Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
Solanki O, Talluri TR, Jhamb D, Kumar S, Pargi KK, Paul N, Kutty M, Dedar RK, Meena M, Gaur M, Bhattacharya TK.The current study evaluated the effect of L-Carnitine supplementation to the stallion semen extender on pre-freeze and post-thaw semen quality and oxidative parameters in Marwari breed stallions. Methods: For this purpose, thirty-six semen ejaculates (6 ejaculates from each stallion) were collected using an artificial vagina (AV) from six adult Marwari stallions. The gel free semen was extended with different concentrations of L-Carnitine (LC) i.e. 0 mM (control), 2.5 mM (T1), 5 mM (T2) and 10 mM (T3). Pre-freeze and post-thaw semen evaluations were conducted for various seminal attributes and...
de Zutter BM, de Paula Freitas-Dell'Aqua C, Dell'Aqua-Junior JA, Monteiro GA, Troncarelli T, Papa FO.This study evaluated the effects of cholesterol, pentoxifylline and casein, with or without skim milk, added to extenders during pre-centrifugation on equine sperm cryosurvival. Seven ejaculates from four stallions (n = 28) were divided into four groups: SM (skim milk), SMP (SM + pentoxifylline), SMCho (SM + cholesterol) and ChoCa (cholesterol + casein). After centrifugation and freezing, sperm kinematics and plasma membrane integrity were assessed immediately and 30 min post-thaw. SMCho and ChoCa showed superior results compared with SM and SMP. These findings indicate that chol...
Brito LFC, Linardi RL, Rosales LAS, Balamurugan NS, Hernández-Avilés C, Ramírez-Agámez L.Efficient use of stallion semen in liquid state is limited by its relatively short shelf-life. A chemically defined extender (Beyond) is now available for long-term liquid semen preservation. The objectives of the present study were to compare Beyond with milk extenders for the preservation of semen at two temperatures, and to evaluate fertility of semen cooled for 4-8 days before artificial insemination. Semen was processed using different extenders: milk, cholesterol (BotuSemen Special); milk-based (INRA 96); and Beyond. Sperm motility, membrane and acrosome integrity, and chromatin struct...
Najjar A, Dhaouadi A, Ezzar S, Benaoun B, Khaldi S.Stallion sexual behavior during semen collection can be influenced by multiple factors, yet the role of sexual arousal intensity remains underexplored. Understanding how arousal modulates behavioral and physiological reproductive traits is essential for improving artificial insemination (AI) efficiency and semen quality. This study aimed to evaluate the effects of sexual arousal intensity, age, origin, and semen collection method on stallion sexual behavior and the quality of fresh and frozen semen. Unassigned: Thirteen Arabian stallions (7 Tunisian, 6 foreign parentage) aged 6-20 years (total...
Aboelmaaty AM, El-Seadawy IE, Kotp MS, Mohamed AA, El-Debaky HA, El-Badry DA, Anwer AM, Tsvetkov T, Daneva T, Elgabry MA.Alpha-tocopherol is one of the non-enzymatic lipophilic antioxidants. Butylated hydroxytoluene (BHT) is a synthetic analog with similar modes of action in protecting the cryopreserved sperms. Objective: This study hypothesized that a certain concentration of any antioxidant is suitable for improving the post-thaw semen quality of stallions. Methods: To determine the optimum BHT concentration, a synthetic antioxidant similar to vitamin E in potency and scavenging oxidative stress power in concentrations of 0.0, 0.25, 0.50, 1.0, 2.0, and 4.0 mM/ml were added to semen extender. The post-thaw sper...
Bugno-Poniewierska M, Bielecka M, Pietras N, Kij-Mitka B, Podstawski Z, Długosz B.The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
Al-Kass Z, Morrell JM, Ntallaris T.Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation)...
Medica A, Aitken RJ, Swegen A, Gibb Z.Context Equine reproductive technologies are crucial for overcoming challenges in natural fertilisation, particularly in sub-fertile stallions and breeding programs focused on genetic conservation and performance enhancement. Assisted reproductive technologies (ARTs), such as artificial insemination (AI), intracytoplasmic sperm injection (ICSI), and in vitro fertilisation (IVF), improve fertility outcomes and enable breeding across geographical distances. Aims This review examines sperm isolation techniques used in ART, evaluating their efficacy, limitations, and potential to enhance reproduct...
Maria Feitosa Ribeiro L, Bersot Gomes JV, Hernando Ortiz Vega W, da Silva Alves A, Netto Machado M, Junio Wutke L, Raquel Quirino C....Stallion breeding is mostly based on desirable phenotypic traits, with little consideration for semen quality. Objective: To identify relationships among semen parameters in stallions using a non-invasive, integrative approach across summer and winter seasons. Methods: Twenty-four stallions were evaluated, in which semen collections, testicular thermography, and Doppler velocimetry of the testicular artery by Doppler ultrasonography were performed, in addition to the measurement of physiological, environmental, and heat stress parameters, both in winter (coat weather) and summer (T-shirt weath...
Wen X, Ren H, He Q, Yi M, Ulaangerel T, Bou G.Horses and donkeys, as integral members of the equine family, exhibit distinct reproductive capabilities and characteristics. Seminal plasma, the fluid component of semen, contains a variety of proteins that play critical roles in sperm function and fertility. This study aimed to systematically compare the protein profiles in the seminal plasma of horses and donkeys, thereby elucidating the molecular differences between these two species. The study utilized 4D-DIA proteomics technology to analyze seminal plasma from horses and donkeys and further validated key proteins through Western blot. Ou...
Usuga A, Vergara AK, Tobón MC, Vargas S, Rojano B, Restrepo G.Maintaining sperm energy homeostasis in vitro is very important to improve the efficacy of stallion sperm preservation. Equine spermatozoa preferentially utilize oxidative phosphorylation over glycolysis to generate ATP. Metformin and rosiglitazone are antidiabetic compounds that enhances metabolic flexibility and glucose utilization. The aim of this study was to evaluate metformin and rosiglitazone supplementation of the freezing medium on quality and oxidative status of thawed stallion semen. A total of 15 ejaculates from five horses were collected and supplemented before freezing with metfo...
Hidalgo M, Ortiz I.Sperm vitrification is a novel-assisted reproductive technique that is increasingly gaining relevance in the last years. This technique allows to cryopreserve sperm from valuable stallions and donkeys without the exposure to permeable cryoprotectants, particularly toxic for the gametes of these species.This chapter aims to describe the current range of methodologies available that are key to ensure sperm quality after vitrification and warming of stallion and donkey sperm.
Miró J, Martínez-Rodero I, Yeste M, Catalán J.Cryopreservation is currently the only strategy for long-term conservation of equine sperm. To get optimal post-thaw sperm survival, carefully following each step of the freezing protocol is crucial. First, one needs to obtain and exhaustively analyze an ejaculate of good sperm quality. Then, the seminal plasma is removed by centrifugation, and the resulting pellet is resuspended in a certain volume of the freezing medium to reach the right sperm concentration. Finally, sperm samples are packaged into 0.5-mL straws, cooled, and frozen using an automatic, controlled-rate freezer. Once the tempe...
Spacek SG, Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Love CC.Cryopreservation of stallion epididymal sperm has become a common clinical procedure after routine castration, euthanasia, or acute death. Unique features of epididymal sperm compared to ejaculated sperm include the requirement to remove sperm from the cauda epididymis, lack of exposure to seminal plasma, and potential recovery of large sperm numbers. In this study, the effect of the flushing technique (Extender (INRA) vs. AIR), freezing extender (LE, CMLE, MFR5, CFR5, or BOTU), and the concentration at which sperm are cryopreserved (200, 400, or 800 × 10 sperm/mL) on post-thaw epididymal s...
Sultan T, Chaudhary AK, Mehta JS, Mehra R, Manuja A, Talluri TR, Bhattacharya TK.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Hidalgo M.Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Guertin JE, Losano JA, Salazar S, Callaham J, Daigneault BW.Bottlenecks to the success of equine assisted reproductive technologies (ART) include suboptimal conditions for prolonged storage of stallion sperm. Shipped stallion sperm are transported in cooling devices designed to maintain temperature for up to 48 h. Increasing the storage time of cooled semen while maintaining acceptable motility would relieve logistical ART challenges. Experiments were conducted to test the hypothesis that external regulation of shipment container temperature would prolong storage time of cooled stallion semen. Initial experiments determined the effect of pre-freezing c...
Luis-Calero M, Muñoz-García CC, Fernández-Hernández P, Macías-García B, González-Fernández L.A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE...
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Caldevilla M, Ferrante A, Neild DM.The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylf...
Becerro-Rey L, Martín-Cano FE, Fabres Robaina Sancler-Silva Y, Gil MC, Ortega-Ferrusola C, Aparicio IM, Gaitskell-Phillips G, da Silva-Álvarez E....Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present i...
Pasch L, Stefanovski D, Dobbie T, Lewis G, Turner RM.Historically, 8 × 0.5 ml straws, containing approximately 800 million sperm and 250 million progressively motile sperm were provided as a single 'breeding dose' of cryopreserved stallion semen. With the use of deep horn artificial insemination, there is a trend to reduce the number of 0.5 ml straws sold as a breeding dose, sometimes down to as little as one straw. Our aims were to determine if the number of straws provided as a breeding dose, as well as other mare, stallion and management factors, have an impact on pregnancy outcome in mares inseminated with cryopreserved semen. Unexpectedly,...
Fanelli D, Moroni R, Sala G, Melanie P, Tarabella I, Telleschi N, Maltinti S, Giorgi M, Barsotti G, Passamonti F, Marmorini P, Rota A, Camillo F....Two studies were conducted to evaluate the use of medical ozone (O) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent...
Nunes MM, Gurgel JRC, Munhoz ML, de Giuli V, Carneiro GF, Miragaya MH, Gallelli MF, Guimaraes JD.The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols wi...
Malaluang P, Niazi A, Guo Y, Nagel C, Guimaraes T, Rocha A, Aurich C, Morrell JM.The microbiome plays a significant role in shaping the health and functioning of the systems it inhabits. The seminal microbiome of stallions has implications for the health of the reproductive tract, sperm quality during preservation and antibiotic use in semen extenders. Diverse bacteria are present on the external genital tract and a mix of commensal microorganisms populates various parts of the reproductive tract, influencing the seminal bacterial content. Other sources of bacteria include the environment, semen collection equipment, and personnel. The bacterial load can adversely affect s...
Kaps M, Wenderoth J, Aurich J, Aurich C.Equine viral arteritis (EVA) can induce a persistent carrier state in stallions which then shed the virus via semen. About 30 years ago, obligatory EVA testing of stallions used for artificial insemination (AI) was implemented in the European Union. Information on the efficacy of these regulations on the prevalence of EVA in stallions are not yet available. Therefore, we retrospectively analyzed results of serological and virus antigen testing for EVA in sires of different age and breed referred to Vetmeduni Vienna for semen preservation or veterinary diagnostic procedures between 2001 and 202...
Catalán J, Yánez-Ortiz I, Martínez-Rodero I, Mateo-Otero Y, Nolis P, Yeste M, Miró J.Previous research revealed that several seminal plasma (SP) metabolites are related to sperm functionality, fertility, and preservation. While it is understood that variations between species exist, whether the SP metabolome differs between donkeys and horses has not been previously investigated. The aim of this work, therefore, was to characterize and compare donkey and horse SP metabolites using nuclear magnetic resonance (NMR) spectroscopy, and relate them to sperm viability and motility. For this purpose, ejaculates from 18 different donkeys and 18 different horses were collected and separ...
Dierberger H, Pieper L, Jung M, Schulze M.Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of dil...
Cavalcanti TP, Pereira AG, Bezerra LGP, Moreira SSJ, da Silva AM, Matos YG, Jorge-Neto PN, Silva AR.The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA-96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA-96 extender as an alternative for the short-term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh ...
Yánez-Ortiz I, Catalán J, Rodríguez-Gil JE, Miró 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...
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...
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 ...
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...
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...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.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...
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 ...
Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín 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...
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...
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...
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 ...
Yeste M, Estrada E, Rocha LG, Marín H, Rodríguez-Gil JE, Miró 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...
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...
Aparicio IM, Martin Muñoz P, Salido GM, Peña 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...
Canisso IF, Panzani D, Miró 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...
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...
Gallardo Bolaños JM, Miró Morán Á, Balao da Silva CM, Morillo Rodríguez A, Plaza Dávila M, Aparicio IM, Tapia JA, Ortega Ferrusola C, Peña FJ.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...
Blommaert D, Sergeant N, Delehedde M, Jouy N, Mitchell V, Franck T, Donnay I, Lejeune JP, Serteyn D.A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentra...
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...
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...
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...
Aurich J, Kuhl J, Tichy A, Aurich C.Differences in the cryotolerance of spermatozoa exist among stallions, but it remains to be determined to what extent such differences are affected by breed. In this study, post-thaw semen quality in stallions presented for semen cryopreservation was analysed retrospectively (1012 ejaculates from 134 stallions of 5 breeds). The percentage of frozen-thawed ejaculates acceptable for artificial insemination (AI) and the number of insemination doses per cryopreserved ejaculate was calculated. Logistic regression analysis revealed sperm motility in raw semen as the most important explanatory variab...
Gallardo Bolaños JM, Balao da Silva CM, Martín Muñoz P, Morillo Rodríguez A, Plaza Dávila M, Rodríguez-Martínez H, Aparicio IM, Tapia JA....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...
Ortega Ferrusola C, González Fernández L, Salazar Sandoval C, Macías García B, Rodríguez Martínez H, Tapia JA, Peña 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...
da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....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 ...
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...
Darr CR, Cortopassi GA, Datta S, Varner DD, Meyers SA.Mitochondrial oxygen consumption is a sensitive indicator of spermatozoal health in the context of cryopreservation. We investigated oxygen consumption of equine sperm mitochondria during incubation in four commercially available sperm cryopreservation extenders: modified INRA 96, BotuCrio, EZ Freezin-"LE" and "MFR5", in addition to several other parameters including motility, reactive oxygen species (ROS) production and viability. All experimental endpoints, with the exception of average path velocity, were affected significantly by freezing extender type after freezing and thawing. Sperm in ...
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...
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%...
