Sperm mitochondrial regulation in motility and fertility in horses

The biological nature of age‐related declines in fertility in males of any species, including stallions, has been elusive. In horses, the economic costs to the breeding industry are frequently extensive. Mitochondrial function in ejaculated sperm, which is essential for sperm motility, is reflected by adenosine triphosphate production, mitochondrial oxidative efficiency and production of reactive oxygen species, and that this balance may become compromised in ageing stallions and during the process of cryopreservation. This presentation will focus on mitochondrial integrity and function as an avenue for understanding the pathophysiology of sperm when undergoing cryopreservation and male ageing. We discuss the importance of understanding the differences and similarities of sperm mitochondria to that of somatic cells regarding structure and mitochondrial biochemistry relating to sperm function. The roles of oxidative phosphorylation and glycolysis in sperm mitochondria are outlined as is the method of determining oxygen consumption and calcium homoeostasis in sperm mitochondria. Further, we outline the role of oxidative stress and reactive oxygen species.     

Relation between respiratory activity and sperm parameters in boar spermatozoa cryopreserved with alpha‐tocopherol and selected by Sephadex

Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo‐osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl‐cyanide‐m‐chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

哺乳动物精子线粒体研究进展

哺乳动物精子线粒体是维持精子活力的关键细胞器，对精子超激活运动、获能、顶体反应及受精等过程起到重要的调节作用。哺乳动物精子线粒体特有的形态特征与特异性酶异构体使其具有独特动力学和调节特性。精子线粒体中发生的氧化磷酸化过程是维持精子运动的重要途径，该过程产生的活性氧对精子功能的维持具有重要作用，但过量可能导致精子损伤，加速精子凋亡。哺乳动物精子质膜磷脂酰丝氨酸外翻和相关半胱氨酸蛋白酶激活级联反应引起细胞凋亡。区别于体细胞线粒体，精子线粒体钙信号可能并未参与精子固有的凋亡途径，作为衡量线粒体功能的敏感指标，其对线粒体膜电位和耗氧量的检测研究至关重要。哺乳动物精子线粒体具有自身的遗传系统，线粒体基因拷贝数可能作为无创衡量精子质量和受精能力的标记。作者重点阐述了哺乳动物精子线粒体的结构、线粒体鞘的形成及其生物功能，包括发生在线粒体中的氧化磷酸化过程、活性氧对精子的利与弊、线粒体参与钙稳态与细胞凋亡过程；介绍了线粒体膜电位和耗氧量的检测，简述了线粒体基因组的研究进展，为进一步探讨线粒体所涉及的精子功能机制奠定基础。     

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential,membrane integrity,sperm motility and in vitro fertilization in bovine epididymal sperm

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post‐mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty‐six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer‐assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre‐freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post‐thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.     

Sperm Susceptibility to Oxidative Stress in the Retuertas Endangered Horse

In order to determine whether the high level of inbreeding of the Retuertas endangered Horses may increase the vulnerability to oxidative stress of the sperm, the susceptibility to lipid peroxidation (LPO) of sperm membranes was evaluated in cooled extended ejaculates. Extended ejaculates (INRA 96) from four Retuertas horses and nine ejaculates from pure Spanish horses were received within 24 hours of collection at our laboratory. An aliquot was analyzed and served as time 0 control. The rest of the ejaculates were split into two additional aliquots: the first aliquot served as an incubation control and the second aliquot received an oxidative insult (Fe₂SO₄). Membrane LPO, motility parameters, membrane integrity, and mitochondrial membrane potential were evaluated. The oxidative insult induced a significant increase in the percentage of sperm with LPO in the Retuertas horses (P < .05). Total and progressive motility were not affected by the oxidative insult in both groups. However, curvilinear velocity (VCL), average velocity (VAP), straightness index (STR), and amplitude of lateral head displacement (ALH) were significantly affected by the treatment (P < .05) in the Retuertas horse population. The membrane integrity of sperm in this group was also affected: the percentage of sperm with intact membranes decreased (57.31% ± 7.76% vs. 47.52% ± 8.28%); and the percentage of necrotic sperm increased (33.27% ± 7.83% vs. 44.85% ± 9.16%) (P < .05). Finally, the oxidative insult increased the population with low mitochondrial membrane potential in these wild horses. We concluded that the high level of inbreeding of this particular breed apparently leads to a higher susceptibility to lipid peroxidation.     

Protective influence of rosiglitazone against time‐dependent deterioration of boar spermatozoa preserved at 17°C

Spermatozoa are highly specialized cells, and energy metabolism plays an important role in modulating sperm viability and function. Rosiglitazone is an antidiabetic drug in the thiazolidinedione class that regulates metabolic flexibility and glucose uptake in various cell types, but its effects on boar sperm metabolism are unknown. In this study, we investigated the potential effect of rosiglitazone against time‐dependent deterioration of boar spermatozoa during liquid preservation at 17°C. Freshly ejaculated semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations of rosiglitazone, and the motility, membrane and acrosome integrity of sperm were detected. Besides, we measured glucose uptake capacity, l ‐lactate production level, mitochondrial membrane potential, adenosine triphosphate (ATP) content and mitochondrial reactive oxygen species (mROS) production of sperm after boar semen had been incubated with or without rosiglitazone, iodoacetate (glycolysis inhibitor) and rotenone (electron transport chain inhibitor) for 5 days. The addition of rosiglitazone significantly enhanced sperm quality and had a strong protective effect on the sperm membrane and acrosome integrity during storage. BTS containing 50 μM rosiglitazone maintained the total motility of liquid‐preserved sperm above 60% for 7 days. Rosiglitazone improved sperm quality by regulating energy metabolism manner of preserved sperm, protected the sperm mitochondrial membrane potential, enhanced sperm ATP production and in the meanwhile reduced mROS through enhancing glycolysis but not oxidative phosphorylation. The data suggested the practical feasibility of using rosiglitazone for improving boar spermatozoa quality during semen preservation.     

The John Hughes Memorial Lecture: Aspects of Sperm Physiology—Oxidative Stress and the Functionality of Stallion Spermatozoa

Spermatozoa are vulnerable to oxidative attack because they contain an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation. In addition, functionally important proteins and DNA are also subject to oxidative modification and adduction by aldehydes, such as 4-hydroxynonenal (4HNE), generated as a consequence of the peroxidative process. The proteins adducted by 4HNE include elements of the mitochondrial electron transport chain, such as succinic acid dehydrogenase. The net result of such electrophilic attack is to stimulate generation of mitochondrial reactive oxygen species (ROS) in a self-perpetuating lipid peroxidation–ROS generation cycle that ultimately triggers the intrinsic apoptotic pathway, leading to a rapid loss of motility and cell death. A major point of difference between apoptosis in spermatozoa and somatic cells is that in the former, nuclear DNA is located in a compartment (the head) separate from the mitochondria and most of the cytoplasm (the midpiece). As a result, nucleases activated and released in the midpiece during apoptosis cannot gain access to the DNA in the sperm head in order to cleave the DNA. However, the ROS generated during apoptosis can readily gain access to the sperm nucleus and generate oxidative base adducts, typically 8-hydroxy, 2′-deoxyguanosine (8OHdG), which are converted into abasic sites by 8-oxoguanine glycosylase (OGG1), the only enzyme of the base excision repair pathway possessed by spermatozoa. These abasic sites subsequently become the foci of DNA fragmentation. Because defective sperm function and DNA damage are frequently associated with oxidative stress, there is a great deal of interest in the use of antioxidants in a therapeutic context. This presentation examines the fundamental relationships between oxidative stress and sperm function and considers the implications of recent findings for the management of sperm function and fertility in stallions.     

Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2‐methyl‐1,4‐naphthoquinone (menadione), or hydroxyl radical formation after FeSO₄ exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8‐iso‐PGF_2α and 4‐hydroxynonenal (4‐HNE). While both treatments induced changes indicative of spermptosis (caspase‐3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.     

Cryopreservation of stallion semen: Effect of adding antioxidants to the freezing medium on sperm physiology

Cryopreservation of stallion semen has not reached the level of efficiency and positive results described in other species. This is mainly due to the greater sensitivity of stallion sperm to the freezing process, showing higher rates of oxidative stress and plasma membrane damage, which trigger the activation of several cell damage pathways that ultimately culminate in DNA fragmentation and cell death. Therefore, finding molecules that improve the efficiency of this technique in stallion by preventing oxidative stress and cell damage is required. Thus, the aim of the present study was to evaluate the effect of adding three antioxidants (MnTBAP, NAC and FeTPPS) to the freezing medium on the quality and functional parameters of stallion sperm. Semen samples from three stallions frozen with the antioxidants were evaluated in two conditions: (a) adding the antioxidants before freezing, and (b) before and after freezing. Plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation, intracellular ROS levels, membrane lipid disorder, DNA damage, sperm motility and binding to the zona pellucida were assessed. The results showed that MnTBAP was the antioxidant treatment that best controlled the oxidative stress process and post-thaw cell damage, showing higher plasma membrane integrity, mitochondrial membrane potential, sperm motility, number of spermatozoa bound to the zona pellucida of bovine oocytes and lower lipid disorder. Additionally, it was determined that a second post-thaw application of antioxidants is detrimental since induced higher cell damage and lower sperm motility, without showing any beneficial effect on the spermatozoa.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

Effects of ascorbic acid 2‐glucoside and alpha‐tocopherol on the characteristics of equine spermatozoa stored at 5°C

The aim of this experiment was to evaluate the effects of adding ascorbic acid 2‐glucoside (AA2G), a water‐soluble antioxidant and stable derivative of ascorbate, to the semen extender and compare it to the addition of vitamin C (Vit. C) and the fat‐soluble antioxidant α‐tocopherol (α‐Toh), both individually and in combination, on the seminal variables of equine sperm submitted to cooling for 72 h. We used two ejaculates from 10 stallions and evaluated them for motility, membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation. In the analysis of lipid peroxidation, the control group showed 2506.2 ± 796.4 ng malondialdehyde/10⁸ sperm, which was higher (P < 0.05) than the groups treated with antioxidants. The average value of motility in the AA2G group was 68.4 ± 18.1%, which was higher (P < 0.05) than that observed in the control group (62.1 ± 16.2%). The variables membrane integrity, chromatin fragmentation and mitochondrial activity did not show significant difference (P > 0.05) between treatments. It was concluded that the antioxidants protected sperm cells from lipid peroxidation and that AA2G was effective during the cooling process of equine semen at 5°C for72 h, providing increased levels of total motility.     

Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant

A soybean lecithin‐based extender supplemented with hyaluronic acid (HA) was assayed for effectiveness to improve the quality of frozen–thawed ram semen. HA has not been tested yet in an extender containing soybean lecithin for freezing ram semen. Thus, the aim of this study was to analyse the effects of soybean lecithin at 1% or 1.5% along with HA at 0, 0.5 and 1 mg ml^‐1 in a Tris‐based extender on the motion characteristics, membrane integrity (HOST), viability, GSH peroxidase (GSH‐PX) activity, lipid peroxidation and acrosomal status after freezing–thawing. Semen was collected from four Mehraban rams during the breeding season and frozen in the six lecithin×HA extenders. The extender containing 1.5% lecithin supplemented with no HA yielded higher total motility (52.5%±1.6), viability (55.8%±1.6) and membrane integrity (44.5%±1.7), but the effects of the lecithin concentration did not reach signification. Linearity‐related parameters, ALH, BCF, lipid peroxidation, GSH‐PX activity, morphology and acrosomal status were not affected by the extender composition. In general, adding HA significantly decreased sperm velocity (1 mg ml^‐1 HA), total motility (only with 1.5% lecithin), viability (1 mg ml^‐1 HA for 1% lecithin; both concentrations for 1.5% lecithin) and membrane integrity. In conclusion, adding HA to the freezing extender supplemented with soybean lecithin failed to improve quality‐related variables in ram semen. Increasing the lecithin content could have a positive effect, but further studies are needed.     

Evaluation of cryopreserved boar semen after supplementation sericin form silkworm (Bombyx mori) in semen extender

Boar cryopreserved semen is scarcely used for artificial insemination due to its quality which is largely reduced by membrane lipid peroxidation. This present study was designed to improve the post‐thawed boar semen quality by determining the optimal level of sericin supplementation (antioxidants) in semen extender. Five levels of sericin supplementation between 0% and 1% (w/v) were examined. Semen was frozen by the liquid nitrogen vapor method, thawed slowly at 5°C for 5 min, and used for the evaluation of sperm quality. The results indicated 0.5%–1% sericin supplementation was more effective on maintenance of sperm viability, acrosome integrity, and mitochondrial functions during freezing–thawing. Moreover, 0.75% sericin supplementation was most protective toward total sperm motility and sperm progressive motility. Additionally, 0.25%–0.75% sericin supplementation significantly suppressed increases in the index of lipid peroxidation. In conclusion, 0.75% sericin is recommended as an alternative component of the freezing extender to improve cryopreserved boar semen. However, further research using AI will be necessary to demonstrate that this indication can be applied to the production of offspring in the farms.     

Sperm Mitochondrial Function is Affected by Stallion Age and Predicts Post-Thaw Motility

Sperm motility and quality decline with stallion age and sperm preservation, but the mechanisms of functional deficit have not been explained. We tested the hypothesis that mitochondrial deficits underlie age- and cryopreservation-related deficits in stallion fertility by measuring mitochondrial function, motility, and reactive oxygen species (ROS) in 88 frozen-thawed ejaculates from 19 stallions of varying ages. As expected with increasing age, total sperm motility, progressive motility, and average path velocity decreased, and ROS production increased. For every unit increase in oxygen consumption, there was a 77% increase in the odds of sperm movement (P < .05), confirming the link between mitochondrial functionality and motility. In addition, the rate of mitochondrial oxygen consumption increased from 4 years of age to a peak at 12 years of age and decreased steadily thereafter (P < .05). This confirms the importance of mitochondrial functionality for overall sperm health and motility, implicating mitochondrial dysfunction as a major contributor to sperm aging and cryopreservation damage.     

First successful frozen semen of the maned wolf (Chrysocyon brachyurus)

This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio^® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Effect of Several Antioxidants on Thawed Ram Spermatozoa Submitted to 37°C up to Four Hours

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N‐acetyl‐cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm , in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP‐Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe²⁺/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm , decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm , rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N‐acetyl‐cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm , DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.     

Electroejaculation Increases Low Molecular Weight Proteins in Seminal Plasma Modifying Sperm Quality in Corriedale Rams

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and 2‐D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.