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.
Nishikawa Y.Retention of high motility of horse spermatozoa preserved at 4 degrees C was improved by a semen extender. Raw semen preserved for 2 to 8 hr at 4 degrees C gave an average conception rate of 67-3% but preservation for 1 to 2 days gave an extremely low conception rate. The conception rate from deep-frozen semen during 8 years was 56-3%.
Lowe JE, Baldwin BH, Foote RH, Hillman RB, Kallefelz FA.Three Quarter-horse stallions were surgically thyroidectomized at 18 months of age. Weekly semen collections were made from these and three control stallions from 25 to 39 months of age. Although the induced state of hypothyroidism in young stallions has a depressing effect on libido and general animation and can be corrected by thyroprotein supplementation, semen characteristics, testicular histology and fertility are not affected by the operation.
Swierstra EE, Pickett BW, Gebauer MR.The cycle of the seminiferous epithelium was divided into eight stages on the basis of meiotic divisions, shape of the spermatid nuclei and location of the spermatids with elongated nuclei. Duration of this cycle was 12-2 days (S.E. +/- 0-1) as determined by [3H]thymidine injections and autoradiography. The life-span of primary spermatocytes was 19-0 days, secondary spermatocytes 0-7 days, spermatids with round nuclei 8-7 days, and spermatids with elongated nuclei 10-1 days. Labelled spermatozoa entered the caput epididymidis 35 days, and appeared in the ejaculate 39-9 days, after the isotope ...
Engle CE, Foley CW, Witherspoon DM, Scarth RD, Goetsch DD.Three experiments were conducted on the metabolism of stallion sperm. In experiment 1, whole and washed sperm were incubated under aerobic and anaerobic enviroments and analyzed before and after controlled incubation for motility, pH, lactic acid, glucose, fructose, and O2 comsumption. In experiment 2, whole and washed sperm were incubated aerobically and anaerobically with and without uterine tubal fluids. Experiment 3 was the same as experiment 2, except added substrates of glucose and lactic acid were studied. The same examinations were made in experiments 2 and 3 as for experiment 1. Motil...
Cermák O.The paper describes the relation of citric acid to other chemical and biological indices of the fertility of stallion sperm. A positive relation was found between citric acid and the density and motility of spermatozoa, to the concentration of ergothioneine, and hemolytic activity, and a negative relation to pH and to the polarographic activity of proteins. Attention is drawn to the important nutritional function of citric acid.
Kozhukharova L.Studied was the antigenic relatedness of hyaluronidase contained in the semen of breeder animals of homologic and heterologic species. The experiments were carried out by means of the immunodiffusion and the immunoelectrophoretic methods. The results obtained showed that the seminal hyaluronidase of bulls, rams and bucks is antigenically related, and that of stallions, boars and rabbits does not exhibit antigenic relatedness. Stallion semen is closely related antigenically with the above-mentioned three animal species' semen as manifested by two precipitation bands, but these are not identical...
Tischner M, Kosiniak K, Bielański W.The emission of stallion semen was studied with the aid of
an `open' Krak\l=o'\w-72Model artificial vagina. The pattern of mating
behaviour was constant in all copulations observed : a mean number of
seven intravaginal thrusts was required to elicit ejaculation. The
pressure within the vestibule of the artificial vagina averaged 66 mmHg
at the beginning of copulation, 142 mmHg just before ejaculation, and
70 mmHg during the emission of semen. Emission appeared to be a more
variable process. Five to ten jets were observed; the mean number was
eight. The early jets occurred under high pr...
Rubessa M, Feugang JM, Kandel ME, Schreiber S, Hessee J, Salerno F, Meyers S, Chu I, Popescu G, Wheeler MB.The capacity for microscopic evaluation of sperm is useful for assisted reproductive technologies (ART), because this can allow for specific selection of sperm cells for in vitro fertilization (IVF). The objective of this study was to analyze the same sperm samples using two high-resolution methods: spatial light interference microscopy (SLIM) and atomic force microscopy (AFM) to determine if with one method there was more timely and different information obtained than the other. To address this objective, there was evaluation of sperm populations from boars and stallions. To the best of our k...
Choi YH, Velez IC, Macías-García B, Riera FL, Ballard CS, Hinrichs K.Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Kenney RM, Cummings MR, Teuscher C, Love CC.Testicular degeneration is a major cause of subfertility in stallions, although an aetiological diagnosis cannot be made in most cases. In the present study, autoimmune testicular degeneration was induced and evaluated in stallions by immunizing stallions with their own spermatozoa mixed with an adjuvant. The factors evaluated included changes in semen quality and testicular histology. A large decrease in sperm number and quality was observed in response to sperm autoantigens. An ELISA test specific for antisperm antibodies was developed which enabled antibody titres in serum, seminal plasma a...
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...
Braun J, Hochi S, Oguri N, Sato K, Torres-Boggino F.Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
De Botton D, Janett F, Burger D, Imboden I, Kähn W, Thun R.The aim of the present study was to investigate the spermatogenic and Leydig cell activity in stallions with impaired semen quality after treatment with equine somatotropin. Experiments were performed using 18 adult clinically healthy stallions with poor semen quality which did not pass breeding soundness evaluation. The animals were randomly divided into a treatment (n = 9) and a control (n = 9) group. Over a period of 90 days, nine stallions received a daily intramuscular injection of 10 mg recombinant equine somatotropin (EquiGen, BresaGen Limited, Adelaide, Australia) and 9 control animals...
Len J, Beehan D, Eilts B, Ebrahimie E, Lyle S.The objective of the study was to investigate if reducing the seminal plasma of stallion extended semen by centrifugation once will suffice to maintain acceptable semen quality for insemination after 4 days of cool storage. Collected semen was extended to 25 × 106 sperm/mL and subjected to one of the following treatments: noncentrifuged (control), centrifuged for 10 minutes at 900 × g and 1800 × g. The supernatant was partially removed, and the sperm pellet, reconstituted and re-extended. It was then placed in a passive cooling device overnight and then transferred to a refrigerator fo...
Held JP, Prater P, Stettler M.A 9-year-old Arabian stallion with a 3-year history of infertility was evaluated for breeding soundness. Both testes were small. Ultrasonography revealed a small amount of free fluid between the tunics of both testes. Results of cytologic examination of the fluid were unremarkable. On semen examination, progressive motility was 10%, and total number of spermatozoa in the ejaculate was 6.6 x 10(9), of which 92% were abnormal. Predominant abnormalities were head defects (75%): 57% of the heads had single or multiple vacuoles, and 60% also had midpiece swelling or bending.
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...
Boye JK, Katzman SA, Kass PH, Dujovne GA.In equids, it is common to inject lidocaine into the testicles at the time of routine castration to provide analgesia. The effects of lidocaine on equine sperm have not been evaluated in vitro or on epididymal sperm collected following castration. The aims of this study were to determine effects of clinically relevant doses of lidocaine on equine spermatozoa in vitro using freshly collected semen and to compare the characteristics of epididymal spermatozoa after routine castration with or without intra-testicular lidocaine administration. We hypothesized that increasing concentrations of lid...
Dietz JP, Sertich PL, Boston RC, Benson CE.Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial conce...
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Roels K, Smits K, Ververs C, Govaere J, D'Herde K, Van Soom A.In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with det...
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...
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...
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...
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...
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...
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...
Aguiar LH, Pinto CRF.Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Luis-Calero M, Muñoz-García CC, Fernández-Hernández P, Macías-García B, González-Fernández L.A protocol for conventional in vitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE...
Parsad R, Bagiyal M, Ahlawat S, Arora R, Gera R, Chhabra P, Sharma U.Donkeys (Equus asinus) have played a vital role in agriculture, transportation, and companionship, particularly in developing regions where they are indispensable working animals. The domestication of donkeys marked a significant turning point in human history, as they became essential for transportation, agriculture, and trade, especially in arid and semi-arid areas where their resilience and endurance were highly valued. In modern society, donkeys are indispensable due to their diversified applications, including meat, dairy, medicine, and functional bioproducts, supporting economic, cultura...
Samper JC, Hernandez Aviles JC, Ramirez-Agamez LF, Love CC, Gonzalez-Marin C, Fleury P, Dini P, De La Fuente A, Foss R, Campos FL, Ross PJ.The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Pozor MA, Macpherson ML, Kelleman AA, Smith H, McNaughten JW.This retrospective study aimed to validate the usefulness of spectral Doppler ultrasonography in diagnosing testicular dysfunction in stallions. The first part of the study included a pony stallion with age-related testicular degeneration (18 - 21 yrs. old). Testicular dimensions, spectral Doppler parameters of testicular blood flow, and semen parameters were obtained numerous times from this pony over four consecutive years. While testicular volume decreased dramatically, and the stallion became azoospermic, there were no changes in any of the spectral Doppler parameters of testicular blood f...
Vitoria A, Barrachina L, Romero A, Fuente S, de Blas I, Gil L, Vázquez FJ.A new surgical technique using a polyether ether ketone (PEEK) anchoring device for testicle-sparing laparoscopic inguinal hernioplasty in stallions was described in 2023 and is known as the PEEK harpoon technique (PHT). In breeding stallions, it is essential that the surgery is effective in preventing inguinal hernia but also that it does not impair the testicular function. This study aims to evaluate whether the PHT may affect testicular function. To achieve that, changes in the testicular blood flow, sperm production and motility characteristics were assessed 28 days after use of the PHT. S...
Alim S, Cheppali SK, Pawar SS, Swamy MJ.Fibronectin type-II (FnII) proteins are major constituents in the seminal plasma of many mammals and play a crucial role in sperm capacitation. Additionally, the seminal FnII proteins from bull and horse exhibit chaperone-like activity (CLA), by acting as small heat shock proteins (shsps). The present work demonstrates that the major FnII protein of donkey seminal plasma, DSP-1 exhibits CLA with broad specificity and protects various client proteins such as alcohol dehydrogenase, lactate dehydrogenase and enolase against thermal and oxidative stress. Binding of phosphorylcholine (PrC) - the he...
Brito LFC.Semen analysis is an important laboratory diagnostic test for stallions. Evaluation of sperm motility is integral to basic semen analysis and results are important for breeding management and clinical practice. Computer-assisted sperm analysis (CASA) allows objective sperm motility evaluation and increases analytical precision. The objective of the present study was to validate a portable CASA system (AndroScope). Fresh/cooled semen samples (33 ejaculates, 18 stallions) and frozen semen (40 ejaculates and one epididymal flush, 27 stallions) were evaluated using the AndroScope and the IVOS II C...
