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). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Physiological role of potassium channels in mammalian germ cell differentiation, maturation, and capacitation.

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 (Kligand ), inwardly rectifying channels (Kir ), and tandem pore domain channels (K2P ). MATERIAL-METHODS: The present review includes recent findings on the role of potassium channels in sperm physiology of mammals. RESULTS-DISCUSSION: While most studies conducted thus far have been focused on the physiological role of voltage- (Kv1, Kv3, and Kv7) and calcium-gated channels (SLO1 and SLO3) during sperm capacitation, especially in humans and rodents, little data about the types of potassium channels present in the plasma membrane of differentiating germ cells exist. In spite of this, recent evidence suggests that the content and regulation mechanisms of these channels vary throughout spermatogenesis. Potassium channels are also essential for the regulation of sperm cell volume during epididymal maturation and for preventing premature membrane hyperpolarization. It is important to highlight that the nature, biochemical properties, localization, and regulation mechanisms of potassium channels are species-specific. In effect, while SLO3 is the main potassium channel involved in the K+ current during sperm capacitation in rodents, different potassium channels are implicated in the K+ outflow and, thus, plasma membrane hyperpolarization during sperm capacitation in other mammalian species, such as humans and pigs. CONCLUSIONS: Potassium conductance is essential for male fertility, not only during sperm capacitation but throughout the spermiogenesis and epididymal maturation.

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 [...].

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 (pHi) 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+/HCO3- transporters and Cl-/HCO3- exchangers. Na+/HCO3- transporters are reported to be essential for the initial and fast entrance of HCO3- that triggers sperm capacitation, whereas Cl-/HCO3- exchangers are responsible for the sustained HCO3- entrance which orchestrates the sequence of changes associated with sperm capacitation. Proton efflux is required for the fast alkalinization of capacitated sperm cells and the activation of pH-dependent proteins; according to the species, this transport can be mediated by Na+/H+ exchangers (NHE) belonging to the SLC9 family and/or voltage-gated proton channels (HVCN1). Herein, we discuss the involvement of each of these channels in sperm capacitation and the acrosome reaction.

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. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

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, O2 consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3α) and protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capacitation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency.

Complete Chromatin Decondensation of Pig Sperm Is Required to Analyze Sperm DNA Breaks With the Comet Assay.

Sperm quality is usually evaluated prior to artificial insemination in farm animals. In addition to conventional semen analysis, other biomarkers, such as mitochondrial activity, integrity and lipid disorder of plasma membrane, generation of reactive oxygen species (ROS) and sperm DNA integrity, have been found to be related to fertility rates in different species. While mounting evidence indicates that the Comet assay is a sensitive method for the detection of DNA breaks, complete sperm chromatin decondensation is required in order to properly analyze the presence of single- and double-strand DNA breaks. In this sense, a previous study showed that longer lysis treatment with proteinase K is needed to achieve complete chromatin decondensation. The current work sought to determine which specific lysis treatment leads to complete chromatin decondensation in pig sperm, as this is needed for the measurement of DNA damage in this species. With this purpose, incubation with a lysis solution containing proteinase K for 0, 30, and 180 min was added to the conventional protocol. The impact of the DNA damage induced by hydrogen peroxide (H2O2; 0.01 and 0.1%) and DNAse I (1U and 4U) was also evaluated. Complete chromatin decondensation was only achieved when a long additional lysis treatment (180 min) was included. Furthermore, olive tail moment (OTM) and percentage of tail DNA (TD) indicated that a higher amount of DNA breaks was detected when hydrogen peroxide and DNAse I treatments were applied (P < 0.05). The comparison of treated and control sperm allowed defining the thresholds for OTM; these thresholds revealed that the percentage of sperm with fragmented DNA determined by the alkaline Comet does not depend on chromatin decondensation (P > 0.05). In conclusion, complete chromatin decondensation prior to alkaline and neutral Comet assays is needed to analyze DNA breaks in pig sperm.

HVCN1 but Not Potassium Channels Are Related to Mammalian Sperm Cryotolerance.

Little data exist about the physiological role of ion channels during the freeze-thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O2-⁻ and H2O2 levels. General blockade of K+ channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O2-⁻ and H2O2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.

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. Sperm cells were subsequently exposed to capacitating medium for 4 h, and then added with progesterone to induce acrosomal exocytosis. Sperm motility, acrosome integrity, membrane lipid disorder, intracellular Ca2+ levels, mitochondrial activity, and tyrosine phosphorylation levels of glycogen synthase kinase-3 (GSK3)α/ß were determined after 0, 2, and 4 h of incubation, and after 5, 30, and 60 min of progesterone addition. Results showed that storing sperm at 17 °C with 15% or 30% seminal plasma led to reduced percentages of viable spermatozoa exhibiting an exocytosed acrosome, mitochondrial membrane potential, intracellular Ca2+ levels stained by Fluo3, and tyrosine phosphorylation levels of GSK3α/ß after in vitro capacitation and progesterone-induced acrosomal exocytosis. Therefore, the direct contact between spermatozoa and seminal plasma during liquid storage at 17 °C modulated their ability to elicit in vitro capacitation and undergo acrosomal exocytosis, via signal transduction pathways involving Ca2+ and Tyr phosphorylation of GSK3α/ß. Further research is required to address whether such a modulating effect has any impact upon sperm fertilizing ability.

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 103, 105, 106, 107 and 108 CFU/mL. A sample in which there was no addition of P. vulgaris was the negative control. Sperm quality was determined by evaluating sperm motility and morphology using the computer-assisted sperm analysis (CASA) system, and plasma membrane and acrosome integrity using flow cytometry at 0, 1, 2, 4, 6, 8 and 10 days of liquid-storage at 17 °C. At the same time points, pH, sperm agglutination, sperm-bacteria interaction and bacterial growth were also assessed. There was impaired sperm motility when the infective doses of P. vulgaris were equal to or greater than 105 CFU/mL with the effect being dose-dependent (P < 0.05). When infective doses of P. vulgaris were of 106 to 108 CFU/mL there were impairments of plasma membrane and acrosome integrity by Day 1 of storage (P < 0.05), and alkalinisation of the storing medium by Day 4 (P < 0.05). Bacterial adhesion increased when infective dose of P. vulgaris was greater and as duration of storage increased. P. vulgaris had a high affinity for the mid- and principal pieces of sperm cells. It is concluded there were alterations in sperm motility in samples infected with P. vulgaris that were associated with bacterial adhesion and medium alkalinisation.

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. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

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. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.

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. perfringens, at infective titer from 101 to 107 CFU/mL. The sperm-bacteria interaction was evaluated by optical and scanning electron microscopy (SEM), through: a) determining the proportion of spermatozoa with either E. coli or C. perfringens adhered and the number and localization of bacteria adhered on the sperm surface; and b) analyzing the effects of bacterial contamination on sperm membrane integrity. While proportions of spermatozoa with bacteria adhered did not differ between E. coli and C. perfringens during the first 3 days of storage, proportions of spermatozoa with E. coli adhered were greater than those with C. perfringens on and subsequent to Day 4 of storage. Both bacterial types adhered non-specifically over the sperm surface; nevertheless, the proportion of spermatozoa with bacteria adhered to the mid-piece was greater with E. coli infection, and of spermatozoa with bacteria adhered to the principal piece greater with C. perfringens infections. Alterations in the sperm plasma membrane induced by C. perfringens affected the acrosomal end and appeared at Day 4 of storage, those induced by E. coli were apparent from Day 7 of storage and affected the acrosomal region and mid-piece. In conclusion, E. coli has a greater capacity to adhere to the sperm surface than C. perfringens during liquid storage at 17 °C, but damage on sperm membrane integrity induced by C. perfringens occurred in a shorter period of time.

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. In the first one, we evaluated the impact of adding three concentrations of each AMP on the bacterial growth and sperm quality of boar semen stored for 10 days. In the second experiment, the ability of these AMPs to control bacterial growth was determined over a 9-day period, following artificial inoculation with Escherichia coli at 107 and 108 CFU mL-1. In both experiments, sperm viability was assessed through flow cytometry, sperm motility was determined with Computer Assisted Sperm Analysis (CASA) and the inhibitory effect on microbial growth was evaluated by bacteria culture on Luria Bertani agar. PBD1 and PBD2 were found to significantly (P < 0.05) decrease sperm motility at 5 µM (% total motile spermatozoa at day 10, Control: 31.6% ±â€¯1.2% vs. PBD1: 6.5% ±â€¯0.3% and PBD2: 5.6% ±â€¯0.4%). Although the highest inhibitory effect on bacterial growth was observed at 3 µM (day 10, PBD1: 1.4 × 106 ±â€¯6.2 × 105 CFU mL-1 and PBD2: 9.1 × 105 ±â€¯2.4 × 105 CFU mL-1) and 5 µM (day 10, PBD1: 1.2 × 105 ±â€¯5.1 × 104 CFU mL-1; PBD2: 8.7 × 104 ±â€¯2.9 × 104 CFU mL-1), the control with antibiotic was found to be more effective (day 10, 8.3 × 103 ±â€¯1.6 × 103 CFU mL-1). In conclusion, PBD1 and PBD2 may be added to antibiotic-free extenders for boar semen at a concentration of 3 µM, but do not completely control all bacterial growth.

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. perfringens, with bacterial loads ranging from 101 to 107cfumL-1. During storage, the changes in sperm quality were determined by assessing pH, sperm viability, sperm motiliy, sperm morphology, sperm agglutination degree, and sperm-bacteria interaction. The infection of seminal doses led to an alkalinization of the medium, which was of higher extend in doses infected with C. perfringens. The effect of contamination on sperm viability and motility relied on bacterial type and load. Therefore, while E. coli was more harmful than C. perfringens in bacterial loads ranging from 101 to 106cfumL-1, the detrimental impact of C. perfringens was more apparent than that of E. coli at a bacterial load of 107cfumL-1. Despite sperm morphology not being affected by either bacterial type or load, sperm agglutination and sperm-bacteria interaction were characteristic of doses infected with E. coli, and increased concomintantly with bacterial load and along storage period. In conclusion, the effects of infection by E. coli on sperm quality were dependent of both bacterial load and storage period, whereas the effects of C. perfringens were mainly dependent on the bacterial load, with a threshold at 107cfumL-1 from which the sperm quality of seminal doses was greatly impaired.

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. Sperm quality was assessed from the analyses of sperm motility and kinetics, sperm viability, expressed as plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, and acrosin activity. Results indicated significant differences between the extenders in the sperm quality of seminal doses. Therefore, the seminal doses diluted in Duragen had higher percentages of progressive motile spermatozoa and membrane-intact spermatozoa than those diluted in Beltsville Thawing Solution throughout all the experimental months. Nevertheless, despite these differences in preserving the sperm quality, pregnancy rates (>90%) and litter sizes (>10 piglets born per litter) were similar between the extenders. Our results had great relevance from a practical point of view because they reported lack of an extender effect on the reproductive performance of seminal doses during short-tem storage.

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. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze-thawing procedures took place, i.e., in the refrigeration step at 15 °C.