Detection of Sperm Membrane Proteins by Immunocytochemistry, Scanning and Transmission Electron Microscopy, and Western Blotting.

Western blotting in combination with scanning and transmission electron microscopy provide accurate and complementary information on the presence and localization of proteins in the plasma membrane, as well as the structural and ultrastructural features of sperm cells. The present chapter describes the procedures to detect the presence of membrane proteins by Western blotting and to localize them in the plasma membrane of sperm cells.

Predictive Indicators of Cryotolerance and Fertility in Bovine Sperm: Evaluating Fresh Semen Quality to Improve AI Outcomes With Frozen-Thawed Sperm.

The success of artificial insemination (AI) with frozen-thawed semen in cattle is influenced by both female factors and sperm quality. In terms of sperm quality, prior studies indicate that the ability of frozen-thawed bovine sperm to fertilise an oocyte is dependent on their quality and resilience to cryopreservation. Cryopreservation induces oxidative stress, leading to ultrastructural damage in the sperm.

Inhibition of forward and reverse transport of Ca via Na/Ca exchangers (NCX) prevents sperm capacitation.

Background: While calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na/Ca exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece.

Clustering of spermatozoa examined through flow cytometry provides more information than the conventional assessment: a resilience to osmotic stress example.

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI).

[Not Available].

Background: Ion channels are essential for differentiation and maturation of germ cells, and even for fertilization in mammals. Different types of potassium channels have been identified, which are grouped into voltage-gated channels (Kv), ligand-gated channels (K), inwardly rectifying channels (K), and tandem pore domain channels (K).

Material-methods: The present review includes recent findings on the role of potassium channels in sperm physiology of mammals.

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals.

Alkalinization of sperm cytosol is essential for plasma membrane hyperpolarization, hyperactivation of motility, and acrosomal exocytosis during sperm capacitation in mammals. The plasma membrane of sperm cells contains different ion channels implicated in the increase of internal pH (pH) by favoring either bicarbonate entrance or proton efflux. Bicarbonate transporters belong to the solute carrier families 4 (SLC4) and 26 (SLC26) and are currently grouped into Na/HCO transporters and Cl/HCO exchangers.

Ion Channels of Spermatozoa: Structure, Function, and Regulation Mechanisms.

Ion transport is essential for sperm physiology, being involved in sperm-cell differentiation and maturation, motility activation, chemotaxis towards the oocyte, and fertilization, as well as in sperm adaptation to the surrounding medium [...

Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus.

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model.

Exogenous Albumin Is Crucial for Pig Sperm to Elicit In Vitro Capacitation Whereas Bicarbonate Only Modulates Its Efficiency.

This work sought to address whether the presence of exogenous bicarbonate is required for pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane potential, O consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3α) and protein kinase A (PKA).

The Presence of Seminal Plasma during Liquid Storage of Pig Spermatozoa at 17 °C Modulates Their Ability to Elicit In Vitro Capacitation and Trigger Acrosomal Exocytosis.

Although seminal plasma is essential to maintain sperm integrity and function, it is diluted/removed prior to liquid storage and cryopreservation in most mammalian species. This study sought to evaluate, using the pig as a model, whether storing semen in the presence of seminal plasma affects the sperm ability to elicit in vitro capacitation and acrosomal exocytosis. Upon collection, seminal plasma was separated from sperm samples, which were diluted in a commercial extender, added with seminal plasma (15% or 30%), and stored at 17 °C for 48 or 72 h.

Long-term storage of boar seminal doses contaminated with Proteus vulgaris: A dose-dependent effect on sperm motility and sperm-bacteria interaction.

This study evaluated how Proteus vulgaris affects sperm quality and sperm-bacteria interaction in stored semen samples. A strain of P. vulgaris resistant to streptomycin, penicillin, lincomycin and spectinomycin was added to boar semen in doses of 10, 10, 10, 10 and 10 CFU/mL.

HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa.

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor.

Elucidating the Role of K Channels during In Vitro Capacitation of Boar Spermatozoa: Do SLO1 Channels Play a Crucial Role?

This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K-channel blocker, also added at 0 min or after 240 min of incubation.

Study of boar sperm interaction with Escherichia coli and Clostridium perfringens in refrigerated semen.

The present study analyses the interaction of boar spermatozoa with Escherichia coli and Clostridium perfringens, in 9-day refrigerated semen samples. Ejaculates from 10 sexually mature boars were inoculated with either E. coli or C.

Evaluation of porcine beta defensins-1 and -2 as antimicrobial peptides for liquid-stored boar semen: Effects on bacterial growth and sperm quality.

The present study evaluated whether two different antimicrobial peptides (AMP): porcine beta defensins-1 (PBD1) and -2 (PBD2) at three concentrations (1.5 μM, 3 μM and 5 μM) could be a suitable alternative to antibiotics in liquid-stored boar semen. Two separate experiments were conducted with liquid-stored boar semen preserved at 17 °C for 9-10 days.

A comparative study of the effects of Escherichia coli and Clostridium perfringens upon boar semen preserved in liquid storage.

The present study compares the sperm quality of boar seminal doses artificially inoculated with Escherichia coli and Clostridium perfringens, and maintained in liquid storage at 15°C for a 9-day period. Seminal doses from 10 sexually mature Piétrain boars were diluted in a Beltsville Thawing Solution (BTS)-based extender and infected either with E. coli or C.

Sperm quality and fertility of boar seminal doses after 2 days of storage: does the type of extender really matter?

The present approach was designed to evaluate the extender effects on sperm quality and fertility of short-term refrigerated seminal doses from Landrace boars lodged in husbandry-controlled conditions. For this purpose, we analyzed the sperm quality of seminal doses diluted in short-term (Beltsville Thawing Solution) and extra-long-term (Duragen) extenders from Days 0 to 2 of storage at 17 °C during an 8-month period. Pregnancy rates and litter size were evaluated from double inseminations within an interval of 12 hours (36 and 48 hours of refrigeration) of multiparous females using seminal doses diluted in each extender type.

Acrosin activity is a good predictor of boar sperm freezability.

The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze-thawing.

The improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability.

Reduced glutathione (GSH) improves boar sperm cryosurvival and fertilising ability when added to freezing extenders. Poor freezability ejaculates (PFE) are known to present lower resistance than good freezability ejaculates (GFE) to cryopreservation procedures. So far, no study has evaluated whether the ability of GSH to counteract the cryopreservation-induced injuries depends on ejaculate freezability (i.

Epididymal maturation and ejaculation are key events for further in vitro capacitation of boar spermatozoa.

Mammalian spermatozoa acquire functionality during epididymal maturation, and the ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to assess the effects of epididymal maturation, ejaculation and in vitro capacitation on sperm viability, acrosome integrity, mitochondrial activity, membrane fluidity, and calcium influx, both as indicators of capacitation status and sperm motility. Results indicated that boar spermatozoa acquired the ability to move in the epididymal corpus; however, their motility was not linear until the ejaculation.