Semen analysis involves the evaluation of semen quality to assess reproductive potential and health in horses. This process typically includes the examination of several parameters such as sperm concentration, motility, morphology, and volume. Semen analysis is used in breeding programs to determine the fertility of stallions and to identify any reproductive issues. The collected data can assist in making informed decisions about breeding management and interventions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, findings, and implications of semen analysis in equine reproduction.
Bulkeley E, Santistevan AC, Varner D, Meyers S.Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been deba...
Shakeel M, Jung H, Yoon D, Yoon M.The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understa...
Orsolini MF, Verstraete MH, van Heule M, Orellana D, Ortega A, Meyers S, Dini P.Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there ...
Yang F, Wu S, Yang W, Yu J, Liu B, Zeng S.To study the effects of exercise on donkey semen parameters, Dezhou donkey (Equus asinus) jackasses (n = 18) were assigned to three groups: control (CN, n = 6), 1-hour exercise (1 h EX, n = 6) and 2-hour exercise (2 h EX, n = 6) groups. They were exercised with a walking machine for 3 days/week for 8 weeks. Semen parameters and reproductive hormones were evaluated weekly. The heart rate, rectal temperature, blood haematology indexes, and the body condition score (BCS) were also measured. Results showed that the ejaculation volume increased while the sperm concentration decreased after ...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Aróstegui LG, Rubio CP, Barranco I, Yeste M, Miró 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, Acácio 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...
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...
Talluri TR, Kumaresan A, Paul N, Sinha MK, Ebenezer Samuel King JP, Elango K, Sharma A, Raval K, Legha RA, Pal Y.Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility ( = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low-...
Da Silva-Álvarez E, Gaitskell-Phillips G, Ortiz-Rodríguez JM, Serres C, García-Rodríguez B, Gutiérrez-Cepeda L, Martín-Cano FE, Echegaray A....This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n ...
Donatsch L, Friker B, Sieme H, Kaeser R, Burger D.A potential source of fertility loss in mares is oviductal dysfunction, potentially caused by masses or debris in the lumen, that may prevent either sperm from reaching the fertilization site or the embryo from reaching the uterus. Recently a novel therapeutic method leading to increased pregnancy results was described by infusing misoprostol, a synthetic prostaglandin E, in the uterus of mares with unexplained fertility problems. In this study, we aimed, after examining the compatibility of misoprostol with semen, to evaluate the pregnancy rate after routine preovulatory deep uterine horn app...
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...
Griffin RA, Swegen A, Baker MA, Ogle RA, Smith N, Aitken RJ, Skerrett-Byrne DA, Fair S, Gibb Z.Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercia...
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 ...
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...
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...
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 ...
Gmel AI, Burger D, Neuditschko M.The use of frozen-thawed semen is an important reproduction tool to preserve the biodiversity of small, native horse breeds such as the Franches-Montagnes (FM). However, not all stallions produce cryotolerant semen with a progressive motility after thaw ≥ 35%. To improve our understanding of the genetic background of male fertility traits in both fresh and frozen-thawed semen, we performed genome-wide association studies (GWAS) on gel-free volume, sperm cell concentration, total sperm count, and progressive motility in fresh and frozen-thawed semen from 109 FM stallions using 335,494 genome-...
Jung H, Yoon M.The production of donor-derived sperm using spermatogonial stem cell transplantation has been studied in various animals including mice, rats, goats, boar, dogs, sheep, and monkeys. However, germ cell transplantation has not been applied in stallions. The objective of this study was to produce donor germ cell-derived sperm using germ cell transplantation in stallions. Donor germ cells were transplanted into the parenchyma of 3 recipient stallions that had been treated with busulfan IV injections of 15 mg/kg body weight. For the preparation of donor single germ cells, tissue (20 g) from each te...
Segabinazzi LGTM, Scheeren VFDC, Freitas-Dell'Aqua CP, Papa FO, Alvarenga MA, Dell'Aqua JA, Canisso IF.The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with t...
Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS.The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using com...
Mahé C, Zlotkowska AM, Reynaud K, Tsikis G, Mermillod P, Druart X, Schoen J, Saint-Dizier M.In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered...
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...
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...
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 ...
Papas M, Catalán J, Fernandez-Fuertes B, Arroyo L, Bassols A, Miró J, Yeste M.While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. T...
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...
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...
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...
Kavak A, Lundeheim N, Aidnik M, Einarsson S.The standard procedure for assessing the breeding potential of a stallion includes the parameter total number of spermatozoa classified as morphologically normal. This study investigated sperm morphology of fresh semen in randomly chosen Estonian (E, n = 8) and Tori (T, n = 7) breed stallions with proven fertility. Two ejaculates were examined from each stallion. An aliquot from each ejaculate was fixed in 1 mL formol-saline immediately after collection and examined with phase-contrast microscope at a magnification 1000x for all types of morphological abnormalities. Furthermore smears were pre...
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...
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...
Moraes CR, Runcan EE, Blawut B, Coutinho da Silva MA.The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen anal...
Töpfer-Petersen E, Ekhlasi-Hundrieser M, Kirchhoff C, Leeb T, Sieme H.Seminal plasma proteins are secretory proteins originating mainly from the epididymis and the accessory sex glands. They are involved in the remodelling of the sperm surface which occurs during sperm transit through the male genital tract and continues later at ejaculation. During this process, collectively called post-testicular sperm maturation, the spermatozoa acquire the ability to fertilise an egg. Seminal plasma proteins have been shown to contribute to early and central steps of the fertilisation sequence, e.g. the establishment of the oviductal sperm reservoir, modulation of capacitati...
Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Hidalgo M, Rodríguez I, Dorado J, Soler C.This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.
Gacem S, Valverde A, Catalán J, Yánez Ortiz I, Soler C, Miró J.This study aimed to characterize the sperm kinematic values with high frames per second, to define the subpopulation structure of a horse and a donkey and compare them. A total of 57 fresh semen ejaculates (26 Spanish and 16 Arabian horse breeds and 10 donkeys) were collected and subsequently analyzed for kinematic parameters using the Computer-aided sperm motility analysis ISAS®v1.2 system and using a Spermtrack® 10-μm depth counting chamber. Sequences were recorded at 250 frames per second, and eight kinematic parameters were automatically evaluated. All kinematic parameters showed signif...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Dorado J, Acha D, Ortiz I, Gálvez MJ, Carrasco JJ, Gómez-Arrones V, Calero-Carretero R, Hidalgo M.The main aim of this study was to evaluate the efficacy of two commercially available liquid stallion semen extenders for the preservation of Andalusian donkey semen at 5°C for up to 72h, and to evaluate the effect of amino acid addition on sperm quality of cooled donkey semen. In addition, this study investigated the effect of seasons on semen characteristics of Andalusian jackasses. Throughout a year, 50 ejaculates were collected from ten adult donkeys and a complete semen evaluation was performed immediately after collection. In Experiment 1, semen samples (n=32) were pooled, divided into ...
Brinsko SP, Love CC, Bauer JE, Macpherson ML, Varner DD.Semen samples were collected from six fertile stallions and seven stallions with unexplained infertility. Percentages of motile sperm (77.5 +/- 11.3 versus 67.5 +/- 12.2, P = 0.2), and progressively motile sperm (70.8 +/- 13.6 versus 60.7 +/- 14.0, P = 0.2) were similar between fertile and subfertile stallions, respectively. Morphologic characteristics in ejaculates of control and affected stallions (% normal: 60.2 +/- 18.2 versus 52.9 +/- 11.3, P = 0.4; % abnormal heads 7.3 +/- 4.8 versus 12.1 +/- 5.0, P = 0.11; and % abnormal acrosomes 1.6 +/- 2.1 versus 3.0 +/- 3.4, P = 0.4) did not differ....
Guasti PN, Souza FF, Scott C, Papa PM, Camargo LS, Schmith RA, Monteiro GA, Hartwig FP, Papa FO.During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 μg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by...
Pagl R, Aurich JE, Müller-Schlösser F, Kankofer M, Aurich C.A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen w...
Varner DD, Gibb Z, Aitken RJ.Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermato...
Peña FJ, Gibb Z.The growing understanding of the mechanisms regulating redox homeostasis in the stallion spermatozoa, together with its interactions with energetic metabolism, is providing new clues applicable to the improvement of sperm conservation in horses. Based on this knowledge, new extenders, adapted to the biology of the stallion spermatozoa, are expected to be developed in the near future. The preservation of semen either by refrigeration or cryopreservation is a principal component of most animal breeding industries. Although this procedure has been successful in many species, in others, substantia...
Li CX, Chang WS, Mitsakos K, Rodger J, Holmes EC, Hudson BJ.Papillomaviruses (PVs) have been identified in a wide range of animal species and are associated with a variety of disease syndromes including classical papillomatosis, aural plaques, and genital papillomas. In horses, 13 PVs have been described to date, falling into six genera. Using total RNA sequencing (meta-transcriptomics) we identified a novel equine papillomavirus in semen taken from a thoroughbred stallion suffering a genital lesion, which was confirmed by nested RT-PCR. We designate this novel virus (EcPV9). The complete 7656 bp genome of EcPV9 exhibited similar characteristics to th...
Aurich C, Ortega Ferrusola C, Peña Vega FJ, Schrammel N, Morcuende D, Aurich J.Spermatozoa contain polyunsaturated fatty acids (PUFA). Cryopreservation damages sperm membranes and they become less functional after thawing. We analysed the lipid composition of spermatozoa from Shetland stallions (n = 15) collected monthly from January to June and hypothesized that sperm lipid patterns change with season. In addition, one ejaculate per month was submitted to cryopreservation. Content of saturated palmytic and stearic acid decreased from January to March (p < 0.001) while content of the PUFA docosapentaenoic (p < 0.001) and arachidonic acid (p < 0.05) and total PUFA...
Restrepo G, Rojano B, Usuga A.Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was ident...
Loomis PR.Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major imped...
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...
Garner DL, Pinkel D, Johnson LA, Pace MM.Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Serafini R, Longobardi V, Spadetta M, Neri D, Ariota B, Gasparrini B, Di Palo R.Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inse...
Gacem S, Papas M, Catalan J, Miró J.The accessory sex glands play a major role in the production of seminal plasma, and testicular artery blood flow seems to strongly influence testicular function. However, very little ultrasound imaging of these organs has been undertaken in donkeys. The present work reports the results of such examinations in five jackasses along the year. The accessory glands were inspected by B-mode ultrasound while the testicular artery blood flow was assessed by colour pulsed-wave Doppler ultrasound. The testicular artery was examined at pampiniform plexus (PPT), supratesticular area (ST) and capsular arte...
