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.
Janett F, Burkhardt C, Burger D, Imboden I, Hässig M, Thun R.The objective of this study was to investigate changes of quality and freezability of stallion semen in response to repeated acute treadmill exercise. Ejaculates from 11 stallions were collected, evaluated and frozen weekly during four periods of 4 weeks each defined as before (period 1), during (period 2) and after (periods 3 and 4) intense exercise. In fresh semen the gel-free volume, sperm concentration, motility, normal sperm and sperm with major defects (acrosome defects, nuclear vacuoles, abnormal heads, midpiece defects and proximal droplets) were evaluated. In frozen-thawed semen, moti...
Baumber J, Ball BA.To determine glutathione peroxidase (GPX) and superoxide dismutase (SOD)-like activities in spermatozoa, seminal plasma, and reproductive tissues (ie, testis, epididymis, bulbourethral gland, prostate, vesicular gland, and ampulla) in horses. Methods: Seminal plasma from 17 stallions, spermatozoa from 5 stallions, and reproductive tissues from 3 stallions. Methods: Activity of GPX was determined by use of assays measuring oxidation of NADPH in the presence of exogenous glutathione, cumene hydroperoxide, and glutathione reductase. Activity of SOD-like enzymes was determined by use of the nitrob...
van Eldik P, van der Waaij EH, Ducro B, Kooper AW, Stout TA, Colenbrander B.Inbreeding is widely believed to negatively affect reproductive performance. Indeed, in some species, high levels of inbreeding are thought to be the major cause of poor semen quality. It is, however, not clear whether inbreeding affects fertility in horses. In this study, the relationship between inbreeding and semen quality was examined in 285 immature Shetland pony stallions submitted for breeding soundness examination in March-April of the years 1992-1997. The majority of stallions examined were 3 years old (85%) and their coefficients of inbreeding ranged from 0 to 25% (mean+/-S.D.: 3+/-4...
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...
McDonnell SM, Hinze AL.Periodic spontaneous erection and penile movements known as masturbation (SEAM) occur normally at approximately 90 min intervals in awake equids. SEAM in horses has traditionally been misunderstood by many horsemen as aberrant behavior that should be eliminated. Accordingly, it is not uncommon for trainers of performance stallions or managers of breeding stallions to punish SEAM in an attempt to eliminate the behavior. Previous clinical observations and preliminary unsystematic trials had suggested that attempts to stop stallion SEAM may lead to an increase rather than a decrease in SEAM, and ...
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...
Leeb T, Sieme H, Töpfer-Petersen E.Our knowledge on the many aspects of mammalian reproduction in general and equine reproduction in particular has greatly increased during the last 15 years. Advances in the understanding of the physiology, cell biology, and biochemistry of reproduction have facilitated genetic analyses of fertility. Currently, there are more than 200 genes known that are involved in the production of fertile sperm cells. The completion of a number of mammalian genome projects will aid in the investigation of these genes in different species. Great progress has been made in the understanding of genetic aberrati...
Peter AT, Colenbrander B, Gadella BM.The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.
Loomis PR, Squires EL.Success with frozen semen requires attention to detail and a basic understanding of the techniques for properly handling and thawing and inseminating frozen semen. Practitioners should also be familiar with strategies used for managing mares for insemination with thawed semen. This manuscript will review those techniques and also present fertility data collected in a commercial setting. Factors that affect pregnancy rates for mares inseminated with frozen-thawed semen such as timing and frequency of insemination were examined for two separate data sets consisting of 332 and 536 mare cycles col...
Pozor M.Although ultrasound evaluation of the reproductive tract of stallions was introduced to veterinary practice long ago, this examination is not always conducted during routine breeding soundness evaluation. The objective of this study was to investigate the clinical relevance of routine ultrasound evaluation of the stallion's reproductive tract. Breeding soundness evaluation of 113 stallions was performed, including ultrasonography of external and internal genitalia. Various pathological conditions were detected using this technique, with the most frequent being varicocele, thickened vaginal tun...
Card C.Morphologic assessment of spermatozoa is one of the most objective measures in a Breeding Soundness Examination of a stallion. There are different systems for morphologic assessment of spermatozoa. The objectives of this article are to review spermatogenesis, describe clinical sample preparation, discuss previous methods of morphologic classification and explain the use of a differential spermiogram. The advantages of the differential spermiogram method of analysis are discussed, along with its use in delineating intrinsic and extrinsic disturbances in spermatogenesis. Case examples of specifi...
Aziz DM, Ahlswede L, Enbergs H.The assay of MTT reduction depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate of a simple and less costly MTT test in determining equine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from 11 stallions (warm blood) were included in this study. Semen was diluted to 100 million cells/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1 an...
Turkstra JA, van der Meer FJ, Knaap J, Rottier PJ, Teerds KJ, Colenbrander B, Meloen RH.Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine HT adjuvant (GnRH/CoVaccine) and in Carbop...
Kreider JL, Ogg WL, Turner JW.Six mature stallions were used to test the effect of prostaglandin F2 alpha (PGF2 alpha ) on sperm production and seminal characteristics. Semen was collected from each stallion twice weekly 1 hr following a 10 mg intramuscular injection of PGF2 alpha or a sham injection. A switchback design was used so that three stallions received PGF2 alpha and three served as controls during the first 9 weeks (period 1). Treatment regimens were reversed during the second 9 weeks (period 2). Treatment of stallions with PGF2 alpha resulted in an increase (P less than .05) in gel free seminal volume and a dec...
Coutinho da Silva MA, Seidel GE, Squires EL, Graham JK, Carnevale EM.The effects of semen extender components on the ability of stallion sperm to bind to the zona pellucida (ZP) and the suitability of using bovine ZP for a ZP-binding assay for stallion sperm were investigated in a series of experiments. In Experiment I, binding of stallion sperm to both bovine and equine ZP was significantly increased when a skim milk-based extender (EZM) was used. In Experiment II, a threefold increase in sperm binding to ZP was observed when sperm were diluted in EZM compared with diluents, which contained no milk (TALP, LAC, and EmCare). In Experiment III, centrifuging the s...
Multigner L, Magistrini M, Ducot B, Spira A.Several reports have suggested that human semen quality has declined throughout the world over the last few decades. Chemicals in the environment acting as endocrine disrupters have been implicated as a possible cause. If this is indeed the case, then similar effects may be observed in animals. We analyzed 1489 ejaculates collected from 390 Breton draught stallions between 1981 and 1996. Semen was collected from all the stallions at a single center, according to standardized semen collection protocols and laboratory methods. Semen volume decreased slightly but significantly and there was an in...
Francl AT, Amann RP, Squires EL, Pickett BW.The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with inseminatio...
Parlevliet JM, Kemp B, Colenbrander B.The semen characteristics and testicular size of 398 3-year-old maiden Dutch Warmblood stallions were studied during February and March. Mean values (+/- SD) of age (1030 +/- 88 days) and testicular size (9.8 +/- 0.9 cm) of the maiden stallions were determined as well as the following semen characteristics (mean of two ejaculates, taken 1 h apart): volume (65 +/- 26 ml), sperm concentration (2.061 +/- 1.685 x 10(8) ml-1), total number of spermatozoa (1.129 +/- 0.71 x 10(10)), percentage of progressively motile spermatozoa (68 +/- 9%), percentage of live spermatozoa with normal morphology (66 +...
Brinsko SP, Van Wagner GS, Graham JK, Squires EL.The aim of the present study was to determine whether there are characteristics of fresh, cooled and frozen-thawed semen samples that can be used to predict the suitability of stallion semen for preservation by cooling or freezing. Each of three ejaculates obtained from 12 stallions was divided into aliquots to be analysed for sperm motility, morphology and membrane integrity as fresh, cooled and frozen-thawed samples. The percentage of morphologically normal spermatozoa was similar in fresh and cooled samples and both were greater than in the frozen samples. There were no strong linear relati...
Parlevliet JM, Colenbrander B.In the selection procedure to acquire a breeding licence, 3-year-old Dutch Warmblood stallions have to undergo a breeding soundness test It is questioned whether this evaluation is predictive of the stallion's fertility results in the first breeding season. Therefore, semen parameters at the beginning of their first breeding season were evaluated and correlated to nonreturn at first cycle and foaling rate of mares bred by stallions (n = 13). The total number of mares inseminated with chilled semen from those stallions was 1055. Semen parameters were recorded on 2 ejaculates, collected 1 h apar...
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 ...
Sancler-Silva YFR, Monteiro GA, Ramires-Neto C, Freitas-Dell'aqua CP, Crespilho AM, Franco MMJ, Silva-Junior ER, Cavalero TMS, Scheeren VFC, Papa FO.Inflammation of the seminal vesicle interferes with fertility and is a persistent problem that is difficult to treat. The aim of this study was to evaluate the semen quality of 5 stallions with seminal vesiculitis before and after local treatment. All stallions were endoscopically treated for seminal vesiculitis during 10 consecutive days. The glandular lumen was accessed and flushed with a Ringer Lactate solution prior to antibiotic infusion. The antibiotic was selected based on the antibiogram from bacterial culture of samples previously collected from the seminal vesicles. The kinetic param...
Scheeren VFC, Sancler-Silva YFR, El-Sheikh Ali H, Kastelic JP, Alvarenga MA, Papa FO.Seminal vesiculitis in stallions reduces fertility and is often underdiagnosed. The most common cause is infection of seminal vesicles by bacteria capable of forming biofilms and a propensity for tissue persistence, for example, Pseudomonas aeruginosa. Achieving a clinical cure is challenging because of a high rate of recurrence. Systemic antibiotic therapy does not reach adequate therapeutic concentrations within the seminal vesicles; one alternative is endoscopy-guided, local antibiotic infusion into the gland lumen, with or without concurrent systemic antibiotics. Current diagnostic and the...
Carver DA, Ball BA.Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during...
Panzani D, Quaresma M, Fanelli D, Camillo F, Moroni R, Rota A, Martins-Bessa A, Nóvoa M, Catalán J, Canisso IF, Conte G, Mirò J.Due to the long courtship needed to attain excitation and erection, donkey semen collection can take up to 90 min. ProstaglandinF2α (PGF2α) has been reported to hasten the onset of erection and ejaculation in domesticated mammals, presumably by inducing smooth muscle contractions in the internal genitalia. However, while it has been anecdotally used in donkeys, it has yet to be critically evaluated. This study aimed to compare behavioral and semen parameters in Catalan, Balearic, Amiata, and Miranda jacks treated with the PGF2α analogue cloprostenol sodium immediately prior to exposure to a...
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 ...
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...
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...
Papaioannou KZ, Murphy RP, Monks RS, Hynes N, Ryan MP, Boland MP, Roche JF.An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (m...
Rigby S, Hill J, Miller C, Thompson J, Varner D, Blanchard T.It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 milli...
Mennella MR, Jones MR.The activity of 5'-nucleotidase (EC 1.3.5), cyclic nucleotide phosphodiesterase (EC 2.1.4.17), non-specific phosphodiesterase (EC 3.1.4.1) and ribonuclease (EC 1.7.7.16)has been investigated in the seminal plasma of whole semen and in the secretions of the seminal vesicle, prostate and epididymis of the bull, boar, ram, stallion, jackass, rabbit and man. Bull seminal plasma showed the highest activity for 5'-nucleotidase, cyclic nucleotide phosphodiesterase and ribonuclease; in contrast, stallion and jackass semen were very poor in these enzymes. Ram, rabbit and boar seminal plasma showed inte...
Jung H, Yoon M.The production of donor-derived sperm using spermatogonial stem cell transplantation has been studied in various animals including mice, rats, goats, boar, dogs, sheep, and monkeys. However, germ cell transplantation has not been applied in stallions. The objective of this study was to produce donor germ cell-derived sperm using germ cell transplantation in stallions. Donor germ cells were transplanted into the parenchyma of 3 recipient stallions that had been treated with busulfan IV injections of 15 mg/kg body weight. For the preparation of donor single germ cells, tissue (20 g) from each te...
de la Cueva FI, Rigau T, Bonet S, Miró J, Briz M, Rodríguez-Gil JE.Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na (+)K (+), ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenney's medium (T...
Hansen PJ, Hoggard MP, Rathwell AC.The ability of stallion seminal plasma to modify phagocytosis of spermatozoa and Streptococcus zooepidemicus was examined. Phagocytosis was monitored indirectly as the H2O2 produced by peripheral blood leukocytes after addition of spermatozoa or bacteria. Hydrogen peroxide production after addition of ejaculated spermatozoa was greater (P less than 0.01) than after addition of epididymal sperm. Furthermore, pre-incubation of epididymal sperm with 6.25-50% seminal plasma caused a dose-dependent increase in subsequent H2O2 production by leukocytes (P less than 0.05). In addition, equine serum wa...
McDonnell SM, Odian MJ.This study is a part of our ongoing work toward developing pharmacological methods for enhancing and inducing ejaculation in stallions with ejaculatory dysfunction. We evaluated a combination treatment of imipramine hydrochloride followed 10 minutes later by xylazine hydrochloride for the induction of ex copula ejaculation. Eight pony stallions each underwent 6 treatment trials conducted at 4-day intervals. The trials were conducted in the animals' stalls, where they were observed for 90 minutes following treatment. To evaluate the effect of pretreatment sexual stimulation on the rate of ejacu...
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...
Sultan T, Chaudhary AK, Mehta JS, Mehra R, Manuja A, Talluri TR, Bhattacharya TK.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Nasiri-Foomani N, Hassani S, Najafi M, Samadi F.The optimization of cryopreservation media to reduce oxidative damage on post-thaw spermatozoa is crucial. This research aimed to assess the antioxidant properties of curcumin-loaded niosomal nanocarriers (CurLNN) on the functional characteristics, the relative expression of apoptotic genes, and flow cytometry assessments of apoptotic-like changes, reactive oxygen species production (ROS), mitochondrial membrane potential, and chromatin integrity in stallion spermatozoa following thawing. Twenty-five ejaculates were diluted in INRA96 freezing media supplemented with 20 μM of either curcumin ...
Bae JW, Hwang JM, Lee WJ, Kim DH, Yi JK, Ha JJ, Oh DY, Kwon WS.The selection of superior sires is paramount for enhancing the efficiency of animal production in the livestock industry. However, semen quality assessment still relies on conventional semen analysis techniques in both animals and humans. Despite extensive efforts to develop various biomarkers for more accurate and precise predictions of male fertility potential, more effective physiological indicators and advance potential biomarkers are needed. Herein, we aimed to develop new potential biomarkers related to sperm motion kinematics for male fertility prediction. We first evaluated sperm motio...
Pickett BW, Faulkner LC, Voss JL.Season had a pronounced effect upon seminal pH and refractometer 'protein', total carbohydrate, dry weight, total N2 and lactic acid in seminal plasma of first and second ejaculates. In addition, total seminal volume, spermatozoa per ml and per ejaculate, non-protein sulphhydryl and glycerylphosphorylcholine of second ejaculates were also influenced. There was a season difference in the concentrations of lactic acid in spermatozoa from first and in total N2 from spermatozoa in second ejaculates. The effects of season on seminal plasma were greater than those on spermatozoa. Spermatozoa in firs...
Dott HM, Foster GC.Semen from boar, bull, ram, rabbit, reindeer and stallion was diluted in formol citrate or formol saline and stained with eosinnigrosin. The proportion of eosinophilic spermatozoa did not differ from that in fresh semen after storage for 48 hr in the formol diluent at temperatures ranging from 4 degrees C to 40 degrees C. Some samples were kept for periods up to 3 weeks with very little increase in the proportion of eosinophilic spermatozoa.
