5 oC (cold shock), as well as during the freeze-thaw process. Increasing the cholesterol content of boar sperm membranes could increase their post-thaw survival, similarly to other species that are cold shock sensitive. Cholesterol can be easily added to sperm membranes using cholesterol-loaded cyclodextrins (CLC). Treating sperm from different species susceptible to cold-shock with CLC before cryopreservation improves sperm cryosurvival. Egg yolk and glycerol are common constituents of extenders used for boar sperm cryopreservation. However, conventional freezing extenders could not be the appropriate for CLC-treated sperm. The aim of this Thesis is to evaluate cryosurvival of CLC or cyclodextrin-treated boar sperm in three different conditions: using conventional freezing extenders, using extenders with alternative concentrations of glycerol and egg yolk and using amides as cryoprotectants. CLC or methyl- s-cyclodextrin treatment (1 mg/120 x 106 sperm) prior to cryopreservation using a conventional freezing extenders provided either slight or no benefit, respectively, to post-thaw sperm plasma membrane integrity (+ 8%; P 0.05). In addition, sperm from both, good and poor freezers, responded similarly to CLC treatment (P > 0.05). Reduction in egg yolk concentration from 20 to 10% was detrimental for post-thaw sperm viability, even in semen treated with CLC (- 12%; P < 0.05). On the other hand, it was observed that traditional concentration of glycerol (3%) was not the appropriate to freeze CLC-treated sperm (- 13% viable sperm compared to control; P < 0.05). Thus, CLC-treated sperm showed a higher tolerance (+ 13 % sperm viability; P < 0.05) to high glycerol concentrations (5%) than non-treated sperm. Regarding the efficacy of amides as cryoprotectants, three of the amides (lactamide, acetamide and formamide) produced deleterious effects in fresh boar sperm (P < 0.05). The other amides (methylformamide, dimethylacetamide and dimethylformamide) efficiently improved post-thaw sperm viability (+ 5 to 15 %; P < 0.05) but negatively affected the sperm motility (- 11 to 16% total motile sperm; P < 0.05) and the sperm fertilizing ability in vitro (dimethylformamide: - 64 % penetration rate; P < 0.05), irrespective of the sperm treatment. On the other hand, CLC-treated samples showed better in vitro fertilizing ability than control samples when glycerol was used as cryoprotectant (+ 2 penetrated spermatozoa/oocyte; P < 0.05). The results obtained in this Thesis suggest that conventional freezing protocols should be optimized for CLC-treated boar sperm in order to obtain the benefit of CLC treatment observed in other species sensitive to cold shock.; [ES] Las inseminaciones artificiales en la especie porcina se realizan habitualmente con semen refrigerado, debido a las bajas…

Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation 1 Running title: Flow cytometry analysis of sperm membrane potential.

Abstract: To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential. In the present work, we used flow cytometry to analyze changes in sperm membrane potential (Em) during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mice sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed by sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K + , by PKA inhibitors and by SLO3 inhibitors in CD1 mice sperm, and undetectable in Slo3 knock-out mice sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3 and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream a

Effect of different cryoprotectants on sperm plasma membrane integrity of osmanabadi and sirohi buck semen

Abstract: Semen was collected from six Osmanabadi and six Sirohi bucks weekly for six weeks by Artificial Vagina method. Semen was diluted using Tris Egg yolk Glycerol (TYG), Tris Egg yolk Ethylene Glycol (TYE) and Tris -Dimethyl sulphoxide (DMSO) dilutors (TYD) and evaluated for sperm plasma membrane integrity by HOS test at pre (50C) and post freezing (-1960C at 24 and 72 hours) stage. At pre freezing (50C) stage in Osmanabadi buck semen there was no significant difference (P>0.05) across different dilutors with respect to HOS positive sperm percentage but in Sirohi bucks significantly higher sperm plasma membrane integrity was observed in TYD and TYG than TYE dilutor. At 24 hours post freezing stage in Osmanabadi buck semen significantly higher plasma membrane integrity was noted in TYD than TYG and TYE dilutors. In Sirohi buck semen significantly higher plasma membrane integrity was noted in TYD than TYE dilutor, TYG being intermediate. At 72 hrs post freezing stage in osmanabadi bucks semen there was no significant difference (p>0.05) across different dilutors with respect to HOS positive sperm percentage. In Sirohi bucks semen significantly, higher (P<0.05) percentage of plasma membrane integrity was observed in TYD than TYE dilutors. It was concluded that in both the breeds higher plasma membrane integrity was observed in TYD dilutor which indicates that DMSO was more effective cryoprotectant than ethylene glycol and glycerol in preserving sperm membrane integrity during cryopreservation of Osmanabadi and Sirohi bucks semen.