Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 106 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques.

Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality.

Sperm cryopreservation represents a powerful tool for livestock breeding. Several efforts have been made to improve the efficiency of sperm cryopreservation in different ruminant species. However, a significant amount of sperm still suffers considerable cryodamage, which may affect sperm quality and fertility. Recently, the use of different "omics" technologies in sperm cryobiology, especially proteomics studies, has led to a better understanding of the molecular modifications induced by sperm cryopreservation, facilitating the identification of different freezability biomarkers and certain proteins that can be added before cryopreservation to enhance sperm cryosurvival. This review provides an updated overview of the molecular mechanisms involved in sperm cryodamage, which are in part responsible for the structural, functional and fertility changes observed in frozen-thawed ruminant sperm. Moreover, the molecular basis of those factors that can affect the sperm freezing resilience of different ruminant species is also discussed as well as the molecular aspects of those novel strategies that have been developed to reduce sperm cryodamage, including new cryoprotectants, antioxidants, proteins, nanoparticles and vitrification.

Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation.

The aim of this study was to investigate the dynamic changes that ram sperm experience during in vitro capacitation before and after cryopreservation. Using flow cytometry and computer assisted sperm analysis system (CASA), protein tyrosine phosphorylation and several functional parameters were evaluated in fresh and cryopreserved ram sperm incubated under capacitating and non-capacitating conditions at 0, 1, 5, 15, 30, 60, 120, 180 and 240 min. A short incubation period (5-30 min) under capacitating conditions was enough to increase mitochondrial activity and tyrosine phosphorylation in cryopreserved sperm, inducing also changes in the motility pattern, which could be related to hyperactivation. However, fresh sperm required a longer incubation (180-240 min) under capacitating conditions to undergo similar modifications. In both types of samples, tyrosine phosphorylation increased in a sequential manner in the midpiece, principal piece and tail at specific time points during in vitro capacitation. Moreover, the proportion of viable sperm with intact acrosome begun to decrease during capacitation, occurring before in cryopreserved sperm. Our findings suggest that cryopreserved ram sperm become competent for fertilization after a short exposure to capacitating conditions as a result of drastic changes inflicted by the freezing-thawing procedure, while prolonged incubations after cryopreservation severely impair sperm quality.

Recombinant SPINK3 improves ram sperm quality and in vitro fertility after cryopreservation.

Capacitation-like changes affect sperm of several species, such as ram, reducing cell survival and fertilizing competence. Proteins from seminal plasma stabilize sperm plasma membranes, being an interesting focus to develop strategies for improving cryopreserved ram semen performance. To date, biotechnologies are focused to reduce damage in frozen-thawed ram spermatozoa through the addition of bioactives. Serine Protease Inhibitor Kazal-type 3 (SPINK3) is a little protein synthesized by mouse seminal vesicle and secreted to seminal plasma. While attached to the sperm, this protein binds to non-capacitated sperm and blocks calcium entry, avoiding a premature physiological capacitation and consequently, acrosome reaction. Due to these characteristics, SPINK3 has been proposed as a decapacitating factor. The aim of this work was to assess whether heterologous SPINK3 is able to protect ram sperm from the well-known cell damages produced by freezing/thawing and to understand the mechanisms by which it is acting. Sperm were supplemented with 13 µM SPINK3 before freezing in an egg yolk-based extender or after thawing and selection. Under both conditions, SPINK3 decreased intracellular calcium content (p < 0.05) and reduced the 25 kDa tyrosine phosphorylated protein demonstrating a decapacitating effect, although the addition of the protein before cryopreservation was not enough to improve other sperm parameters. However, the addition of SPINK3 post thawing was able to significantly ameliorate viability, motility, mitochondrial status and to avoid the increase of lipid peroxidation (p < 0.05). Moreover, sperm treated with SPINK3 was not only still capable to fertilize, but also improved it, as evidenced by an increase in the oocyte cleavage rate (p < 0.05) although, the embryo development and embryo quality were not affected. Our findings would contribute to develop a strategy for improving sperm quality by using decapacitating proteins. In fact, the outcomes of this work demonstrate that SPINK3 is able to reduce sperm cryo-injuries when is added after thawing, improving functionality and thus in vitro fertilization results.

Freezing-Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation.

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

Quality Standards for Allergen Immunotherapy Clinics in Spain: Consensus Document.

BACKGROUND AND OBJECTIVE: Allergen immunotherapy clinics (AITCs) in Spain differ widely in terms of structure, organization, resources, and portfolio of services. Therefore, it is essential to unify treatment criteria and define quality standards for the most complex AITCs. Objective: To establish a series of recommendations that make it possible to guarantee quality and safety in the administration of immunotherapy and define quality standards for the most complex AITCs. METHODS: This project began with an online survey of 65 allergy departments/units throughout Spain in 2013. Next, a 2-phase consensus process was carried out. In the first phase, 10 experts defined and agreed on the standards using the RAND/UCLA Appropriateness method; in the second, the agreements were validated by means of a 2-round Delphi consultation with 84 experts. RESULTS: Consensus was reached on minimum safety and quality criteria in the administration of allergen immunotherapy, and 2 levels of highly complex AITCs were defined: accredited AITCs and accredited AITCs with excellence. Consensus was also reached on quality standards and accreditation criteria for both levels. CONCLUSIONS: This project is pioneering in terms of its purpose (the definition of quality standards for AITCs) and of the use of structured participation techniques (combination of the RAND/UCLA and Delphi methods). It enabled the design of minimum standards for quality and safety in administering AIT, as well as quality criteria for accreditation of AITCs supported by a broad panel of experts from the Spanish Society of Allergology and Clinical Immunology.

Quality standards for allergen immunotherapy clinics in Spain: consensus document

BACKGROUND: Allergen immunotherapy clinics (AITCs) in Spain differ widely in terms of structure, organization, resources, and portfolio of services. Therefore, it is essential to unify treatment criteria and define quality standards for the most complex AITCs. OBJECTIVE: To establish a series of recommendations that make it possible to guarantee quality and safety in the administration of immunotherapy and define quality standards for the most complex AITCs. METHODS: This project began with an online survey of 65 allergy departments/units throughout Spain in 2013. Next, a 2-phase consensus process was carried out. In the first phase, 10 experts defined and agreed on the standards using the RAND/UCLA Appropriateness method; in the second, the agreements were validated by means of a 2-round Delphi consultation with 84 experts. RESULTS: Consensus was reached on minimum safety and quality criteria in the administration of allergen immunotherapy, and 2 levels of highly complex AITCs were defined: accredited AITCs and accredited AITCs with excellence. Consensus was also reached on quality standards and accreditation criteria for both levels. CONCLUSIONS: This project is pioneering in terms of its purpose (the definition of quality standards for AITCs) and of the use of structured participation techniques (combination of the RAND/UCLA and Delphi methods). It enabled the design of minimum standards for quality and safety in administering AIT, as well as quality criteria for accreditation of AITCs supported by a broad panel of experts from the Spanish Society of Allergology and Clinical Immunology

ANTECEDENTES: Las unidades de inmunoterapia (UIT) en España son muy diferentes en cuanto a estructura, organización, recursos y cartera de servicios. Por ello, resulta esencial homogeneizar criterios de actuación y definir estándares de calidad para las UIT de mayor complejidad. OBJETIVO: Establecer recomendaciones que permitan garantizar la calidad y seguridad en la administración de la inmunoterapia y definir estándares de calidad para las UIT de mayor complejidad. MÉTODOS: Proyecto iniciado (año 2013) con una encuesta on-line a 65 servicios o unidades de alergología de toda España. Posteriormente, se desarrolló un proceso de consenso en dos fases. En la primera, diez expertos definieron y consensuaron los estándares mediante el método RAND/UCLA; en la segunda, los acuerdos se validaron mediante una consulta Delphi a dos rondas con 84 expertos. RESULTADOS: Se consensuaron criterios mínimos de seguridad y calidad en la administración de inmunoterapia con alérgenos (ITA) y se definieron dos niveles de UIT de mayor complejidad: las UIT acreditadas (UITA) y las UIT acreditadas con excelencia (UITAE), consensuándose también los estándares de calidad y criterios de acreditación para ambos niveles. CONCLUSIONES: Proyecto pionero en su objetivo - definición de estándares de calidad de UIT- y en el empleo de técnicas de participación estructuradas -combinación de los métodos RAND/UCLA y Delphi-. El resultado es la definición de unos mínimos de calidad y seguridad para administrar ITA, y un conjunto de criterios de calidad para la acreditación de las UIT que cuenta con el respaldo de un amplio panel de expertos de la SEAIC

Cinnamtannin B-1, a novel antioxidant for sperm in red deer.

Cinnamtannin B-1 (CNB-1) is a naturally occurring trimeric A-type proanthocyanidin contained in several plants such as cinnamon (Cinnamomum zeylanicum). It is considered to be a potent antioxidant. The protective effect of CNB-1 against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from six stags were thawed, pooled and extended to 400 × 106 sperm/ml in BGM (bovine gamete medium). After being aliquoted, the samples were supplemented with different concentrations of CNB-1 (0, 0.1, 1, 10 and 100 µg/mL), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. This experiment was replicated six times. Spermmotility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (TUNEL) were assessed. After 4 h of incubation, CNB-1 prevented the deleterious effects of oxidative stress, thus improved sperm progressivity and velocity (P<0.05). Furthermore, 1 and 10 µM CNB-1 improved sperm linearity, even when compared to those samples that had not been subjected to oxidative stress (P<0.05). The greatest concentration, 100 µM, prevented sperm lipoperoxidation and reduced ROS production in samples subjected to oxidative stress.

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software.

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 × 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.

Immunohistochemical investigation of predictors of response or aggressiveness of bowen disease after photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a good option for the treatment of Bowen's disease (BD). However, BD occasionally can progress into a squamous cell carcinoma (SCC) after PDT. OBJECTIVE: Find predictors of aggressiveness of BD after PDT METHODS: Two biopsies of patients with BD treated with PDT with progression to SCC within three months were selected for immunohistochemical (IHC) studies. Conventional PDT was applied. IHC analysis was performed together with hematoxylin-eosin in the biopsies prior to and after treatment with PDT. RESULTS: Among the IHC markers studied, none showed different expressions between pre-treatment and post-treatment biopsies except for HSP70 CONCLUSIONS: The expression of Hsp70 in BD may predict future aggressive behaviour in BD when treated with PDT. Nevertheless, due to the small number of biopsies studied, further investigations are required to draw conclusions.

Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa.

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 µM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

An In Vitro Evaluation of Biochemical Processes Involved in Lead-Induced Changes on Ram Spermatozoa.

Lead (Pb(2+) ) is a toxic heavy metal which interferes with several physiological processes regulated by Ca(2+) , including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb(2+) /ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb(2+) on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb(2+) have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb(2+) action. On the contrary, Pb(2+) -incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb(2+) + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb(2+) in spermatozoa; thus, the enhancement of Pb(2+) effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb(2+) by spermatozoa. However, the increase of intracellular Pb(2+) in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Influence of semen collection method on sperm cryoresistance in small ruminants.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Reduced glutathione addition improves both the kinematics and physiological quality of post-thawed red deer sperm.

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2h, 39°C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1mM or 5mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<0.001). After 2h of incubation at 39°C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4±1.1% and 11.7±0.9%) than GSH1, GSH5 (15.2±1% and 14.6±1.1%) and Control samples (16.9±1.2%) (P<0.001). Use of GSH at both concentrations (1 and 5mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates.

Sperm head phenotype and male fertility in ram semen.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.