Equid semen refers to the reproductive fluid produced by male horses, which contains sperm cells necessary for fertilization. The quality and viability of equid semen are important for successful breeding programs and can be influenced by various factors, including genetics, age, diet, and environmental conditions. Key parameters used to assess semen quality include volume, concentration, motility, and morphology of sperm cells. Techniques for collecting, evaluating, and preserving equid semen, such as cryopreservation, are critical for artificial insemination practices. This page compiles peer-reviewed research studies and scholarly articles that explore the physiology, assessment, and management of equid semen in the context of equine reproduction.
Claes A, Stout TAE.In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yield...
Fanelli D, Tesi M, Monaco D, Diaz-Jimenez M, Camillo F, Rota A, Panzani D.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, Gutiérrez V, López ME, Pérez 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/...
Jakop U, Müller K, Müller P, Neuhauser S, Callealta Rodríguez I, Grunewald S, Schiller J, Engel KM.On their way to the oocyte, sperm cells are subjected to oxidative stress, which may trigger the oxidation of phospholipids (PL). Applying MALDI-TOF MS, HPTLC and ESI-IT MS, we comparatively analyzed the PL compositions of semen and blood of species differing in their reproductive systems and types of nutrition (bull, boar, stallion, lion and man) with regard to the sensitivity to oxidation as well as the accumulation of harmful lyso-PL (LPL), transient products of lipid oxidation. In addition, the protective capacity of seminal fluid (SF) was also examined. The PL composition of erythrocytes ...
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...
Quiñones-Pérez C, Martínez 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...
Stelletta C, Alberti S, Cil B, Tekin K, Tirpan MB, Arganaraz M, Akcay E, Daskin A.The identification of various substances in seminal plasma has opened the way to study their functionality. It was aimed to identify the electrophoretic protein profile (EPP) and biochemical parameters (BP) of seminal plasma (SP) as predictors of semen quality and fertility in stallion. Forty-six ejaculates from 7 fertile stallions, aged between 6-26 years, were collected from May to July and 117 mares were used to obtain fertility data. For each ejaculate, volume, sperm motility, concentration were determined and seminal plasma samples were collected to perform one- -dimensional electrophores...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Arostegui LG, Rubio CP, Yeste M, Miró 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...
Monteiro RA, Cunha RM, Guerra MMP, de Almeida VM, Peña-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...
Lançoni R, Celeghini ECC, Gonella-Diaza AM, Júnior 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...
Fernández-Hernández P, García-Marín LJ, Bragado MJ, Domingo A, González-Fernández L, Macías-García B.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...
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 ...
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...
Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró 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...
Khanam A, Swelum AA, Khan FA.Pharmacologically induced ex copula ejaculation is a method used for collection of semen when the traditional methods of semen collection are not feasible. Common indications for this method include health issues that either preclude the physical act of mating or result in impaired erection and ejaculation. The method also offers an alternative when there is a lack of equipment and facilities required for semen collection using the conventional artificial vagina method. A variety of pharmacological protocols for ex copula ejaculation in stallions have been evaluated in both research and clinic...
Alim S, Cheppali SK, Laitaoja M, Talluri TR, Jänis J, Swamy MJ.Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the prot...
Moura TCM, Arruda LCP, Araújo 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...
Bugno-Poniewierska M, Kij-Mitka B, Podstawski Z, Długosz B, Lasek O, Mrowiec P, Tischner M.The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, u...
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 ...
Pruß 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...
Eterpi M, Magistrini M, Couty I, Gavin-Plagne L, Aguirre-Lavin T, Schmitt E, Carion O.Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes pr...
Ramirez-Perez H, Guerrero-Netro HM, Torres-Rodríguez P, Díaz-Durán M, Boeta-Acosta AM, Diaw M.The objective of this study was to evaluate the effects of caffeine and taurine on the motility and viability of chilled equine semen. Methods: A total of 12 ejaculates were collected from three mature stallions with proven fertility during the breeding season. The gel-free spermatic fraction of each ejaculate was divided into two aliquots and diluted with a semen extender (either INRA 96® or BotuSemen Gold®). The aliquots were then split and assigned to one of the six treatment groups: control (no supplement), caffeine (2 and 4 mM), taurine (25 and 50 mM), and a combination of caffeine (2 m...
Perrett J, Harris IT, Maddock C, Farnworth M, Pyatt AZ, Sumner RN.Over the past five decades, there has been increasing evidence to indicate global declines in human semen quality. Parallel adverse trends measured in male animals indicate a potential environmental aetiology. This study evaluated the progressive motility (PM) of stallion ejaculate through a systematic review and meta-analysis. A total of 696 estimates of equine PM from 280 studies, which collected semen samples between the years 1990 and 2018, were collated for meta-analysis. The method of motility analysis, breed, season of collection, and geographical location were extracted. Simple linear ...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, da Silva-Álvarez E, Gil MC, Ortega-Ferrusola C, Peña FJ.Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our lab...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, da Silva-Álvarez E, Masot J, Redondo E, Gil MC, Ortega-Ferrusola C, Peña FJ.Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm anal...
Al-Kass Z, Spergser J, Aurich C, Kuhl J, Schmidt K, Morrell JM.Bacteria contaminate semen during collection and handling. The objective of this study was to identify the bacteria in pony stallion semen, the effects of antibiotics included in commercial semen extenders (lincomycin and spectinomycin) and the effect of modified single layer centrifugation (MSLC), on bacterial load. Ejaculates from six pony stallions, 3 ejaculates per animal, were extended in EquiPlus extender either with or without antibiotics. Aliquots were processed by MSLC to form four treatment groups: control and MSLC with antibiotics (CA and SA, respectively) and control and MSLC witho...
Bucci D, Isani G, Spinaci M, Tamanini C, Mari G, Zambelli D, Galeati G.Spermatozoa, as other eukaryotic cells, need hexoses to produce energy to maintain membrane homeostasis, to move along the female genital tract and to carry the male genome to the female gamete. GLUTs are a family of proteins that permit and improve the passive transport of hexoses inside cells. This study was aimed at investigating the presence and localization of GLUTs 1, 2, 3 and 5 in boar, stallion and dog spermatozoa by both immunofluorescence and western blotting. GLUTs exhibited a peculiar distribution along the sperm cell depending on the isoforms considered, the hexose they transport ...
Jung H, Roser JF, Yoon M.Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of sp...
Pillet E, Duchamp G, Batellier F, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Hen egg yolk is normally used as a cryoprotective agent in semen freezing extenders, but its use has sanitary and practical disadvantages. Moreover the protection afforded by egg yolk has not yet been completely elucidated. The objective of this study was to compare the egg yolk plasma fraction to whole egg yolk in stallion freezing extender. Plasma contains mainly Low Density Lipoproteins (LDL), which are widely presumed to be the cryoprotective agent in egg yolk. Plasma can be produced on an industrial scale, sterilised by gamma-irradiation and incorporated in a ready-to-use extender (our ul...
Ortega Ferrusola C, González Fernández L, Macías García B, Salazar-Sandoval C, Morillo Rodríguez A, Rodríguez Martinez H, Tapia JA, Peña FJ.The ability of stallion spermatozoa to produce nitric oxide (NO) before (fresh) and after freezing and thawing (FT) was evaluated by means of flow cytometry after loading the sperm suspension with the probe, 4,5-diaminofluorescenin diacetate. The presence of NO synthase (NOS) was investigated by Western blotting using anti-NOS1, anti-NOS3, or anti-universal NOS antibodies (Abs). While NO was detected both in fresh and FT sperm suspensions, its production increased after cryopreservation only when egg yolk was removed from the extender. Anti-NOS1 Ab intensively labeled a single band with an app...
Mateo-Otero Y, Zambrano F, Catalán J, Sánchez R, Yeste M, Miro J, Fernandez-Fuertes B.In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. Objective: To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in respon...
Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F.In recent years, there has been an increased interest in new preservation techniques that facilitate sperm storage and distribution, with freeze-drying (FD) having been proposed as an alternative method for sperm preservation and maintenance of genetic resources in different animal species. FD is a method in which frozen material is dried by sublimation of ice, thereby involving a direct transition from a solid (ice) to a vapour (gas) phase. One of the main advantages of FD is that nitrogen and dry ice are no longer required for the storage and shipment of frozen sperm, which can be stored at ...
Love CC, Thompson JA, Brinsko SP, Rigby SL, Blanchard TL, Lowry VK, Varner DD.Relationships between sperm motility parameters and viability were evaluated using two fluorescent staining techniques in fresh extended semen (fresh and after 24 h storage at 5 degrees C) that had various concentrations of dead sperm added to simulate different levels of viable and nonviable sperm. Both protocols incorporated SYBR-14 and propidium iodide (PI) while the second protocol added the mitochondrial probe JC-1. The relationship between total sperm motility and percent viable sperm was high between staining protocols (r = 0.98). Time (0 h versus 24 h, P<0.0001) and treatment (0, 10, 2...
Jasko DJ, Little TV, Lein DH, Foote RH.Information pertaining to evaluation of single ejaculates of semen and records for 2 consecutive breeding seasons were obtained. In all, data for 99 individual breeding seasons (n = 43 Standardbreds and 56 Thoroughbreds) were evaluated. Included in each semen evaluation was examination of semen characteristics and computer-aided analysis of spermatozoal movement characteristics. On the basis of the analysis of breeding records for 4,175 mares (7,017 estrous cycles), a per-estrous cycle fertility rate was calculated from data for 96 of the breeding seasons. Stallions with lower fertility than t...
Turner RM, McDonnell SM.Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additio...
Bolaños JM, Morán AM, da Silva CM, Dávila MP, Muñoz PM, Aparicio IM, Tapia JA, Ferrusola CO, Peña FJ.To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the ...
Al-Kass Z, Guo Y, Vinnere Pettersson O, Niazi A, Morrell JM.Bacteria colonize stallion semen during collection and processing which may cause disease in inseminated females or negatively affect sperm quality during storage prior to insemination. Antibiotics are added to semen extenders to control the growth of these bacteria but may induce antimicrobial resistance. Research into alternatives to antibiotics for this purpose requires knowledge of which bacteria are present in semen. Not all bacteria in semen, however, can be identified by conventional microbiological techniques. The objectives of the study were to: i) determine which bacteria are present...
Martin Muñoz P, Ortega Ferrusola C, Vizuete G, Plaza Dávila M, Rodriguez Martinez H, Peña FJ.Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard tech...
Bubenickova F, Postlerova P, Simonik O, Sirohi J, Sichtar J.Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to hepar...
Trimeche A, Yvon JM, Vidament M, Palmer E, Magistrini M.The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Felix MR, Turner RM, Dobbie T, Hinrichs K.Methods for standard in vitro fertilization have been difficult to establish in the horse. We evaluated whether prolonged sperm pre-incubation would support subsequent fertilization. Fresh sperm were pre-incubated with penicillamine, hypotaurine, and epinephrine (PHE) for 22 h. Co-incubation of cumulus-oocyte complexes (COCs) for 6 h yielded 43% fertilization; culture of presumptive embryos yielded 21% blastocysts. Sperm incubated similarly, but without PHE, did not fertilize oocytes. Use of extended semen in the system yielded 54% blastocysts and was applied in subsequent experiments. Trans...
Kankofer M, Kolm G, Aurich J, Aurich C.Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the...
Talluri TR, Mal G, Ravi SK.To investigate various biochemical components of seminal plasma in Marwari stallions and Poitou Jacks and to find out their correlation with that of the seminal characteristics. Methods: In this study, semen was collected from six Marwari stallions and six Poitou jacks aged from 4 to 6 years and with known fertility status. The semen collection from the stallions were collected during the breeding season, i.e., between the months of April and June. From the collected semen ejaculates, we estimated the values of some biochemical components, viz., total protein content, total lipid content, and ...
Troedsson MH, Liu IK, Crabo BG.Following the deposition of semen in the mares uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mares reproductive tract. Fertilizable sperm are present in the oviduct within 4 hours after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that sperm...
Macías García B, Morrell JM, Ortega-Ferrusola C, González-Fernández L, Tapia JA, Rodriguez-Martínez H, Peña FJ.The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E). Sperm motility, sperm chromatin structure, membrane integrity and mitochondrial membrane potential were studied in filtered and non-filtered spermatozoa. Single-layer centrifugation (SLC) using Androcoll-E significantly improved all the sperm parameters studied, implying...
Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MH, Bischof JC, Roberts KP.Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm a...
van Eldik P, van der Waaij EH, Ducro B, Kooper AW, Stout TA, Colenbrander B.Inbreeding is widely believed to negatively affect reproductive performance. Indeed, in some species, high levels of inbreeding are thought to be the major cause of poor semen quality. It is, however, not clear whether inbreeding affects fertility in horses. In this study, the relationship between inbreeding and semen quality was examined in 285 immature Shetland pony stallions submitted for breeding soundness examination in March-April of the years 1992-1997. The majority of stallions examined were 3 years old (85%) and their coefficients of inbreeding ranged from 0 to 25% (mean+/-S.D.: 3+/-4...
Vilés K, Rabanal R, Rodríguez-Prado M, Miró J.Artificial insemination (AI) involving the placing of frozen-thawed semen directly into the jenny uterine body is associated with very low pregnancy rates. This might be because of an exacerbation of the acute response of the endometrium to sperm, as seen in mares with persistent induced mating endometritis. Pregnancy rates can be increased in such mares, however, by including anti-inflammatory treatments in the insemination protocol (Bucca S, Carli A, Buckley T, Dolci G, Fogarty U. The use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced e...
Blottner S, Warnke C, Tuchscherer A, Heinen V, Torner H.The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were eva...
Baumber J, Ball BA.To determine glutathione peroxidase (GPX) and superoxide dismutase (SOD)-like activities in spermatozoa, seminal plasma, and reproductive tissues (ie, testis, epididymis, bulbourethral gland, prostate, vesicular gland, and ampulla) in horses. Methods: Seminal plasma from 17 stallions, spermatozoa from 5 stallions, and reproductive tissues from 3 stallions. Methods: Activity of GPX was determined by use of assays measuring oxidation of NADPH in the presence of exogenous glutathione, cumene hydroperoxide, and glutathione reductase. Activity of SOD-like enzymes was determined by use of the nitrob...
Del Prete C, Stout T, Montagnaro S, Pagnini U, Uccello M, Florio P, Ciani F, Tafuri S, Palumbo V, Pasolini MP, Cocchia N, Henning H.During cold storage stallion spermatozoa experience undergo oxidative stress, which can impair sperm function and fertilizing capacity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the main endogenous enzymatic antioxidants in stallion seminal plasma, and counteract reactive oxygen species. Semen dilution reduces the endogenous antioxidant concentrations. The aim of this study was to investigate whether addition of 15 IU/mL each of SOD, CAT, and GPX to diluted stallion semen would ameliorate a reactive oxygen-mediated decrease in semen quality during 72 h o...
Sieme H, Martinsson G, Rauterberg H, Walter K, Aurich C, Petzoldt R, Klug E.The objective of this research was to improve the techniques in processing chilled and frozen-thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim-up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 ...
Calvete JJ, Raida M, Gentzel M, Urbanke C, Sanz L, Töpfer-Petersen E.In the bovine, seminal plasma heparin-binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin-like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin- and phosphorylcholine-binding proteins of stallion and boar seminal plasma. Horse seminal plasma proteins HSP-1 and HSP-2, and boar protein pB1, belong to the same family as the bull heparin- and phosphorylcholine-binding proteins BSP-A1/2, BSP-A3, and BSP-30K. We have determined the amino acid sequence and posttrans...
Pasing SS, Aurich C, von Lewinski M, Wulf M, Krüger M, Aurich JE.An important factor influencing stallion fertility is the microbial contamination of semen. Aims of this study were to investigate changes in the microbiological population of the genital mucosa and semen in artificial insemination stallions (n=16) from before to after one breeding season (February-August). MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) was used for identification of microbial agents. For bacteriology, swabs from the urethral opening, urethral fossa and penile sheath as well as semen were collected at 4-week-intervals. For semen mot...
Varner DD, Scanlan CM, Thompson JA, Brumbaugh GW, Blanchard TL, Carlton CM, Johnson L.Three experiments were conducted to evaluate the effects of different antibiotics in a milk-glucose semen extender on motility of equine sperm and elimination of bacteria following storage of extended semen in vitro. In Experiment 1, 7 antibiotics were compared: amikacin, gentamicin, streptomycin, potassium penicillin, sodium penicillin, ticarcillin, and polymixin B. In Experiment 2, 3 antibiotic treatments were compared: potassium penicillin G, amikacin, or a combination of potassium penicillin G and amikacin. In Experiment 3, 3 antibiotic treatments were compared: potassium penicillin G-amik...
