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.
Bogle OA, Carrasco RA, Ratto MH, Singh J, Adams GP.The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) in male reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in...
Nouri H, Shojaeian K, Jalilvand G, Kohram H.The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation wer...
Hartmann C, Gerner W, Walter I, Saalmüller A, Aurich C.In the mare, early pregnancy loss is common, but involvement of the maternal immune system in the pathogenesis of this condition has not been investigated in detail so far. In the present study, we assessed effects of exposure of the endometrium to semen or seminal plasma in oestrous mares on the response of regulatory T lymphocytes (Tregs) in the peripheral circulation as well as in the endometrium. Raw semen, seminal plasma or PBS (control) were introduced into the uterus of oestrous mares (n = 12). Blood was collected immediately before insemination or PBS infusion (time 0), and 12, 24 ...
Ferrer MS, Miller LMJ.Antisperm antibodies (ASAs) have been associated with infertility in stallions. The objectives of this study were to investigate the frequency of ASA-positive semen samples in satisfactory and non-satisfactory breeder stallions, the association between ASA binding and semen quality, and factors that may affect the diagnosis. Breeding soundness examinations were performed in 21 stallions and the percentage of IgG- and IgA-bound spermatozoa was evaluated using flow cytometry. Median IgG and IgA binding did not differ between the first and second ejaculates. The percentage of IgA-bound spermatozo...
Gautier C, Schmidt K, Aurich J, Aurich C.The hypothesis in this study was continuous treatment of stallions with the GnRH agonist deslorelin inhibits reproductive functions. A 2-week pre-experimental period was followed by an 11-week deslorelin implant treatment. Stallions received 4.7 (D1, n = 7), or 18.8 mg deslorelin (D2, n = 5) or remained untreated (C, n = 5). Libido, sperm motility, membrane integrity, DNA fragmentation, estrogen receptors, basal plasma testosterone and Anti Muellerian hormone (AMH) concentrations were evaluated once weekly during the treatment period. The testosterone response to the GnRH agonist buserelin a...
Filho JS, Corcini CD, Santos FCC, Anciuti AN, Gatti NLS, Anastacio E, Mielke R, Nogueira CEW, Curcio BR, Varela AS. BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. Objective: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. Methods: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. R...
Ferreira HN, Ferreira-Silva JC, Rocha JM, Farras MC, Calixto M, Moura MT, Alvarenga MA, Oliveira AL.Semen cryopreservation causes DNA damage, thus requiring continuous monitoring. Objective: To compare two assays for sperm DNA fragmentation (SDF) from stallions with contrasting semen freezability. Methods: Thirteen stallions were classified as good semen freezers (GSF) or bad semen freezers (BSF). Ejaculates were cryopreserved with three diluents. Semen was subject to SDF evaluation using the sperm chromatin structure assay (SCSA) and Halomax after thawing (0 h) and after a 4 h thermoresistance test. Results: On semen of BSF, analysis by SCSA was similar between evaluations, but Halomax show...
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...
Love CC.Sperm quality assays have increased in number in the last 10 years. Most of these assays are flow cytometry based in application and are modified from assays that have been developed to measure somatic cell function. The goal of any sperm quality assay should be to advance the clinicians/researchers understanding of sperm cell function and the relationship to fertility. While these assays appear to measure somatic cell-like functions in sperm there tends to be little understanding how the results of these assays relate to fertility.
Jiménez-Trejo F, Coronado-Mares I, Boeta M, González-Santoyo I, Vigueras-Villaseñor R, Arriaga-Canon C, Herrera LA, Tapia-Rodríguez M.Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several re...
Carossino M, Dini P, Kalbfleisch TS, Loynachan AT, Canisso IF, Shuck KM, Timoney PJ, Cook RF, Balasuriya UBR.Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the dow...
Johannisson A, Figueiredo MI, Al-Kass Z, Morrell JM.An improved fertility prediction for stallions is of importance for equine breeding. Here, we investigate the potential of a combined staining of stallion spermatozoa for superoxide and mitochondrial membrane potential (MMP) for this purpose. Semen samples were analysed immediately after arrival at the laboratory, as well as after 24 h. Superoxide was measured by MitoSOXRed, while MMP was measured with JC-1. Menadione was used to stimulate superoxide production. In addition, other parameters of sperm quality, namely motility, membrane integrity, chromatin integrity, sperm kinematics and Hoec...
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...
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...
Jeannerat E, Marti E, Berney C, Janett F, Bollwein H, Sieme H, Burger D, Wedekind C.The major histocompatibility complex (MHC) has repeatedly been found to influence mate choice of vertebrates, with MHC-dissimilar mates typically being preferred over MHC-similar mates. We used horses (Equus caballus) to test whether MHC matching also affects male investment into ejaculates after short exposure to a female. Semen characteristics varied much among stallions. Controlling for this variance with a full-factorial within-subject experimental design, we found that a short exposure to an MHC-dissimilar mare enhanced male plasma testosterone and led to ejaculates with elevated sperm nu...
Pérez-Marín CC, Requena FD, Arando A, Requena L, Requena F, Agüera EI.The aim of this study was to determine the effects of various abiotic factors, such as light, physical stress (pipetting) and thermal shock, on the quality of fresh and cooled equine sperm. In experiment I, four sperm aliquots were subjected to different light exposures: (i) protected control samples (CTRL), (ii) exposed to UV light at 10 cm (UV10), (iii) exposed to UV light at 20 cm (UV20) and (iv) exposed to laboratory lighting (LAB). In experiment II, four semen aliquots were subjected to repeated pipetting for 0, 10, 20 and 30 times (CTRL, P10, P20 and P30, respectively). In experiment I...
Del Prete C, Tafuri S, Ciani F, Pasolini MP, Ciotola F, Albarella S, Carotenuto D, Peretti V, Cocchia N.Stallion semen is damaged by oxidative stress during cooling and transport. Semen processing and extenders have been tested to improve the fertilizing capacity of semen and to preserve semen during transport. Dietary supplementation with natural antioxidants has been proposed to prevent oxidative damages. In this study, for the first time, the effect of dietary supplementation with Lepidium meyenii (Maca) on the characteristics of fresh and chilled stallion semen was evaluated. Maca is a traditional Andean crop used as a nutraceutical for the fertility-enhancing properties that are linked with...
Brom-de-Luna JG, Canesin HS, Wright G, Hinrichs K.Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1,...
Murcia-Robayo RY, Jouanisson E, Beauchamp G, Diaw M.Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification dif...
Shojaeian K, Nouri H, Kohram H.Overproduction of reactive oxygen species during sperm freeze-thawing process leads to membrane lipid peroxidation, DNA damage, motility loss, and subsequent death. This oxidative stress can be alleviated by the addition of some antioxidants to semen extenders prior to freezing. This study was performed to evaluate the in vitro effectiveness of MnTBAP (a cell permeable antioxidant) on stallion sperm freezability and in vivo fertility rate. Twenty-one ejaculates were, collected with missouri model artificial vagina (n = 3 stallions, seven ejaculate each), and diluted (1:2 v/v) with phosphoc...
Aurich C, Ortega Ferrusola C, Peña Vega FJ, Schrammel N, Morcuende D, Aurich J.Spermatozoa contain polyunsaturated fatty acids (PUFA). Cryopreservation damages sperm membranes and they become less functional after thawing. We analysed the lipid composition of spermatozoa from Shetland stallions (n = 15) collected monthly from January to June and hypothesized that sperm lipid patterns change with season. In addition, one ejaculate per month was submitted to cryopreservation. Content of saturated palmytic and stearic acid decreased from January to March (p < 0.001) while content of the PUFA docosapentaenoic (p < 0.001) and arachidonic acid (p < 0.05) and total PUFA...
Rui BR, Angrimani D, Bicudo LC, Losano J, Nichi M, Pereira R.Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high-precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For thi...
Ghallab AM, Shahat AM, Fadl AM, Ayoub MM, Moawad AR.The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooli...
Pricking S, Bollwein H, Spilker K, Martinsson G, Schweizer A, Thomas S, Oldenhof H, Sieme H.Accurate determination of the testes volume and prediction of the daily sperm output (DSO) is valuable information for reproductive management of a stallion. The aim of this study was to compare different methods for measuring the testes volume, including caliper, 2D and 3D ultrasound. Special emphasis was on feasibility of 3D volume analysis. First, 22 castrated testes were measured and derived volumes were compared with volumes determined via volume displacement in a graded cylinder with saline solution. Then, during the breeding season, testes sizes of 52 stallions were measured in vivo an...
Rota A, Sgorbini M, Panzani D, Bonelli F, Baragli P, Ille N, Gatta D, Sighieri C, Casini L, Maggiorelli MM, Aurich C, Camillo F.Reproductive management of male donkeys employed for artificial breeding has been poorly studied. The aim of this study was to evaluate the effect of housing system, with the animals grouped together in a paddock or kept in individual boxes, on sexual behaviour, cortisol and testosterone concentration and seminal characteristics of adult male donkeys. The study included four Amiata donkey jacks (stallions) from which ejaculates, saliva and blood were collected during two distinct 3 weeks periods, one in the group and one in the box housing system. Time needed for semen collection was shorter ...
Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF.In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cool...
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 ...
Wach-Gygax L, Burger D, Malama E, Bollwein H, Fleisch A, Jeannerat E, Thomas S, Schuler G, Janett F.In this study annual fluctuations of DNA fragmentation and quality of cold-stored equine sperm were evaluated. Ejaculates were collected weekly during one year from 15 stallions. Ejaculate volume, sperm concentration and total sperm count were determined and semen was then extended and cold-stored for 48 h. Sperm motility was evaluated by CASA before and after 24 as well as 48 h of cold storage. In addition, the percentages of sperm with intact plasma membrane and acrosome (PMAI %) and with low intracellular Ca2+ level were determined in cold-stored semen (24 h, 48 h). SCSA™ was performe...
Schmidt K, Deichsel K, de Oliveira RA, Aurich J, Ille N, Aurich C.We hypothesized that housing of stallions in a thermoneutral temperature zone during autumn and winter does not only influence metabolism and hair shedding but also improves the characteristics of raw and processed semen. Fertile Shetland pony stallions were followed from October to June. This time coincided with the seasons autumn, winter and spring. Ponies were kept in outside paddocks (group CON, n = 8) or in indoor stables (group ST, n = 8) from October to March when ST stallions returned to outdoor paddocks, but ponies remained in the same groups. The rectal temperature was measured o...
Jeannerat E, Janett F, Sieme H, Wedekind C, Burger D.The theory of ejaculate economics was mainly built around different sperm competition scenarios but also predicts that investments into ejaculates depend on female fecundity. Previous tests of this prediction focused on invertebrates and lower vertebrate, and on species with high female reproductive potential. It remains unclear whether the prediction also holds for polygynous mammals with low female reproductive potential (due to low litter size and long inter-birth intervals). We used horses (Equus caballus) to experimentally test whether semen characteristics are adjusted to the oestrous cy...
Love CC.Stallion semen evaluation is an important part of the breeding soundness evaluation. The results of the semen evaluation cannot be interpreted without a thorough knowledge of the mare and management effects that may have played a role or may affect the potential fertility of the stallion evaluated. There are considerations and limitations that the clinician should understand about each test. Any sperm quality test must be interpreted with a clear understanding of how it relates to fertility.
Love CC, Noble JK, Standridge SA, Bearden CT, Blanchard TL, Varner DD, Cavinder CA.The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Dowsett KF, Pattie WA.A study of stallion fertility was conducted on commercial studs in eastern Australia over 4 breeding seasons (1974/75-1977/78). Data from 47 stallions (66 stallion seasons) aged 2-26 years and representing 7 breeds and 1664 mares were used to relate seminal characteristics to fertility. Percentage pregnancies per service was the most sensitive measure of fertility with stallions on commercial studs. Breed and age of stallion, breeding season and the referral status of the stallion ('suspect' or 'normal') had significant (P less than 0.01) effects on fertility. The semen characteristics that we...
Sieme H, Oldenhof H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonl...
Ghaoui Rel-H, Thomson PC, Evans G, Maxwell WM.Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen...
Merkl M, Ertl R, Handschuh S, Aurich C, Schäfer-Somi S.In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen ...
de la Torre J, Sánchez-Martín P, Gosálvez J, Crespo F.Sperm quality was assessed in normozoospermic human (n = 10) and Spanish breed stallion (n = 10) after sperm fractionation during ejaculation. The first ejaculated fraction was separated from the second. A third sample was reconstituted using equivalent proportion of both fractions (RAW). Fraction 1, Fraction 2 and RAW semen were incubated for 30 min at 37°C to homogenise the impact of iatrogenic damage between both species. Sperm concentration, motility and sperm DNA damage were assessed in each fraction and RAW semen. The results showed two important facts: (i) spermatozoa confined at ...
Nogueira BG, Sampaio BF, Souza MI, Costa E Silva EV, Zúccari CE.Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with...
Cary JA, Madill S, Farnsworth K, Hayna JT, Duoos L, Fahning ML.The purpose of this study was to evaluate 2 methods of semen collection that could be used as terminal procedures in stallions with irreparable conditions, such as fractures or colic. Electroejaculation was attempted under general anesthesia. Forty-eight hours later, the ponies were castrated and 2 different epididymal sperm collection techniques were attempted by using a flushing or floating method. Additionally, the effect of supplemental seminal plasma was evaluated. Experimentally, electroejaculation was found to be a safe but ineffective method of terminal semen collection. Viable sperm c...
Miszczak F, Shuck KM, Lu Z, Go YY, Zhang J, Sells S, Vabret A, Pronost S, Fortier G, Timoney PJ, Balasuriya UB.This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.
Deichsel K, Palm F, Koblischke P, Budik S, Aurich C.Lipid peroxidation contributes to the damage of the sperm plasma membrane. In different species, dietary supplementation with antioxidants has been shown to improve semen quality. Therefore, we tested effects of dietary supplementation with antioxidants and l-carnitin on semen quality in Shetland pony stallions (n=6). Semen was collected twice a week over a time period of 16 weeks. From weeks 5 to 12, a special diet for stallions containing a variety of antioxidants (STALLION, Pavo Pferdenahrung GmbH, Goch, Germany; tocopherol 300 mg/day; ascorbic acid 300 mg/day; l-carnitin 4000 mg/day; folic...
Canisso IF, Carvalho GR, Morel MC, Guimarães JD, McDonnell SM.The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser ...
Bulkeley E, Santistevan AC, Varner D, Meyers S.Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been deba...
Vitoria A, Romero A, Fuente S, Barrachina L, Vazquez FJ.This report describes a technique for standing laparoscopic vasectomy in stallions through a prospective descriptive study. A preliminary study was carried out with two experimental intact male horses and subsequently the procedure was performed in two clinical cases. These horse owners want to keep their animals in the most possible natural way, preserving its stallion behaviour in a herd without generating offspring. The horses were sedated and restrained in stocks and laparoscopic vasectomy was performed using three portal sites in both paralumbar fossae recording surgical times. A 4-cm seg...
Griffin RA, Swegen A, Baker MA, Ogle RA, Smith N, Aitken RJ, Skerrett-Byrne DA, Fair S, Gibb Z.Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercia...
Dias GM, Retamal CA, Tobella L, Arnholdt AC, López ML.'The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatoz...
Wu Z, Zheng X, Luo Y, Huo F, Dong H, Zhang G, Yu W, Tian F, He L, Chen J.The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess w...
Wnuk M, Lewinska A, Oklejewicz B, Bartosz G, Tischner M, Bugno-Poniewierska M.Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS(•+) decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox s...
Graham JK.This article describes some of the basic methodology for conducting stallion semen evaluations. Not all of these assays will likely be conducted on every semen sample collected. Routine evaluations should include determination of semen volume, sperm concentration, and an estimation of the percentage of progressively motile sperm, at a minimum. Other assays can be used if a seminal problem is discerned at the beginning of the breeding season or after a stallion has recovered from an illness or injury. Additionally, laboratory assays are particularly important to conduct on cryopreserved spermat...
Tearle JP, Smith KC, Boyle MS, Binns MM, Livesay GJ, Mumford JA.Six Welsh Mountain pony colts were infected intranasally with the Ab4 isolate of EHV-1. Clinical and virological monitoring demonstrated mild upper respiratory tract disease, with nasal shedding of virus and establishment of a cell-associated viraemia. Detailed pathological examination of the urogenital tract was performed post mortem on days 4-9 post-infection (PI). EHV-1 was isolated from the epididymis on day 8 and the testis on day 9 PI, with viral replication in endothelial cells of these organs and an associated necrotizing vasculitis and thrombosis. Productive viral infection of germina...
Hebia-Fellah I, Léauté A, Fiéni F, Zientara S, Imbert-Marcille BM, Besse B, Fortier G, Pronost S, Miszczak F, Ferry B, Thorin C, Pellerin JL....In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility ...
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...
Hurtgen JP, Johnson LA.During a 2-year period, 7 stallions were identified as having ejaculated spermatozoa characterized by a high incidence (27-74%) of acrosomal abnormalities. The most frequent abnormality of the acrosome was the 'knobbed sperm' defect which was observed in nigrosin--eosin, Giemsa, and haematoxylin and eosin stained semen smears under light microscopy, in buffered formal--saline and in glutaraldehyde-fixed wet mounts under phase contrast and differential interference microscopy, and in glutaraldehyde-fixed spermatozoa with electron microscopy and scanning electron microscopy. The defect was visib...
Pojprasath T, Lohachit C, Techakumphu M, Stout T, Tharasanit T.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integ...
Varner DD, Blanchard TL, Brinsko SP, Love CC, Taylor TS, Johnson L.Numerous techniques may be used for evaluation of the different reproductive disorders of the stallion. Approaches may vary from real-time ultrasonography and biopsy for evaluating testicular tumors to use of special assays for evaluating sperm or plasma for presence of antisperm antibodies. This communication addresses techniques used to evaluate five relatively uncommon, but perplexing, disorders of breeding stallions: (1) seminal vesiculitis, (2) hemospermia associated with idiopathic urethral defects, (3) acrosomal dysfunction, (4) abnormal spermatozoal chromatin, and (5) azoospermia.
Gonzalez-Castro RA, Amoroso-Sanches F, Stokes JE, Graham JK, Carnevale EM.Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal p...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Ortiz I, Arenas R, Morrell JM, Hidalgo M.Stallion sperm was vitrified using straws in comparison with spheres and conventional freezing. Vitrification was performed plunging 30 μL of sperm (spheres) or 0.5 mL straws into liquid nitrogen (LN) and conventional freezing using 0.5 mL straws frozen in LN vapors. Sperm vitrified in straws were submitted to different warming procedures (42°C/20 seconds; 60°C/15 seconds) and single-layer centrifugation (SLC). Total (TM, %) and progressive sperm motility (PM, %), plasma membrane (IMS, %) and acrosome integrity (AIS, %) were statistically compared between treatments (mean ± SEM). Signif...
Papas M, Catalán J, Recuero S, Morrell JM, Yeste M, Miró J.This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid dis...
Alamaary MS, Haron AW, Hiew MWH, Ali M.Present study aimed to investigate the effect of adding antioxidants, cysteine and ascorbic acid on the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvate (GPT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (GGT) enzymes of post-thawed stallion sperm. Ten ejaculates were collected each from four healthy stallions and cryopreserved using HF-20 freezing extender containing either 0 mg/ml cysteine or ascorbic acid, 0.5 mg/ml cysteine and 0.5 mg/ml ascorbic acid. All samples in freezing extender containing cysteine or ascorbic acid or ...
Oldenhof H, Bigalk J, Hettel C, de Oliveira Barros L, Sydykov B, Bajcsy ÁC, Sieme H, Wolkers WF.In this study, modeling and experimental approaches were used to investigate the interplay between cooling rate and protectant concentration for cryopreservation of stallion sperm. Glycerol (GLY), ethylene glycol (EG), dimethylformamide (DMF), propylene glycol (PG), and dimethyl sulfoxide (DMSO) were tested as cryoprotective agents (CPAs), using concentrations up to 1500 mM and cooling rates ranging from 5°C to 55°C min. Modeling of the extent of sperm dehydration during freezing was done using previously determined values of the sperm membrane permeability to water to predict optimal cool...
