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.
Elkhawagah AR, Donato GG, Poletto M, Martino NA, Vincenti L, Conti L, Necchi D, Nervo T.This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated ...
Ren H, Wen X, He Q, Yi M, Dugarjaviin M, Bou G.The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins...
Fanelli D, Moroni R, Sala G, Melanie P, Tarabella I, Telleschi N, Maltinti S, Giorgi M, Barsotti G, Passamonti F, Marmorini P, Rota A, Camillo F....Two studies were conducted to evaluate the use of medical ozone (O) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent...
Ing NH, Konganti K, Ghaffar N, Johnson CD, Forrest DW, Love CC, Varner DD.Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs i...
Fuentes F, Contreras MJ, Arroyo-Salvo C, Cabrera P P, Silva M, Merino O, Arias ME, Felmer R.Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidi...
Peña FJ, Martín-Cano FE, Becerro-Rey L, Ortega-Ferrusola C, Gaitskell-Phillips G, da Silva-Álvarez E, Gil MC.We are currently experiencing a period of rapid advancement in various areas of science and technology. The integration of high throughput 'omics' techniques with advanced biostatistics, and the help of artificial intelligence, is significantly impacting our understanding of sperm biology. These advances will have an appreciable impact on the practice of reproductive medicine in horses. This article provides a brief overview of recent advances in the field of spermatology and how they are changing assessment of sperm quality. This article is written from the authors' perspective, using the sta...
Fracaro PL, Corcini CD, Souza Gatti NL, Filho JS, Acosta IB, Filho JS, Hartwig FP, Rosa Curcio BD, Varela Junior AS.Glycerol is a cryoprotectant widely used in the freezing of mammalian semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. Objective: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. Methods: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Anal...
Díaz Rojas E, Carrillo Moreno DI, Contreras Villarreal V, Arellano Rodríguez F, Alvarado Espino AS, Ángel García O.The use of reproductive biotechnologies in equine practice has shown that some stallions are subfertile, so ways to improve fertility have been sought. This study aimed to evaluate the effect of nutraceutical supplementation on improving semen quality in Quarter Horse stallions. Semen from six Quarter Horse stallions was assessed for 4 months every 20 days using the computer-assisted semen analysis system. They were evaluated for 60 days before supplementation; then, the same stallions were re-evaluated for 60 days with nutraceutical supplementation (30 g/day). Volume showed no significant...
Egyptien S, Deleuze S, Ledeck J, Ponthier J.Stallion sperm analysis is indicated for infertility diagnosis, pre-sale expertise, production of fresh or frozen doses, and frozen straw quality control. Various collection methods are described, and numerous assays can be performed on semen. Determining an approach for each of these cases is challenging. This review aims to discuss how to obtain relevant clinical results, answering stallion owners' concerns. Semen can be collected with an artificial vagina on a phantom or a mare, by electro-ejaculation under anesthesia, or after pharmacological induction. The collection method influences the...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Köhne M, Kirch F, Tönissen A, Martinsson G, Rabe U, Sieme H, Schuberth HJ.To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups...
Muñoz E, Castro M, Aguila L, Contreras MJ, Fuentes F, Arias ME, Felmer R.Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed ...
Mogielnicka-Brzozowska M, Woźniak MJ, Cichowska AW, Fraser L, Kraziński B, Strzezek R, Zielińska D.Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content ...
Gonçalves AR, Telo da Gama L, Antunes L, Guimarães H, Bliebernicht M, Duarte JC, Cosinha C, Duarte Rego B, Ferro da Costa P, Guimarães T, Rocha A....The objectives of this study were to establish baseline information for seminal traits in Lusitano stallions, to assess the impact of inbreeding, interval between collections and age on semen quality during the breeding and non-breeding seasons, and to estimate the corresponding genetic parameters. A total of 2129 ejaculates by 146 Lusitano stallions used for artificial insemination, obtained from four equine reproduction centers distributed throughout Portugal, over a period of 14 years (2008-2021), were included in the study. The seminal traits analyzed, and the corresponding means and stand...
Miller LMJ, Woodward EM, Campos JR, Squires EL, Troedsson MHT.Sperm protein at 22 kDa has been associated with fertility. Objective: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration. Methods: Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. Results: Histopathology confirmed ...
Medica AJ, Lambourne S, Aitken RJ.This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cell...
Silva GF, Cunha R, Carvalho F, Ribeiro M, Rocha A, Amorim I, Guimarães T.A 30-year-old Lusitano stallion presented with an enlarged right epididymis. The ultrasound scan revealed a cyst-like formation and the histopathological examination was compatible with epididymal cyst located at the body/tail transition, epididymal spermatocele and sperm granuloma and epididymitis. However, these conditions did not seem to affect the animal's reproductive performance, nor did the semen parameters analyzed over the 8 years after the diagnosis show significant changes. Nevertheless, since the ejaculate contains mostly sperm cells from the tail of the epididymis, where fertile s...
Jhamb D, Talluri TR, Sharma S, Juneja R, Nirwan SS, Yadav D, Pargi KK, Tanwar A, Kumar P, Kumar R, Mehta SC, Parashar M, Gaur M.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing...
Hensel B, Jakop U, Schmicke M, Schröter F, Jung M, Schulze M.Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions with...
Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Beckham AMN, Varner DD, Love CC.Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High...
Hatami M, Qasemi-Panahi B, Daghigh Kia H, Moghaddam G, Janmohammadi H.Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, β-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β-carotene. Subse...
Freitas ML, Viana JHM, Dode MAN, Maggiotto SR, Pivato I, Braga TRC, Lim AIPG, de Oliveira RA.The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a lati...
Al-Kass Z, Ntallaris T, Morrell JM, Johannisson A.Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sc...
Assumpção TI, Lançoni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Aparicio IM, Rojo-Domínguez P, Castillejo-Rufo A, Peña FJ, Tapia JA.Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In so...
Prasinou P, De Amicis I, Fusaro I, Bucci R, Cavallini D, Parrillo S, Caputo M, Gramenzi A, Carluccio A.Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid cont...
Vigolo V, Gautier C, Falomo ME, Aurich C.The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen-thawed semen from 15 stallions that were...
Ferrer MS, Hurley DJ, Norton N, Ellerbrock RE.The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 10 PMN/ml, 3) 0.5 × 10 PMN/ml, 4) 2.5 × 10 PMN/ml, 5) 5 × 10 PMN/ml, 6) 10 × 10 PMN...
Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC.The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 ...
Aguiar CS, Barros CHSC, Machado WM, Allaman IB, Leite AO, Barbosa LP, Snoeck PPDN.The aim of this study was to evaluate the association of different concentrations of Trolox and the addition of a fixed concentration of DHA in the freezing of semen of Mangalarga Marchador stallions. To that end, 16 ejaculates were frozen in the following extenders: E1) BotuCrio (BC; Control); E2) BC + 50 ngml DHA + 30 µM Trolox (BCDHA30T); E3) BC + 50 ngml DHA + 40 µM Trolox (BCDHA40T); E4) BC + 50 ngml DHA + 50 µM Trolox (BCDHA50T). All the tested extenders were similar in preserving different kinematic parameters, cell functional integrity, compacted DNA, and high and intermediate mitoc...
Troedsson MH.Endometritis is a common cause of infertility in broodmares. In the past, the condition was believed to be exclusively the result of bacterial contamination of the uterus. Treatment strategies were focused on preventing bacteria from entering the uterus and on treating mares with signs of endometritis with antibiotics. More recent research on uterine defense mechanisms has increased our understanding of the pathophysiology of equine endometritis. Additional causative agents have been identified, and we have learned to separate uterine infections and a physiologic breeding-induced endometritis ...
Timoney PJ, McCollum WH, Murphy TW, Roberts AW, Willard JG, Carswell GD.The carrier state has been confirmed virologically in Thoroughbred and non-Thoroughbred stallions naturally infected with equine arteritis virus (EAV). Short-term or convalescent and long-term carriers occur. The frequency rate of the long-term carrier state in Thoroughbreds was high, averaging 33.9% among the three groups of stallions under study. While the convalescent carrier state only lasted a few weeks after clinical recovery, the long-term carrier state could persist for years. There was evidence, however, that not all such carriers might remain persistently infected for life. Carrier s...
Morrell JM, Johannisson A, Dalin AM, Hammar L, Sandebert T, Rodriguez-Martinez H.Artificial insemination is not as widely used in horses as in other domestic species, such as dairy cattle and pigs, partly because of the wide variation in sperm quality between stallion ejaculates and partly due to decreased fertility following the use of cooled transported spermatozoa. Furthermore, predictive tests for sperm fertilising ability are lacking. The objective of the present study was to assess sperm morphology and chromatin integrity in ejaculates obtained from 11 warmblood breeding stallions in Sweden, and to evaluate the relationship of these parameters to pregnancy rates to i...
Adams GP, Ratto MH.Ovulation in mammals involves pulsatile release of GnRH from the hypothalamus into the hypophyseal portal system with subsequent release of LH from the anterior pituitary into systemic circulation. Elevated circulating concentrations of LH induce a cascade of events within the mature follicle, culminating in follicle rupture and evacuation. The broad classification of species as either spontaneous or induced ovulators is based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus. In spontaneously ovulating species (e.g., human, sheep, cattle, horse, pigs), relea...
Cocchia N, Pasolini MP, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G, Mancini A.New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: ...
Ortiz-Rodriguez JM, Anel-Lopez L, Martín-Muñoz P, Álvarez M, Gaitskell-Phillips G, Anel L, Rodríguez-Medina P, Peña FJ, Ortega Ferrusola C.Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values ...
Loomis PR.Clinical reproduction in the horse more closely parallels human clinical reproduction than in other domestic farm animals. Horse breeders rarely include fertility as a selection criterion when making mating decisions; in most breeds, there is no licensing or approval of stallions. This has led to a significant number of stallions in the breeding pool that possess desirable performance traits but are subfertile for a variety of reasons, some of them genetically transmitted between generations. Therefore, semen characteristics can vary greatly among stallions within the breeding population. A ch...
Stradaioli G, Sylla L, Zelli R, Chiodi P, Monaci M.The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen ev...
Vidament M, Vincent P, Martin FX, Magistrini M, Blesbois E.A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and...
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...
Janett F, Stump R, Burger D, Thun R.The aim of this study was to evaluate the effects of an anti-GnRH vaccine on testosterone concentration, antibody titer, scrotal width, semen quality and sexual behavior in the stallion. Adverse reactions to the vaccine were also determined. Eight clinically healthy sexually experienced stallions aged between 6 and 15 years from the National Stud in Avenches (Switzerland) were used. Five stallions were immunized 3 times at an interval of 4 and 8 weeks, respectively, with 200 microg of a GnRH-protein conjugate (Equity, CSL Limited, Australia) intramuscularly in the neck and 3 control animals re...
Al-Essawe EM, Wallgren M, Wulf M, Aurich C, Macías-García B, Sjunnesson Y, Morrell JM.Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from "good" (GF) or "bad" (BF) freezer stallions on sperm cells' fertilizing ability. "Good freezers" refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst "bad freezer" stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the bind...
Höfner L, Luther AM, Waberski D.Ongoing progress in proteomic characterization of seminal plasma has stimulated research on the identification of biomarkers for male fertility and sperm preservability. So far, many studies have evaluated the benefits of reconstituting cryopreserved or sex-sorted semen with seminal plasma. Less information is available about the effect of remaining or added seminal plasma in liquid preserved semen. The interaction between seminal plasma and spermatozoa is species -specific, and within species often complex and ambiguous. This article aims to review the action of seminal plasma on sperm functi...
Hoogewijs MK, de Vliegher SP, Govaere JL, de Schauwer C, de Kruif A, van Soom A.Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objective: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and re...
Swegen A, Lambourne SR, Aitken RJ, Gibb Z.Media used for equine sperm storage often contain relatively high concentrations of glucose, even though stallion spermatozoa preferentially utilize oxidative phosphorylation (OXPHOS) over glycolysis to generate ATP and support motility. Rosiglitazone is an antidiabetic compound that enhances metabolic flexibility and glucose utilization in various cell types, but its effects on sperm metabolism are unknown. This study investigated the effects of rosiglitazone on stallion sperm function in vitro, along with the possible role of AMP-activated protein kinase (AMPK) in mediating these effects. Sp...
Barrier Battut I, Kempfer A, Becker J, Lebailly L, Camugli S, Chevrier L.Several laboratories routinely use flow cytometry to evaluate stallion semen quality. However, objective and practical tools for the on-field interpretation of data concerning fertilizing potential are scarce. A panel of nine tests, evaluating a large number of compartments or functions of the spermatozoa: motility, morphology, viability, mitochondrial activity, oxidation level, acrosome integrity, DNA integrity, "organization" of the plasma membrane, and hypoosmotic resistance, was applied to a population of 43 stallions, 33 of which showing widely differing fertilities (19%-84% pregnancy rat...
Miró J, Lobo V, Quintero-Moreno A, Medrano A, Peña A, Rigau T.The Sperm-Class Analyzer detected four subpopulations of spermatozoa with different motility characteristics in the ejaculate of the Catalonian donkey. Significant differences (P < 0.001) in the distribution of these subpopulations, as well as in total sperm number and percentage total motility, were seen in the diluted semen of four sampled donkeys. All the ejaculates evaluated showed excellent semen quality characteristics; the sperm they contained was more rapid than horse sperm. Principal components analysis showed sperm l-lactate production to be a good predictor of semen condition. This,...
Peña FJ, García BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO.We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.
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 ...
Aurich C.The spermatozoon is a highly specified cell that has the abilities of active motility and fertilization of the ovum. Damage to the sperm plasma membrane results in the irreversible loss of its functions. Because of the high content of unsaturated fatty acids in the plasma membrane, mammalian sperm are sensitive to oxidative stress. While mild peroxidation appears to promote capacitation of the sperm cell, excessive peroxidation will damage the plasma membrane and results in loss of motility and fertility. The functional integrity of the sperm plasma membrane can be determined by functional tes...
Garner DL, Evans KM, Seidel GE.The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some specie...
Arienti G, Carlini E, De Cosmo AM, Di Profio P, Palmerini CA.Human semen contains membranous vesicles called prostasomes. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin, and Ca2+. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. No prostasome-like vesicles have been described in horse semen up to now. We have demonstrated the presence of prostasome-like vesicles in the equine semen and characterized them as to size, morphology, and lipid composition; we have found that they are similar to human prostasomes in many respects. We prop...
Hoffmann B, Landeck A.To gain further information on gonadal function of the stallion, concentrations of testicular steroids in blood plasma (bpl) and seminal plasma (spl) and their distribution in the ejaculate were determined. Blood and semen samples from a total of 11 stallions were collected from November to July. Estrone (E1), estrone sulfate (E1S), estradiol-17beta (E2beta) and testosterone (T) were determined in bpl and spl, and in addition androstenedione (A), dehydroepiandrosterone (DHEA) and 5alpha-dihydrotestosterone (5alpha-DHT) were measured in spl. At certain points of time, aliquots of an ejaculate w...
Córdova A, Strobel P, Vallejo A, Valenzuela P, Ulloa O, Burgos RA, Menarim B, Rodríguez-Gil JE, Ratto M, Ramírez-Reveco A.This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
Contri A, De Amicis I, Molinari A, Faustini M, Gramenzi A, Robbe D, Carluccio A.In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after suppl...
Varela E, Rey J, Plaza E, Muñoz de Propios P, Ortiz-Rodríguez JM, Álvarez M, Anel-López L, Anel L, De Paz P, Gil MC, Morrell JM....Contaminating bacteria present in stallion ejaculates may compromise sperm quality during storage. Different procedures have been used to reduce the load of microorganisms in semen and avoid bacterial growth during storage. The aims of this study were: 1) to evaluate different techniques to eliminate bacteria in semen 2) to study the relationship between total microflora load (TML) and ROS production; and 3) to determine if TML affects the functionality of cool-stored sperm. Ejaculates from 11 stallions were split and processed in 3 ways: A. extended semen; B. conventional centrifuged semen, a...
Kareskoski M, Katila T.Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.
Oldenhof H, Wolkers WF, Sieme H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
Neild D, Chaves G, Flores M, Mora N, Beconi M, Agüero A.The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and ...
