Showing papers on "Sperm plasma membrane published in 2019"

Update on mammalian sperm capacitation: how much does the horse differ from other species?

Abstract: In contrast to various other mammalian species, conventional in vitro fertilization (IVF) with horse gametes is not reliably successful. In particular, stallion spermatozoa fails to penetrate the zona pellucida, most likely due to incomplete activation of stallion spermatozoa (capacitation) under in vitro conditions. In other mammalian species, specific capacitation triggers have been described; unfortunately, none of these is able to induce full capacitation in stallion spermatozoa. Nevertheless, knowledge of capacitation pathways and their molecular triggers might improve our understanding of capacitation-related events observed in stallion sperm. When sperm cells are exposed to appropriate capacitation triggers, several molecular and biochemical changes should be induced in the sperm plasma membrane and cytoplasm. At the level of the sperm plasma membrane, (1) an increase in membrane fluidity, (2) cholesterol depletion and (3) lipid raft aggregation should occur consecutively; the cytoplasmic changes consist of protein tyrosine phosphorylation and elevated pH, cAMP and Ca2+ concentrations. These capacitation-related events enable the switch from progressive to hyperactivated motility of the sperm cells, and the induction of the acrosome reaction. These final capacitation triggers are indispensable for sperm cells to migrate through the viscous oviductal environment, penetrate the cumulus cells and zona pellucida and, finally, fuse with the oolemma. This review will focus on molecular aspects of sperm capacitation and known triggers in various mammalian species. Similarities and differences with the horse will be highlighted to improve our understanding of equine sperm capacitation/fertilizing events.

Egg yolk-free cryopreservation of bull semen.

Abstract: Egg yolk is a common ingredient of mammalian semen extender to protect sperm against initial cold shock. However, egg yolk has biosecurity risks. Our main objectives were to cryopreserve bull semen without egg yolk using exogenous cholesterol and to study the protective role of glycerol in egg yolk-free semen extender. Other objectives were to compare protein profiles and in vitro fertilization potential of bull sperm frozen with and without egg yolk. In first experiment, semen was either diluted in conventional tris-egg yolk glycerol (TEYG control) extender or first treated with cholesterol-cyclodextrin complex (CC, 2 mg/ml semen) followed by dilution in egg yolk-free tris-glycerol (TG) extender (collectively called as “CC+TG”) at 22°C or 4°C, and frozen. Post-thaw sperm motion characteristics were similar between CC+TG and TEYG control extenders, and temperature of glycerol addition. In second experiment, semen was frozen in CC+TG extender varying in glycerol concentration (7 to 0%; v/v). Post-thaw sperm quality decreased with the decline in glycerol concentration in TG extender, even higher concentration of CC complex (3 or 4 mg/ml semen) could not protect sperm in the absence of glycerol in TG extender. In third experiment, SDS electrophoresis of proteins from fresh sperm and sperm frozen in CC+TG, and TEYG control extenders was conducted. Protein profiles in fresh sperm and CC+TG frozen sperm were almost similar. Egg yolk proteins bound tightly with sperm plasma membrane. In fourth experiment, in vitro fertilization potentials of sperm frozen in TEYG control and CC+TG extenders were tested. Cleavage and blastocyst rates of semen frozen in CC+TG and TEYG control extenders were similar. In conclusion, cholesterol-cyclodextrin replaced egg yolk from the semen extender; glycerol remained essential for egg yolk-free sperm cryopreservation; and CC+TG extender did not modify sperm plasma membrane CC+TG whereas egg yolk extender changed the plasma membrane composition of bull sperm.

Compartmentalization of the proteasome-interacting proteins during sperm capacitation

Abstract: Ubiquitination is a stable, reversible posttranslational modification of target proteins by covalent ligation of the small chaperone protein ubiquitin. Most commonly ubiquitination targets proteins for degradation/recycling by the 26S proteasome in a well-characterized enzymatic cascade. Studies using human and non-human mammalian spermatozoa revealed the role of the ubiquitin-proteasome system (UPS) in the regulation of fertilization, including sperm-zona pellucida (ZP) interactions as well as the early events of sperm capacitation, the remodeling of the sperm plasma membrane and acrosome, and for the acquisition of sperm fertilizing ability. The present study investigated the activity of UPS during in vitro capacitation of fresh boar spermatozoa in relation to changes in sperm proteome. Parallel and sequential treatments of ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions were used to isolate putative sperm proteasome-associated sperm proteins in a compartment-specific manner. A differential proteomic approach employing 1D PAGE revealed differences in accumulated proteins at the molecular weights of 60, 58, 49, and 35 kDa, and MS analysis revealed the accumulation of proteins previously reported as proteasome co-purifying proteins, as well as some novel proteins. Among others, P47/lactadherin, ACRBP, ADAM5, and SPINK2 (alias SAAI) were processed by the proteasome in a capacitation dependent manner. Furthermore, the capacitation-induced reorganization of the outer acrosomal membrane was slowed down in the presence of proteasomal inhibitors. These novel results support the proposed role of UPS in sperm capacitation and open several new lines of inquiry into sperm capacitation mechanism.

Diabetes and Sperm DNA Damage: Efficacy of Antioxidants

Abstract: Diabetes mellitus (DM) represents one of the major threats to human health all over the world, affecting almost every system of the body. Its prevalence is escalating globally and is associated with reproductive impairment in both males and females. Reports on male infertility due to DM is lacking since most affected individuals are unaware of their infertility condition due to the late onset of DM. Glucose metabolism is an imperative occasion in spermatogenesis, causing adverse effects on male fertility, principally on sperm DNA quality, motility, and ingredients of seminal plasma. DM is coupled with an increased oxidative stress (OS), causing sperm nuclear and mitochondrial DNA damage. Reactive oxygen species (ROS) hassles the fluidity of sperm plasma membrane, decreases sperm motility and ability to fuse with oocyte as well as altering the sperm DNA integrity. Lamentably, spermatozoa cannot repair the damage initiated by excessive ROS as they do not have the cytoplasmic enzymes required to achieve the repair. Diabetes may impact the epigenetic change during spermatogenesis which may be inherited through male gamete to more than one generation increasing the risk of diabetes in offspring. Administration of antioxidants in male infertility has started to pull in significant intrigue. Many studies have shown that antioxidants amazingly reduce the oxidative stress markers and boost the antioxidant enzymes. Currently treatment strategies are aimed at lowering ROS levels to maintain normal cell function. This review highlights the potential impact of DM on sperm DNA integrity, epigenetic dysregulation and efficacy of antioxidant therapy.

The role of galectin-3 in spermatozoa-zona pellucida binding and its association with fertilization in vitro.

Abstract: Human spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.

Extenders modify the seminal plasma ability to minimize freeze-thaw damage on ram sperm.

Abstract: Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze-thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk-based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen-thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin-based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze-thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non-capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.

Sperm characterization of the Amazonian freshwater cururu stingray Potamotrygon wallacei (Potamotryogonidae): basic knowledge for reproduction and conservation plans.

Abstract: This study aimed to describe the morphology and sperm quality of free-living adult males of cururu stingray Potamotrygon wallacei, endemic from the Rio Negro basin, Brazilian Amazon. The sperm was collected in loco from the seminal vesicle region and fixed in buffered saline formaldehyde solution for further evaluation of morphometry, sperm plasma membrane integrity and sperm concentration. The spermatozoa presented a total length of 138.25 ± 1.82 μm with a helical shape and a long head. A high percentage of cells with intact membrane (98 ± 2%) and normal spermatozoa (92 ± 1%) were observed. The cell concentration was 0.34 ± 0.05 × 1010 spermatozoa/ml of semen. These observations are unprecedented for potamotrygonid species and will serve as a basis for future management and conservation strategies.

Determination of a Robust Assay for Human Sperm Membrane Potential Analysis.

Abstract: Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.

Antioxidants, Dietary Fatty Acids, and Sperm: A Virtual Reality Applied Game for Scientific Dissemination

Abstract: Fatty acid (FA) profile appears to be critical to infertility, and the effects of dietary FAs on sperm FA content are a current focus of studies in the field of nutrition and reproduction. Starting from a validated "OXISTRESS" model in which modification of FA content results to influence reactive oxygen species, antioxidants, isoprostanes, cytokines, sperm kinetic, and acrosome reaction, we developed a virtual reality game where the player, in order to improve the health of some virtual spermatozoa, is called to take dietary choices and then discover their consequences on the main biological aspects. In the LabVR of the University of Siena, a team of VR environment designer and developer used Unity development engine to make the experience run on Oculus Quest and a wireless 6DOF (six degrees of freedom of movement in 3D space) VR Headset. In the game, the player is immersed in the epididymis and observes closer how dietary n-3 may change the sperm plasma membrane and consequently modify sperm traits. A simulation game in the virtual reality may represent a tool to give greater visibility to scientific data in the relevance of appropriate dietary habits in the human health.

Low-mobility sperm phenotype in the domestic turkey: Impact on sperm morphometry and early embryonic death

Abstract: The sperm mobility assay measures the ability of sperm to swim through a dense layer of Accudenz® , and the sperm mobility phenotype has been shown to predict fertility and other sperm performance traits in roosters and turkeys. In this study, we examined turkey sperm morphometry and rates of early embryonic death associated with high- and low-mobility semen. We also assessed whether the hypo-osmotic stress test, which evaluates the structural integrity of the sperm plasma membrane, may be used as a faster and simpler assay for sperm mobility and viability. We confirmed previous work that found that high-mobility sperm are faster and swim more linearly than low-mobility sperm, and that mobility traits were repeatable within males. In contrast to previous studies, we did not find higher rates of fertility, but low-mobility sperm was associated with higher rates of early embryonic death, though this trend was not significant. High-mobility sperm had longer sperm heads, explained by longer nuclei, despite shorter acrosomes. Although these sperm were faster, midpiece length and flagellum length did not differ between high- and low-mobility sperm. Finally, mobility was not found to be associated with sperm performance in the hypo-osmotic stress test.

Cryodamage of plasma membrane and acrosome region in chicken sperm.

Abstract: Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen-thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil+ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non-permeating cryoprotectants: trehalose (KEG-TRE) or glycine (KEG-GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo-PRO-1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG-GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome-sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo-PRO-1) was noted in the KEG-GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.

EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalus) BULL SEMEN

Abstract: This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (≥ 3 mass motion, > 70% progressive motility and viability, 1 x 10 9 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F 2I± (PGF 2I± ) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher ( p < 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable ( p ≥ 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant ( p < 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen. Key words : buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

Abstract: Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4oC and 16oC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4oC and 16oC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Action of cholesterol-loaded cyclodextrin on viability of ram semen.

Abstract: Background : Studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. The mechanism of action of CLC is not well understood, however, it seems to involve sperm protection during the freezing and thawing process. Studies show that its use enhancing increased osmotic tolerance and reduced premature sperm capacitation reaction. In this sense, studies report that cyclodextrins have the property of carrying cholesterol to the membrane, but in some cases can also remove this cholesterol from the plasma membrane. Improvements were reported in the sperm parameters of buffaloes, bulls, stallions and sheep. Ram naturally present less lipids in their membrane, on average 27%, while bulls have 31%, rabbits 62%, and humans 50%. The aimed of the present study was to evaluate the use of cholesterol-loaded cyclodextrin (CLC), a commercial diluent, in the kinetics and viability of frozen and thawed ram spermatozoa. Materials, Methods & Results : Five ejaculates, from five rams of Dorper breed were collected and divided into three groups: control, 1 mg CLC and 2 mg CLC. Semen was diluted in different concentrations of CLC (0, 1, and 2 mg/120×10 6 spermatozoa), and incubated at room temperature (21°C) for 10 min. Samples were conditioned in 0.5 mL straws and incubated at 5°C for 4 h, exposed to LN 2 vapor for 10 min and storing a cryogenic container. The parameters as spermatic kinetics, plasma membrane, acrosomal membrane (MPAI, %), and intracellular levels of superoxide anion ( O 2 - ) were evaluated. Sperm progressive motility (PM), rapid spermatozoa percentage (RAP), linearity (LIN, %), average path velocity (VAP, μm/s) and MPAI (%) were more satisfactory with the use of 1 mg compared to 2 mg ( P < 0.05). In addition, 1 mg CLC showed decreased levels of superoxide anion formation (O 2- ), a free radical detrimental to spermatozoa ( P < 0.05). The use of 2 mg of CLC reduce VAP ( P < 0.05) and did not have any beneficial effect on the evaluated parameters. Discussion : Authors did not observe improvement in the parameters of progressive motility when using 1 mg of CLC in goat semen and 2 mg in bull semen with the slow freezing protocol. This differs from our work, as we found that 1 mg of CLC improved the PM parameters, but not at the concentration of 2 mg CLC. Additionally, authors verified that cyclodextrin at 3 mg concentration was effective in protecting the sperm against the deleterious effects of H 2 O 2 . They obtained superior plasma membrane motility, viability, and integrity of the CLC-treated samples compared to the control group. The superoxide anion (O 2 - ) is a free radical formed from molecular oxygen by the addition of an electron. It is generated spontaneously, mainly in the membrane of the mitochondria, by the respiratory chain and by flavoenzimes, lipoxygenases, and cicloxygenases. In our study, we found a difference between the study group with 1 mg CLC and the control group. Thus, we suggest that CLC may have a beneficial effect in stabilizing the sperm plasma membrane. Use of CLC at a concentration of 1 mg was found to be effective for the improvement of parameters of sperm progressive motility, rapid sperm percentage, and plasma and acrosomal membrane integrity. In addition, the study group with 1 mg of CLC showed decreased levels of superoxide anion formation, a free radical detrimental to spermatozoa.

Effects of Low Seminal Plasma Antioxidant Potential on Semen Quality and Male Fertility

Abstract: It is very important to identify the factors which affect normal sperm functions. New Bio-chemical parameters in seminal plasma were analyzed to determine the biochemical factors that affect normal sperm function. The research groups consists of infertile groups Asthenozoospermia (n=31) and Normozoospermia (n=27) with a healthy men as a control (n=24). The patients have been selected and examined according to the World Health Organization (2010). In addition the seminal fluid analysis the biochemical parameters were analyzed in the seminal plasma of each sample including; Total antioxidant capacity, Catalase, Glutathione Reductase , glutathione and Malondialdehyde.Results showed a decrease in seminal plasma total antioxidant capacity of Astheno-zoospermia and Normozoospermia , and a significance decrease in catalase level for both infertile groups, Malondialdehyde level significantly increased in both infertile groups and Glutathione level significantly decreased in Asthenozoospermia group as compared to control group. Antioxidants have an important role in sperm protection via disturbing the balance in ROS production which destroys sperm plasma membrane causing loss sperm activity and it's ability to fertilize the egg.

The effects of IAM38 blocking or CD4 blocking on the binding of exogenous DNA in rabbit sperm

Abstract: The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.

Changes in the phospholipid asymmetry of buffalo sperm plasma membrane in relation to cryopreservation technology.

Abstract: Received: Revised: Accepted: Published online: July 28, 2018 November 06, 2018 November 09, 2018 January 28, 2019 This study evaluates the integrity of the plasma membrane (PM), and the mitochondrial transmembrane potential and their relationship with the motility and kinematic parameters of buffalo spermatozoa frozen in pellets and straws. Ejaculates (n=20) from eight Bulgarian Murrah buffaloes were frozen in pellets (Nagaze-Niva; medium GH22L) and in straws (Cassou; medium Triladyl) at final sperm concentration 80×10/ml. The comparative CASA analysis of the motility and kinematic parameters of spermatozoa showed significant differences in the rapid motility and in the VCL values in favor of pellets vs straws (P<0.05). The molecular changes in PM phospholipid asymmetry (assessed by double staining Annexin V-Cy3 Apoptosis Detection Kit) were higher after freezing – thawing, when compared to the fresh semen (P<0.05). No significant differences were observed in the number of live and functional spermatozoa with intact PM (6CF+/Ann V Cy3-) and the number of apoptotic spermatozoa (6CF+/Ann V Cy3+) between the two freezing technologies. The percentage of spermatozoa with well preserved and functioning mitochondria visualized by Rhodamine123 (Rh123) in the fresh semen was higher, when compared to spermatozoa after cryopreservation (P<0.05). An increase in the percentage of cells with nonfunctional mitochondria (Rh123or Rh123+) was observed after cryopreservation using both freezing technologies. In conclusion, the freezing of buffalo sperm in pellets, using GH22L protective medium, preserves high sperm motility, kinematics, PM integrity, and mitochondrial transmembrane potential, which is a guarantee of good fertilization capacity of the spermatozoa. ©2018 PVJ. All rights reserved