Quirino M, Jakop U, Mellagi APG, Bortolozzo FP, Jung M, Schulze M.For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rh...
Zeitoun MM, Ateah MA, Almaiman AT, Mansour MM.The present study attempted to investigate the effect of various concentrations of spirulina platensis additions to the semen extender on Arabian stallion spermatozoa quality. Semen samples were collected with artificial vagina from five fertile stallions and diluted with an extender containing spirulina (2, 4, 6, and 8 mg/100 mL) or without spirulina (control). Aliquots of diluted semen were cooled (5°C, 90 minutes) and frozen (-196°C, 7 days), then physical traits of thawed spermatozoa were examined. Furthermore, antioxidant activities of superoxide dismutase (SOD), glutathione reductase (...
Podico G, Canisso IF.This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentratio...
Catalu00e1n J, Yu00e1nez-Ortiz I, Tvarijonaviciute A, Gonzu00e1lez-Aru00f3stegui LG, Rubio CP, Barranco I, Yeste M, Miru00f3 J.The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to h...
Nogueira BG, Pereira RR, Bitencourt JL, Milan B, Reis WVA, Junior MV, Acu00e1cio BR, Sampaio BFB, I L Souza M.The objective of the experiment was to evaluate the effect of the addition of different concentrations of the antioxidants Coenzyme Q10 (CoQ10) and melatonin to equine semen freezing diluent, alone or in combination, during the cryopreservation process. Twenty ejaculates (n = 5 stallions) were divided in groups: Control (C, without the addition of antioxidants), melatonin 0.75 mM (MEL1), melatonin 1.5 mM (MEL2), CoQ10 40 μg/mL (Q1), CoQ10 200 μg/mL (Q2), and CoQ10 40 μg/mL+ 0.75 mM melatonin (Q1 +MEL1). Q1 and Q2 groups demonstrated intact plasma membrane and high mitochondrial me...
Contreras MJ, Arias ME, Silva M, Cabrera P, Felmer R.Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and asses...
Albrizio M, Lacalandra GM, Cinone M.In this study, we defined the composition of the culture medium that yield a significant percentage of alive and functional equine spermatozoa during enough time before artificial insemination. The effects of sodium bicarbonate were analyzed in fresh and frozen semen in respect to sperm viability, capacitation, spontaneous acrosome reaction and several kinetic parameters such as total motility, progressive motility, VCL, VSL, ALH, BCF, LIN. Moreover, employing Bayk-6844 and Nifedipine, the involvement of L-type voltage-gated calcium channels in the modulation of intracellular calcium concentra...
In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, ...
Usuga A, Gutiu00e9rrez V, Lu00f3pez ME, Pu00e9rez LF, Jaramillo L, Rojano B, Restrepo G.Microbial growth in semen may cause a decline of sperm quality and fertility; however, the addition of antifungals to semen extender has been shown to impair the overall fertility of the sperm. The aim of this study was to evaluate the antifungal activity of conventional and natural compounds, and their effect on the motility and kinetics of cooled stallion semen. A total of 15 ejaculates from five stallions were collected using the artificial vagina. Each ejaculate was supplemented with: fluconazole at 12.5 (F1), 25 (F2) and 50 (F3) mg/ml; amphotericin-B at 6.5 (A1), 12.5 (A2) and 25 (A3) mg/...
Nikitkina EV, Dementieva NV, Shcherbakov YS, Atroshchenko MM, Kudinov AA, Samoylov OI, Pozovnikova MV, Dysin AP, Krutikova AA, Musidray AA....The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), p...
Quiu00f1ones-Pu00e9rez C, Martu00ednez A, Ortiz I, Crespo F, Vega-Pla JL.Despite the advances in reproductive technology, there is still a considerable number of low sperm quality cases in stallions. Recent studies in humans have detected several seminal microflora-spermatozoa associations behind some idiopathic infertility cases. However, no studies are available on horses, and there is limited information on the microflora present in stallion ejaculates. Accordingly, the objective of this study was to examine associations to the presence of bacteria families with five sperm quality parameters: concentration, total number of spermatozoa, total and progressive moti...
Catalu00e1n J, Yu00e1nez-Ortiz I, Tvarijonaviciute A, Gonzu00e1lez-Arostegui LG, Rubio CP, Yeste M, Miru00f3 J, Barranco I.This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into tw...
Andrade AFC, Knox RV, Torres MA, Pavaneli APP.When preserving sperm in the liquid or cryopreserved state, seminal plasma (SP) components within ejaculates can alter fertilizing capacity of these gametes. Depending on the species or how semen is collected, volume and concentration of SP components varies considerably. The SP contains substances essential for maintenance of sperm viability and fertility; however, these components can be deleterious depending on quantity, or duration of time before there is removal of SP from sperm in semen processing. Substances that impair (e.g., BSP - bull; HSP-1 - stallion; Major seminal plasma protein P...
Pruu00df D, Oldenhof H, Wolkers WF, Sieme H.The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calc...
Monteiro RA, Cunha RM, Guerra MMP, de Almeida VM, Peu00f1a-Alfaro CE, Silva SV.The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. Af...
Lanu00e7oni R, Celeghini ECC, Gonella-Diaza AM, Ju00fanior VG, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very l...
In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three s...
Macu00eado IN, Arruda LCP, de Santana BB, de Moura TCM, Guerra MMP, Bezerra DG, Carneiro GF, Silva SV.The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O/mL. The semen samples were diluted (200 x 10 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by ...
Zuidema D, Kerns K, Sutovsky P.Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use ...
Lagares MA, da Silva GC, Cortes SF, Moreira FHM, Neves FCD, Alves NC, Viegas RN, Diniz TF, Lemos VS, de Rezende ASC, Freitas MM, Stahlberg R....The objective of this study was to enhance the in vitro sperm quality and in vivo fertility of frozen-thawed equine semen by the addition of l-carnitine (LC) to post-thawed semen. Different concentrations of LC were added to thawed samples to obtain four treatments control and 0.5, 1 and 2 mM LC. In the in vitro experiments, sperm motility and kinematics, membrane integrity and intracellular calcium ion concentration ([Ca ] ) were investigated, and the antioxidant bioactivity of LC was assessed by measuring hydrogen peroxide and nitrite concentrations (NO ). The fertility rate was assessed v...
Yu00e1nez-Ortiz I, Catalu00e1n J, Rodru00edguez-Gil JE, Miru00f3 J, Yeste M.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, p...
Yu00e1nez-Ortiz I, Catalu00e1n J, Delgado-Bermu00fadez A, Carluccio A, Miru00f3 J, Yeste M.In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed pri...
Moura TCM, Arruda LCP, Arau00fajo Silva RAJ, Silva RPF, Oliveira AS, Tobal LFM, Batista AM, Carneiro GF, Guerra MMP.The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 10 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (-196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasm...
Orsolini MF, Meyers SA, Dini P.As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towa...
Orsolini MF, Meyers SA, Dini P.As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.
Al-Khaldi K, Yimer N, Sadiq MB, Firdaus Jesse Bin Abdullah F, Salam Babji A, Al-Bulushi S.Diluents and various biological products have been used in different animal species, with promising outcomes in post-thaw sperm quality. Nevertheless, only a few reports are available for the semen of Arabian horses. Edible bird's nest (EBN) - a product of the salivary secretions of swiftlet species is widely known to have both antioxidant and anti-inflammatory properties. Presently, there is no data available on the role of EBN supplemented in different extenders and its effect on semen quality in stallion semen. Two in vitro experiments were conducted to examine the effects of edible bird's ...
Pruu00df D, Oldenhof H, Wolkers WF, Sieme H.The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared v...
Aitken RJ, Gibb Z, Mitchell LA, Lambourne SR, Connaughton HS, De Iuliis GN.The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic ...
White IG.When sperm of the ram, bull, boar and stallion are cold-shocked by rapid cooling to near freezing point, motility and metabolic activity are irreversibly depressed and the acrosome and plasma membrane disrupted. Ram sperm become susceptible to cold shock in the proximal corpus region of the epididymis when the cytoplasmic droplet has moved backwards to the distal portion of the sperm midpiece. The membrane constituents phospholipids and cholesterol are important in cold shock which causes loss of lipid from sperm. The susceptibility of sperm to cold shock is linked with a high ratio of unsatur...
Ball BA.Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and f...
Ortega-Ferrusola C, Sotillo-Galu00e1n Y, Varela-Fernu00e1ndez E, Gallardo-Bolau00f1os JM, Muriel A, Gonzu00e1lez-Fernu00e1ndez L, Tapia JA....The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
Timoney PJ, McCollum WH, Roberts AW, Murphy TW.The chronic carrier state was virologically confirmed in 15 thoroughbred stallions naturally infected with equine arteritis virus based on the results of test matings and, or, isolations of the virus from semen. Carrier stallions were shown to shed equine arteritis virus in the semen for at least one to two years. Existence of a short-term or convalescent carrier state was also demonstrated in five additional stallions. The frequency of the long-term carrier state in stallions naturally infected with equine arteritis virus was 35 per cent; it varied considerably between groups of stallions on ...
Kotilainen T, Huhtinen M, Katila T.The objective of this study was to investigate the inflammatory reaction induced in the equine uterus by insemination with fresh and frozen semen. Eleven groups (6 to 8 mares per group) were studied during 2 breeding seasons. The mares were inseminated using raw semen, frozen semen, extended fresh and frozen semen, concentrated fresh semen, seminal plasma and seminal extenders only. One group was bred naturally. Six hours after insemination, the uteri were flushed with 50 ml of phosphate-buffered saline (PBS). Seventeen out of 104 samples (16%) exhibited slight bacterial growth. Neutrophil con...
Ball BA, Vo AT, Baumber J.To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC, Foster DN.Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm-neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3h, but as little as 10% SP increas...
Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K.Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sper...
Canisso IF, Panzani D, Miru00f3 J, Ellerbrock RE.Donkeys are nonseasonal, polyestrous, territorial, and nonharem breeders. Although there are many similarities between horses and donkeys, there are also reproductive features that differ, from the longer cervix in the jenny to spermatogenic efficiency in the jack. Mules display reproductive cyclic activity but are rarely fertile. Frozen donkey semen has high pregnancy rates in mares, but lower rates in jennies. This article reviews key aspects of donkey and mule reproductive physiology, reproductive medicine, and assisted reproductive techniques that are useful for practitioners offering assi...
Troedsson MH, Loset K, Alghamdi AM, Dahms B, Crabo BG.Insemination of mares with bacteria-free equine spermatozoa results in an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen. In vitro studies have demonstrated that equine spermatozoa activate complement, resulting in cleavage of factors C5a and C3b. Since uterine secretion is rich in complement, it is likely that an interaction between spermatozoa and uterine secretion results in C5a-mediated chemotaxis and migration of PMNs into the uterine lumen. Once in the uterine lumen, the PMNs phagocytize bacteria and spermatozoa, which is an important part of sperm elimination from...
Sieme H, Oldenhof H, Wolkers WF.Native sperm is only marginally stable after collection. Cryopreservation of semen facilitates transport and storage for later use in artificial reproduction technologies, but cryopreservation processing may result in cellular damage compromising sperm function. Membranes are thought to be the primary site of cryopreservation injury. Therefore, insights into the effects of cooling, ice formation and protective agents on sperm membranes may help to rationally design cryopreservation protocols. In this review, we describe membrane phase behaviour of sperm at supra- and subzero temperatures. In a...
Loomis PR, Graham JK.One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen ...
Gallardo Bolau00f1os JM, Miru00f3 Moru00e1n u00c1, Balao da Silva CM, Morillo Rodru00edguez A, Plaza Du00e1vila M, Aparicio IM, Tapia JA....Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Ortega Ferrusola C, Gonzu00e1lez Fernu00e1ndez L, Salazar Sandoval C, Macu00edas Garcu00eda B, Rodru00edguez Martu00ednez H, Tapia JA, Peu00f1a FJ.In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active cas...
Aparicio IM, Martin Muu00f1oz P, Salido GM, Peu00f1a FJ, Tapia JA.Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of au...
Aitken JB, Naumovski N, Curry B, Grupen CG, Gibb Z, Aitken RJ.This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptop...
Peu00f1a FJ, O'Flaherty C, Ortiz Rodru00edguez JM, Martu00edn Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C.Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine resid...
Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ.The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mito...
da Silva CM, Macu00edas-Garcu00eda B, Miru00f3-Moru00e1n A, Gonzu00e1lez-Fernu00e1ndez L, Morillo-Rodriguez A, Ortega-Ferrusola C....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
Yeste M, Estrada E, Rocha LG, Maru00edn H, Rodru00edguez-Gil JE, Miru00f3 J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Suh TK, Schenk JL, Seidel GE.Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 microm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 5...
Aurich C, Schreiner B, Ille N, Alvarenga M, Scarlet D.Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ej...
Moore AI, Squires EL, Graham JK.Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sper...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically norma...
Moore AI, Squires EL, Graham JK.Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80%...
Brinsko SP, Varner DD, Love CC, Blanchard TL, Day BC, Wilson ME.Eight stallions were used in 2 x 2 crossover study to determine if feeding a nutriceutical rich in docosahexaenoic acid (DHA) would improve semen quality. Stallions were randomly assigned to one of two treatment groups (n = 4 per group). Stallions were fed their normal diet (control) or their normal diet top-dressed with 250 g of a DHA-enriched nutriceutical. Feeding trials lasted for 14 week, after which a 14-week washout period was allowed and the treatment groups were reversed for another 14 week feeding trial. Feeding the nutriceutical resulted in a three-fold increase in semen DHA levels ...
Gallardo Bolau00f1os JM, Balao da Silva CM, Martu00edn Muu00f1oz P, Morillo Rodru00edguez A, Plaza Du00e1vila M, Rodru00edguez-Martu00ednez H....AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, w...
Kareskoski M, Katila T.Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.
Del Prete C, Tafuri S, Ciani F, Pasolini MP, Ciotola F, Albarella S, Carotenuto D, Peretti V, Cocchia N.Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with...
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