J. D. A. Losano

Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm2 of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r2 = 0.91 and CASA: r2 = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Role of residual cytoplasm on oxidative status during sperm maturation in dogs.

During maturation sperm cells acquire their fertilizing ability; however, this event also produces reactive oxygen species and as such local antioxidant protection is required. Thirteen epididymis from dogs were used, and sperm samples were collected from different segments of the epididymis (i.e. caput, corpus and cauda). The samples were evaluated for motility and vigor, permeability of plasma membrane, presence of cytoplasmic droplet, acrosome integrity and the activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). Samples collected from the cauda showed higher motility, vigor and lower permeability of plasma and acrosomal membranes in relation to the corpus and caput, indicating different levels of maturity. The enzyme activity remained unchanged in the samples. A significant positive correlation was observed in the epididymal caput, between distal cytoplasmic droplet and SOD, and a negative correlation, in the epididymal cauda, between proximal cytoplasmic droplet and GPx, indicating the physiological need of the specific antioxidants in these segments.

The Stimulated Glycolytic Pathway Is Able to Maintain ATP Levels and Kinetic Patterns of Bovine Epididymal Sperm Subjected to Mitochondrial Uncoupling

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Fatty acid content in epididymal fluid and spermatozoa during sperm maturation in dogs

BackgroundDuring sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis (lipids were extracted, transmethylated and analyzed by chromatography).ResultsCaput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.ConclusionsThese findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.

Lipid composition of the canine sperm plasma membrane as markers of sperm motility

The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll® , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.

Susceptibility of Stallion Spermatozoa to Different Oxidative Challenges: Role of Seminal Plasma

Abstract Seminal cryopreservation provides several advantages in horse breeding. However, improvement on post‐thaw sperm survival is still necessary. One of the main factors known to impair post‐thaw quality of stallion’s sperm is the oxidative damage caused by reactive oxygen species (ROS). In this context, the aim of this study was evaluated the effects of ROS on stallion sperm and the possible influence of seminal plasma (SP). Toward this aim, 13 ejaculates from adult stallions (n = 13) were divided into two aliquots which were centrifuged (600g/10 minutes), and the SP was removed and reserved. Pellets were suspended in physiological saline solution (A) or SP (B). Each of these solutions was divided into four aliquots and subjected to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical [OH−]) and the toxic by‐product of lipid peroxidation (malondialdehyde [MDA]). Samples were then evaluated for the susceptibility to lipid peroxidation (thiobarbituric acid reactive substances) plasma membrane integrity (eosin–nigrosin), acrosome integrity (Fast Green/rose bengal), mitochondrial activity (3′3 diaminobenzidine), and DNA integrity. Samples incubated in the presence of SP were highly impaired by OH− with regard to motility, plasma membrane integrity, mitochondrial activity, and DNA integrity. On the other hand, in the absence of SP, MDA was highly deleterious, especially with regard to motility, plasma membrane integrity, and mitochondrial activity. Thus, these results indicate that SP may have an important role on the protection of stallion sperm against the damages caused by MDA, an important product of lipid peroxidation. HighlightsSeminal plasma (SP) has a protective effect against oxidative damages.Hydroxyl radical in the presence of SP is the most deleterious reactive oxygen species (ROS) to equine spermatozoa.Malondialdehyde is more deleterious than ROS to equine spermatozoa in the absence of SP.Hydroxyl radical is extremely deleterious to equine sperm DNA.Malondialdehyde impaired membrane integrity and mitochondrial activity.

Comparison of Cryopreservation Protocols (Single and Two-steps) and Thawing (Fast and Slow) for Canine Sperm.

ABSTRACT In addition to the existence of several cryopreservation protocols, no systematic research has been carried out in order to confirm the suitable protocol for canine sperm. This study aims to assess the effect of adding 5% glycerol during cryopreservation at 37°C (one-step) and 5°C (two-steps), in addition of testing two thawing protocols (37°C for 30 seconds, and 70°C for 8 seconds). We used 12 sperm samples divided into four experimental groups: Single-Step - Slow Thawing Group; Two-Step - Slow Thawing Group; Single-Step - Fast Thawing Group; and Two-Step - Fast Thawing Group. Frozen-thawed samples were submitted to automated analysis of sperm motility, evaluation of plasmatic membrane integrity, acrosomal integrity, mitochondrial activity, sperm morphology, sperm susceptibility to oxidative stress, and sperm binding assay to perivitellinic membrane of chicken egg yolk. Considering the comparison between freezing protocols, no statistical differences were verified for any of the response variables. When comparison between thawing protocols was performed, slow thawing protocol presented higher sperm count bound to perivitelline membrane of chicken egg yolk, compared to fast thawing protocol. Regardless of the freezing process, the slow thawing protocol can be recommended for the large scale cryopreservation of canine semen, since it shows a consistent better functional result.

Carnosine as malondialdehyde scavenger in stallion seminal plasma and its role in sperm function and oxidative status

Semen biotechniques may impair sperm quality due to excessive production of reactive oxygen species (ROS). Additionally, products of the oxidative reaction, especially involving lipids (e.g., malondialdehyde - MDA), may be even more harmful to sperm. Carnosine, previously reported to be present in seminal plasma of several species, may be a key factor on sperm tolerance to biotechniques by counterattacking the deleterious influence of MDA. Therefore, the aim of this study was to measure the levels of carnosine present in equine seminal plasma and relate these findings with sperm function and oxidative status during cooling and cryopreservation. Thus, semen samples were collected from 40 stallions in duplicate (N = 80) and then submitted to cooling and cryopreservation. Samples were then allocated into groups of high and low tolerance to refrigeration and cryopreservation (bad cooler and good cooler/bad freezer and good freezer, respectively), and in groups of different concentrations of carnosine (High, Medium-high, Medium-low and Low carnosine). Samples were evaluated for sperm kinetics patterns, function of sperm structures and oxidative status. In good cooler samples, it was observed higher concentrations of carnosine (Good cooler: 224.98 ± 19.16 ng/mL; Bad cooler: 159.72 ± 15.99 ng/mL; p = 0.0056), ROS production (Good cooler: 26.40 ± 18.33%; Bad cooler: 18.33 ± 1.84%; p = 0.001) and lipid peroxidation rates (Good cooler: 193.23 ± 18.22 ng/mL; Bad cooler: 131.92 ± 12.25; p = 0.0064). Groups of samples with higher carnosine concentrations had lower levels of malondialdehyde (High: 79.33 ± 6.72 ng/mL; Medium-high: 140.45 ± 11.70 ng/mL; Medium-low: 202.57 ± 16.30 ng/mL and Low: 231.02 ± 32.35 ng/mL; p < 0.05), demonstrating that carnosine was effective in removing lipid peroxidation products. Due to the removal of seminal plasma during the cryopreservation process, no differences occurred in carnosine levels between bad and good freezer groups. In this context, this study provides relevant data for future therapies using carnosine during cryopreservation, aiming to replace the levels lost due to the necessary removal of seminal plasma.

Induced sperm oxidative stress in dogs: Susceptibility against different reactive oxygen species and protective role of seminal plasma

Oxidative stress (OS) is characterized by an unbalance between increased levels of reactive oxygen species (ROS) and/or impaired antioxidant protection. In this context, the composition of seminal plasma (SP) plays a key role in protecting sperm against OS. However, reproductive biotechnologies applied to dogs recommend the removal of SP. Thus, antioxidant therapy may be an important alternative when applying biotechniques such as semen cryopreservation in this specie. However, in order to be efficient, the choice of the ideal antioxidant in each condition is essential since each ROS is preferably neutralized by different antioxidant systems. Therefore, this study aims to evaluate the susceptibility of canine spermatozoa to different oxidative challenges (superoxide anion [O2-], hydrogen peroxide [H2O2], hydroxyl radical [OH-] and malondialdehyde [MDA]) in the present or absence of SP. We used ejaculates of eight dogs and submitted to induce oxidative challenges (with or without SP). After incubations, samples were evaluated for the susceptibility to lipid peroxidation, motility, mitochondrial activity and function, DNA integrity, plasma membrane and acrosome integrity. Sperm with SP had mitochondrial function preserved against ROS. However, in the absence of SP, H2O2 reduced mitochondrial membrane potential. In addition, regardless on SP, H2O2 was deleterious to sperm kinetics and plasma/acrosomal membranes. Incubation with OH- reduced mitochondrial activity and increased DNA fragmentation also independent on the absence of presence of SP. Furthermore, samples with SP were more resistant to lipid peroxidation (i.e., decreased concentration of TBARS). In conclusion, H2O2 and OH- appears to be the most deleterious ROS to dog sperm and SP protects the spermatozoa against mitochondrial injuries and lipid peroxidation.

Effect of senescence on morphological, functional and oxidative features of fresh and cryopreserved canine sperm

Abstract The present research aimed to compare the hormonal profile, sperm quality and freezability of young and senile dogs. Dogs were assigned into Young Group (n = 11) and Senile Group (n = 11), additionally divided into Fresh Semen Group and Cryopreserved Semen Group. Males were evaluated for libido score and blood estrogen and testosterone assay. Sperm morphofunctional evaluations were performed based on Computer Assisted Sperm Analysis, morphology, mitochondrial activity, mitochondrial membrane potential, plasma and acrosomal membrane integrity, and DNA fragmentation. Sperm oxidative features were: protein oxidation, lipid peroxidation and production of advanced glycation end-products. Young dogs had higher libido score, sperm velocity average pathway, linearity of motility and mitochondrial activity index and lower percentage of major defects, total defects and proximal cytoplasmic droplet, despite the lack of difference between hormone profile of aged dogs. Fresh semen of senile dogs had increased percentage of spermatozoa with high mitochondrial membrane potential compared to young dogs and to cryopreserved sperm. Cryopreserved semen of young dogs had higher acrosomal membrane integrity compared to the Senile Group. In conclusion, sperm of aged dogs have reduced quality, signaled by higher morphological defects, ultimately altering sperm mitochondrial function and sperm kinetics. Furthermore, spermatozoa from senile dogs are more sensible to cryoinjury.