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.
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...
Pereira RR, Nogueira BG, Milan B, Acacio BR, Freitas-Dell'Aqua CP, Souza MI, Sampaio BF.The aim of this study was to determine an ozone dosage capable of inducing pro-oxidation, and to verify its action on sperm cells during the process of cooling and cryopreservation of equine semen. In this study, we evaluated the ozone concentrations of 2µg/mL,15µg/mL, 30µg/mL e 60 µg/mL added in equine semen cooling and freezing extenders. Samples were evaluated for sperm kinetics patterns, function of sperm structures and lipid peroxidation. In the experiment, the concentration of 15 µg/mL showed higher total and progressive motility when comparing to control (60.3±3 and 40.7±3.4 vs. ...
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...
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-...
Wiebke M, Hensel B, Nitsche-Melkus E, Jung M, Schulze M.This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques for cooled storage of semen from livestock animals. The first part describes the current state of the art of liquid semen preservation in boars, bulls, and stallions, including the diluents, use of additives, processing, temperature, and cooling of semen. The species-specific physiology and varying extents of cold shock sensitivity are taken into consideration. In addition, factors influencing the quality of cooled-stored semen are discussed. Methods, trends, and the most recent a...
Medica AJ, Aitken RJ, Nicolson GL, Sheridan AR, Swegen A, De Iuliis GN, Gibb Z.Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stall...
Blommaert D, Sergeant N, Delehedde M, Donnay I, Lejeune JP, Franck T, Serteyn D.The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant di...
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...
Ferrer MS, Canisso IF, Podico G, Ellerbrock RE, Hurley DJ, Palomares R.Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen usi...
Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H.Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spe...
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...
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...
Pruß D, Yang H, Luo X, Liu D, Hegermann J, Wolkers WF, Sieme H, Oldenhof H.Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined si...
Miró J, Catalán J, Marín H, Yánez-Ortiz I, Yeste M.While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. ...
Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips GL, Silva A, Ortega-Ferrusola C, Gil MC, Peña FJ.Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were pr...
Bazzano M, Laus F, Spaterna A, Marchegiani A.Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from differ...
Lançoni R, Celeghini ECC, Giuli V, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batc...
Tongu EAO, Segabinazzi LGTM, Alvarenga ML, Monteiro A, Papa FO, Alvarenga MA.This study aimed to evaluate the effects of mesenchymal stem cell-conditioned medium (MSC-CM) on sperm parameters, intrauterine polymorphonuclear neutrophils (PMN), intrauterine fluid accumulation (IUF), and fertility in mares. In experiment 1, two ejaculates from ten stallions were extended to 50 million sperm/mL using a milk-based extender. Thereafter, 20 mL of extended semen was added of MSC-CM as follows: 0, 5, 10, 15, and 20 mL. Sperm kinetics and plasma membrane integrity were evaluated immediately after dilution (T0) and 2 h post-incubation at 37 °C (T2). In experiment 2, mares cha...
Olğaç KT, Akçay E.In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared w...
Al-Kass Z, Brown A, Johannisson A, Ntallaris T, Morrell JM.Although single layer centrifugation (SLC) selects robust spermatozoa from stallion semen, the effect of individual variation has not been studied in detail. The objective of this study was to determine the variation among stallions in the effects of SLC on sperm quality during cooled storage for up to 48 hr. Semen samples from seven stallions (18 ejaculates) were split, with one portion being used for SLC and the other serving as a control (CON). Sperm quality (kinematics, reactive oxygen species (ROS) production, membrane integrity (MI) and chromatin integrity) were analysed at 0, 24 and 48...
Ortiz-Rodriguez JM, Nerozzi C, Bucci D, Mislei B, Mari G, Tamanini C, Peña FJ, Spinaci M, Galeati G.Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfas...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a spli...
Li N, Yu J, Yang F, Shao Y, Wu S, Liu B, Li M, Wang T, Li J, Zeng S.The aim of this study was to evaluate the effects of L-proline on the extender quality of frozen and post-thawed jackass semen. Jackass (n = 6) semen samples were collected and cryopreserved in gradient concentrations (0-80 mM) of L-proline in extenders; post-thawed semen samples were cultured in L-proline medium for 10 hours at 37°C. For cryopreservation experiment I, the motile parameters, mitochondrial membrane potential (MMP), and plasma membrane, acrosome, and chromatin structure integrities of post-thawed semen were assessed. For culture experiment II, additional ROS contents were ana...
Kareskoski M, Vakkamäki J, Laukkanen K, Palviainen M, Johannisson A, Katila T.Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of...
Yang F, Li N, Liu B, Yu J, Wu S, Zhang R, Yang W, Ji C, Sun Q, Ma J, Li M, Zhou J, Zhou X, Pietrani M, Losinno L, Zeng S.With the expansion of the donkey industry, timed artificial insemination (TAI) is becoming increasingly important in the reproductive management of jennies, however, TAI has not been widely investigated in donkeys. Objective: To develop efficient TAI protocols for cooled or frozen semen in jennies, based around ovulation induction with a GnRH analogue. Methods: Experimental exploratory study. Results: In experiment 1, the effects of different GnRH analogue (deslorelin) doses, follicle diameter (FD) at induction, repeated use of a GnRH analogue, and the influence of season on induction efficien...
Pasch L, Schmidt A, King W.The use of autologous blood processing tools including platelet-rich plasma (PRP) devices is increasingly widespread in veterinary medicine. In equine reproduction, a number of studies have explored the effects of intrauterine infusion of PRP on persistent mating-induced endometritis. Artificial insemination with thawed frozen semen incites an intrauterine inflammatory response and we sought to extend the applications of intrauterine PRP to normal mares being inseminated with frozen semen. We investigated a subset of our normal breeding population to observe the clinical effects of prebreeding...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh ...
Lüttgenau J, Imboden I, Wellnitz O, Romer R, Scaravaggi I, Neves AP, Borel N, Bruckmaier RM, Janett F, Bollwein H.Persistent breeding-induced endometritis (PBIE) is a leading cause of infertility in mares. The objective of the study was to assess genital perfusion and endometrial gene expression of inflammatory cytokines in mares classified as susceptible (n = 5) or resistant (n = 5) to PBIE. Ten mares were examined daily during estrus until 6 d after hCG-induced ovulation for two estrous cycles. Twenty-four hours after application of 1500 IU hCG, 4 mL of killed (by repeated freezing in liquid nitrogen and thawing at 50 °C) deep-frozen semen or sterile saline was instilled into the uterine body an...
Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we e...
Papin J, Stuhtmann G, Martinsson G, Sieme H, Lundeheim N, Ntallaris T, Morrell JM.Removal of seminal plasma facilitates stallion sperm survival during storage, but washing may damage sperm chromatin. Therefore, sperm quality was compared in samples following single-layer centrifugation (SLC) or sperm washing and controls (extension only) in two extenders, INRA82 and INRA96. Ejaculates from six stallions were split among six treatments: SLC, sperm washing, and controls, in INRA82 and INRA96. Sperm motility and acrosome status were evaluated at 0, 24, 48, 72 and 96 hours; morphology at 0, 24, 48, 72 hours and chromatin integrity at 0 and 96 hours, with storage at 6°C. Sperm ...
Janett F, Thun R, Niederer K, Burger D, Hässig M.The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawe...
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...
Ortiz-Rodriguez JM, Ortega-Ferrusola C, Gil MC, Martín-Cano FE, Gaitskell-Phillips G, Rodríguez-Martínez H, Hinrichs K, Álvarez-Barrientos A....Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-...
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...
Sieme H, Harrison RA, Petrunkina AM.Success in cryopreserving stallion semen has been very variable. Several different freezing regimes have been published. However, because extenders and procedures used in each regime have differed, direct comparison of these techniques has been very difficult, and controlled studies comparing different techniques have not been reported. A number of different factors affect sperm cryosurvival. In this article we review briefly current cryopreservation procedures for stallion semen, and then in more detail cryobiological determinants of sperm function, and mechanisms of cryoinjury and cryoprotec...
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 ...
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...
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...
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...
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...
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...
Baumber J, Ball BA, Linfor JJ.To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. Methods: 2 ejaculates collected from each of 5 stallions. Methods: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motili...
Schulman ML, May CE, Keys B, Guthrie AJ.Recent CEM outbreak reports reflect a novel epidemiologic manifestation with a markedly different risk association for transmission via artificial reproduction and subsequent to inadvertent importation of unapparent carrier stallions. Artificial breeding has an increased association with horizontal or fomite-associated transmission. Reported risk factors include inadequate biosecurity protocols at centralised breeding facilities associated with stallion management and methods of semen collection, processing and transport. Detection of carriers is based on traditional bacteriology from genital ...
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...
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...
Medica AJ, Aitken RJ, Nicolson GL, Sheridan AR, Swegen A, De Iuliis GN, Gibb Z.Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stall...
Muñoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Peña FJ.Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the prese...
Carneiro JAM, Canisso IF, Bandeira RS, Scheeren VFC, Freitas-Dell'Aqua CP, Alvarenga MA, Papa FO, Dell'Aqua JA.This study aimed to evaluate the antioxidant properties of coenzyme Q10 (CoQ10) during cryopreservation of semen obtained from stallions having good and bad semen freezing ability (GFA vs. BFA, respectively). Forty ejaculates (n = 20 stallions) were split into five centrifugation and five freezing extenders containing different concentrations of CoQ10 (0, 25, 50, 75 and 100 μmols/L). If CoQ10 was added to the centrifugation extender, the freezing extender had no CoQ10 added; similarly, if CoQ10 was added to the freezing extender, the centrifugation extender had no CoQ10. Semen cryoprese...
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 ...
Spizziri BE, Fox MH, Bruemmer JE, Squires EL, Graham JK.Irreversible damage occurs to spermatozoal membranes, during the phase transition, when spermatozoa are cooled from room temperature to 5 degrees C. Some of this damage can be ameliorated by adding cholesterol to the membrane, thereby altering membrane lipid composition. Adding cholesterol-loaded cyclodextrins (CLCs) to stallion spermatozoa prior to freezing, increases cell cryosurvival. However, the fertilizing potential of CLC-treated stallion spermatozoa is unknown. To address this, experiments were conducted which evaluated the ability of CLC-treated stallion spermatozoa to capacitate, acr...
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 ...
Squires E.Numerous reproductive technologies have been developed in the past several decades, which have dramatically changed the way mares are bred. This review will focus on embryo recovery and transfer, cooled-shipped embryos, embryo freezing, oocyte freezing, oocyte collection and transfer, intracytoplasmic sperm injection (ICSI), and sexed semen. Embryo transfer procedures have been constant for many years and the costs have not changed. The major change has been the ability to store embryos at 5 C for 12-24 hours and transport them to recipient stations. Embryo freezing has become more common us...
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...
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...
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...
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
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...
