Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

Slow Freezing of Preserved Boar Sperm: Comparison of Conventional and Automated Techniques on Post-Thaw Functional Quality by a New Combination of Sperm Function Tests.

The slow freezing of boar sperm is the only way to preserve genetic material for extended periods; this can be achieved with exposure to liquid nitrogen vapors (conventional) or by using automated freezing equipment. The aim was to compare the effect of both techniques on post-thaw functionality. Boar sperm devoid of seminal plasma and resuspended in lactose-egg yolk-glycerol medium were cryopreserved. Conventional: straws were exposed to LN2 vapors; automated: using a drop curve of -39.82 °C·min-1 for 113 s from -5 to -80 °C during the critical period; and subsequent immersion in NL2. Cell viability, cholesterol flow, mitochondrial membrane potential (MMP), lipid peroxidation, peroxynitrite, superoxide anion levels, phosphatidylserine translocation, and caspase activation were evaluated by flow cytometry. In addition, total motility (TM) and progressive motility (PM) were determined by the SCA system immediately (T0), 60 (T60), and 120 min (T120) post-thawing. Automated freezing significantly reduces cholesterol flow and free radical and lipid peroxidation levels, making it possible to preserve motility for 120 min of incubation. At the same time, viability, acrosome integrity, MMP, and caspase activation did not differ from the conventional technique. In conclusion, controlling the temperature drop curve using automated freezing equipment reduces oxidative/nitrosative stress, preserving membrane fluidity and sperm motility.

Mammals' sperm microbiome: current knowledge, challenges, and perspectives on metagenomics of seminal samples.

Bacterial growth is highly detrimental to sperm quality and functionality. However, during the last few years, using sequencing techniques with a metagenomic approach, it has been possible to deepen the study of bacteria-sperm relationships and describe non-culturable species and synergistic and antagonistic relationships between the different species in mammalian animals. We compile the recent metagenomics studies performed on mammalian semen samples and provide updated evidence to understand the importance of the microbial communities in the results of sperm quality and sperm functionality of males, looking for future perspectives on how these technologies can collaborate in the development of andrological knowledge.

Adverse Effects of Single Neutrophil Extracellular Trap-Derived Components on Bovine Sperm Function.

Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 µg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 µg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. I: Protective effect of melatonin and butylhydroxytoluene on sperm function.

The addition of antioxidants to the cryopreservation medium has been shown to exert a positive effect on the quality of frozen-thawed sperm in different species. The objective of the present study was to evaluate the effects of supplementing the freezing medium with butylhydroxytoluene (BHT) and melatonin (MEL) in frozen-thawed pig spermatozoa. With this purpose, six ejaculates coming from six separate boars were cryopreserved in traditional freezing medium (i.e. lactose/egg-yolk/glycerol; Control) supplemented with 1.0 mM BHT (BHT-1), 2.0 mM BHT (BHT-2), 0.01 µM MEL (MEL-1) and 1.0 µM MEL (MEL-2). We evaluated sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidation of thiol groups, and levels of total reactive oxygen species (ROS), peroxynitrite and superoxide anion (·O2-). We also analysed total (TM) and progressive sperm motilities (PM), and kinetic parameters at post-thaw (T0, T30 and T60). The BHT-2 and MEL-2 groups presented higher viability and acrosome integrity, and lower levels of peroxynitrite, ·O2- and lipid peroxidation than the control (P < 0.05), whereas MEL-2 diminished the levels of total ROS (P < 0.05). TM and PM were not affected by the treatment, while, LIN and STR shows differences between experimental groups. In conclusion, the addition of BHT and MEL to cryopreservation medium diminishes oxidative and nitrosative stress markers, which has repercussions for the integrity of plasma and acrosomal membranes of frozen-thawed spermatozoa.

Antioxidants and their effect on the oxidative/nitrosative stress of frozen-thawed boar sperm.

In swine, the use of frozen-thawed boar sperm for artificial insemination remains a suboptimal reproductive technology. Among the negative effects of cryopreservation on sperm cells, it is worth highlighting that cryopreservation causes irreversible alterations in motility and components of the sperm membrane as a result of dramatic changes in temperature (cooling/freezing curve) and osmolality. In addition, freeze-thawing may induce oxidative stress and increase the generation of reactive oxygen species (ROS) and nitrogen reactive species (RNS). While boar sperm cryopreservation has been reported to increase lipid peroxidation and the intracellular levels of hydrogen peroxide, less research on its impact on RNS has been conducted. Furthermore, previous studies have investigated the effects of supplementing cryopreservation media with antioxidants to counteract the deleterious effects of ROS and RNS. Antioxidants of synthetic origin or natural extracts have been used, with some showing noticeable and positive effects on functional sperm parameters both in vitro and in vivo. The aim of this review is to provide an update on the effect of different molecules with antioxidant capacity on the function of cryopreserved boar sperm.

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. II: Effect of the addition of saccharides to freezing medium on sperm function.

Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O2●-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O2●- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.

Multiparameter Flow Cytometry Assay for Analysis of Nitrosative Stress Status in Human Spermatozoa.

Male infertility is an increasing health problem, and oxidative/nitrosative stress plays an important role in the etiology of this condition. Nitrosative stress due to excessive levels of reactive nitrogen species (RNS) is associated with impaired male fertility. Flow cytometry may be a useful tool for semen evaluation, but the availability of multiparameter assays for analysis of sperm quality is limited. The present study standardized a multiparameter flow cytometry analysis for nitrosative stress status in human spermatozoa in a single assay. A suitable multicolor fluorochrome panel was designed and consisted of fluorescein-boronate to detect peroxynitrite, a highly RNS, propidium iodide to analyze viability, tetramethylrhodamine methyl ester perchlorate to detect mitochondrial membrane potential (MMP) and monobromobimane to analyze thiol oxidation. Proper positive and negative controls for each fluorochrome were used to establish the technique, and sperm cells of different qualities and spermatozoa subjected to cryopreservation were analyzed. The results showed that the controls clearly discriminated between the high and low fluorescence intensities for each fluorochrome. The analysis of sperm cells of different quality demonstrated that the assay properly detected differences in all parameters analyzed according to sperm quality. The results may be reported as the mean fluorescence intensity of each fluorochrome and the percentage of cells exhibiting different characteristics. In conclusion, a protocol was standardized to analyze nitrosative stress status, including peroxynitrite production, viability, MMP, and thiol oxidation, in a single analysis using flow cytometry. This protocol may be applied to research approaches and clinical andrology to improve the evaluation of sperm quality and provide a promising tool to increase the use of clinical flow cytometry. © 2020 International Society for Advancement of Cytometry.

Rest days and storage of boar semen at 17°C: Effect on motility and sperm concentration.

Boar fertility is an important factor in farm production; it is therefore of interest to determine factors which reduce the fertilising capacity of semen samples stored at 17°C for use in intrauterine insemination. This work evaluated the effect of the number of rest days between each mounting of the boar, and the number of days that the semen was stored at 17°C, on sperm motility and semen concentration. We also analysed whether the boar's age influenced the sperm concentration. The results showed that only the total motility diminished as the storage time at 17°C increased (p < .05). A low negative correlation was observed between the variables' rest days and total and progressive motility. The sperm concentration presented no relation with rest days or the boar's age. The boars' rest days had no effect on motility and sperm concentration in the males studied, allowing them to be used with the frequencies described with no effect on these parameters.

LED-based red light photostimulation improves short-term response of cooled boar semen exposed to thermal stress at 37°C.

Pre-treatment of boar semen with a red light photostimulation procedure increases its "in vivo" fertilising ability. However, "in vitro" conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short-term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620-630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca2+ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca2+ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short-term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.

Preservation of boar semen: An update.

In the pork industry, artificial insemination and the storage of boar semen in liquid at 17°C are routinely applied to optimize the ejaculate and bring about rapid genetic changes that are reflected in the animal protein. Although the results are satisfactory, they are below what occurs with natural mating. It is currently possible to preserve boar semen with storage at 17°C and slow freezing, since to date there is only one study on vitrification, with negative results applicable only in the case of implementing an intracytoplasmic sperm injection. In both methods and due to the sensitivity of boar sperm to osmotic and temperature changes, there is a loss in the quality of the initial sample; however, slow freezing in boar semen has greater deleterious effects on the sample that are reflected in the pregnancy rates and number of live births. Therefore, only 1% of all inseminations are done with frozen semen. The aim of this review is to provide advances and results of studies conducted on the preservation of boar semen, delving more deeply into the critical points that each of the preservation techniques presents, including bacterial contamination, extender components, temperature, ice nucleation, use of additives in extenders and the main deleterious effects on sperm quality.

Criopreservación de espermatozoides de canino con extracto de hojas de arándano (Vaccinium corymbosum L. ) / Cryopreservation of canine spermatozoa with blueberry leaf extract (Vaccinium corymbosum L. )

La criopreservación espermática induce daño por estrés oxidativo en las células, lo que conlleva a un deterioro de la calidad del semen descongelado. Los espermatozoides pueden ser protegidos de este daño, por la adición de antioxidantes al medio de congelación. El objetivo de este estudio fue determinar el efecto de la adición de extracto de hojas de arándano (EHA) al medio de congelación, sobre la calidad de espermatozoides de canino criopreservados. Espermatozoides desprovistos del plasma seminal fueron congelados con diferentes concentraciones de EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) adicionadas al medio de congelación. Post descongelación se evalúo la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR-14/PI) e integridad de la membrana acrosomal (FITC-PNA/PI) por citometría de flujo. La motilidad progresiva fue similar al control con las concentraciones de EHA1y EHA4 (P >0,05), mientras que con las concentraciones de EHA2 y EHA6 se observó una disminución significativa de este parámetro comparado con el control (P <0,01 y P <0,001 respectivamente). La adición de EHA1, EHA2 y EHA4 al medio de congelación no presentó diferencias significativas respecto al control sobre la viabilidad e integridad de la membrana plasmática (P >0,05); por el contrario, con la adición de EHA6 se observaron valores significativamente menores (P <0,001). Los valores de integridad de la membrana acrosomal, con las diferentes concentraciones de EHA no presentaron diferencias significativas respecto al control. En conclusión, los resultados obtenidos en este estudio revelaron que las concentraciones de EHA utilizadas no fueron eficaces en mejorar la calidad del semen canino descongelado.

During cryopreservation, oxidative stress damage leads to a deterioration of the quality of thawed semen, which could be reduced by the addition of antioxidants to freezing extender. This study was designed to determine the effect of the addition of blueberry leaf extract (EHA) to freezing extender, on quality of cryopreserved canine sperms. Sperm devoid from seminal plasma were frozen with different concentrations of EHA (0 %, control; 1 %, EHA1; 2 %, EHA2; 4 %, EHA4 y 6 %, EHA6) added to freezing extender. Post-thawing progressive motility was evaluated; the viability and plasma membrane integrity (SYBR-14/PI) and acrosomal membrane integrity (FITC-PNA/PI) were assessed by flow cytometry. Progressive motility was similar to the control with concentrations of EHA1 and EHA4 (P >0.05); at concentrations of EHA2 and EHA6 a significant decrease of this parameter compared to control (P <0.01and P <0.001, respectively) was observed. The addition of EHA1, EHA2 and EHA4 to the freezing extender showed no significant differences with respect to the control on viability and plasma membrane integrity (P >0.05); however with the addition of EHA6 values significantly lower (P <0.001) were exhibited. The concentrations of EHA used showed no significant differences with respect to the control on acrosome membrane integrity. In conclusion, the results of this study revealed that none of the concentrations of EHA used were effective in improving canine thawed semen quality.

Validación de sybr-14 y 6-cfda para evaluar la viabilidad e integridad de la membrana plasmática en espermatozoides caninos de raza chihuahua / Validation of sybr-14 and 6-cfda to evaluate the viability and plasma membrane integrity in sperm of chihuahua canine breed

En la aplicación de técnicas reproductivas es importante determinar in vitro la capacidad fecundante de los espermatozoides, para ello se utilizan combinaciones de tinciones para evaluar los diferentes parámetros de función espermática, aumentando así la precisión de la estimación de la muestra. El objetivo del estudio fue comparar la efectividad de la utilización de los fluorocromos 6-CFDA y SYBR-14 combinados con PI para determinar la viabilidad e integridad de la membrana plasmática por citometría de flujo. Se utilizó semen fresco de caninos (n=5) de raza Chihuahua, con una concentración espermática >150x106 esp/ml y motilidad progresiva >80%. Tres protocolos fueron ensayados: grupo 1: SYBR-14/PI, grupo 2: 6-CFDA/PI y grupo 3: PI. La integridad de la membrana plasmática de los espermatozoides fue similar entre grupos 1 y 2, independiente del fluorocromo utilizado (37,26±13,9 y 33,8±14,6, respectivamente; p=0,4601). Asimismo, la viabilidad espermática entre los grupos 1, 2 y 3 (62,7±13,9, 66,1±14,6 y 66,4±13,3, respectivamente; p=0,8987). En conclusión, no se evidenció diferencias en la efectividad para determinar la viabilidad e integridad de la membrana plasmática mediante la utilización de SYBR-14 y 6-CFDA, ambas tinciones pueden ser incorporadas al análisis de rutina de semen canino de raza Chihuahua.

In applying reproductive techniques in vitro it is important to determine the fertilizing capacity of the sperm, for this a combination of dyes were used to assess different parameters of sperm function, thereby increasing the accuracy of the estimation of the sample. In dogs (Canis lupus familiaris) Chihuahua breed there is no precedent for evaluating sperm function parameters. The aim was to assess the viability and plasmatic membrane integrity, basic parameters of sperm function. Propidium iodide (PI) was used, a fluorescent dye-specific DNA, which combined with fluorochromes permeable acts as marker of the sperm membrane integrity. The effectiveness of the use of 6-CFDA and SYBR-14 fluorochromes combined with PI was also compared to determine viability and sperm membrane integrity using flow cytometry. Fresh semen of dogs (n=5) Chihuahua breed was used with a concentration of >200x106 sp/ml and progressive motility >80%. Three protocols were performed: group 1: SYBR-14/PI, group 2: 6-CFDA/PI and group 3: PI. The plasma membrane integrity of sperm was similar, independent of the fluorophore used between groups 1 and 2 (13.9±37.26 and 33.8±14.6, respectively, p=0.4601). This also applied to sperm viability between groups 1, 2 and 3 (62.7±13.9, 66.1±14.6 and 66.4±13.3, respectively, p=0.8987). No difference was demonstrated in effectiveness to determine the viability and integrity of the sperm membrane using SYBR-14 and 6-CFDA, both dyes can be incorporated in to routine analysis of semen in canine Chihuahua breed.