Spermatogenesis in horses is the process by which spermatozoa are produced in the testes. This complex sequence of events involves the proliferation and differentiation of germ cells, supported by Sertoli cells and influenced by hormonal regulation. Key stages include spermatogonial mitosis, meiosis, and spermiogenesis, resulting in the formation of mature sperm cells. Hormones such as testosterone and follicle-stimulating hormone (FSH) are integral to regulating this process. This topic encompasses research studies and scholarly articles that explore the mechanisms, hormonal influences, and factors affecting spermatogenesis in horses, as well as its implications for equine reproduction and breeding management.
Ruiz AJ, Castaneda C, Raudsepp T, Tibary A.This case report describes spermatogenic arrest and azoospermia in a stallion with a unique Y chromosome-autosome translocation. Clinical diagnosis of azoospermia was based on history of infertility and evaluation of ejaculates collected for artificial insemination. Clinical and ultrasonographic evaluation of the external and internal genitalia did not reveal any abnormalities except for smaller than normal testicular size. Azoospermia of testicular origin was confirmed by determining alkaline phosphatase concentration in semen. Histological evaluation of testicular tissue after castration con...
Sales FA, Ferreira-Silva JC, Vieira JI, Chaves MS, Caldas EL, Filho JP, Freitas VJ, Oliveira MA.Addition of extenders to thawed semen could improve fertility. Objective: To determine the efficiency of extenders to increase viability of thawed semen, measured by sperm parameters in vitro and pregnancy rates after artificial insemination (AI). Methods: Sperm motility and acrosin activity were measured during a thermoresistance test (TRT). Results: Progressive motility decreased (P<0.05) after 30 min in thawing semen treated with saline solution (SS) and only after 60 min with Tyrode's solution (TS) or freezing diluent (FD). The total motility decreased (P<0.05) after 60 min in thawed semen...
Tafuri S, Cocchia N, Carotenuto D, Vassetti A, Staropoli A, Mastellone V, Peretti V, Ciotola F, Albarella S, Del Prete C, Palumbo V, Esposito L....The present study was conducted to assess the chemical composition of Yellow Maca () and its biological activity on stallions following oral administration of hypocotyl powder. Maca was subjected to methanolic extraction and the chemical analysis was carried out by LC-MS-QTOF (liquid chromatography-mass spectrometry). Our results showed that Maca contains some effective antioxidants, a high percentage of glucosinolates, and other important components with a high antioxidant capacity. To evaluate the plant biological activity in stallions fed with Maca powder for 60 days, the redox status and s...
Griffin RA, Baker M, Aitken RJ, Swegen A, Gibb Z.Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10-14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assay...
Ghallab ARM, Abou-Ahmed MM, Fad AM, El-Badry DA, Shahat AM, Moawad AR.Cryopreservation of Arabian stallion semen is important in order to improve the function and fertility of frozen/thawed semen in this breed. Objective: To investigate the effects of centrifugation, type of semen extenders, and type of cryoprotectants on the quality of frozen/thawed Arabian stallion spermatozoa. Methods: Semen samples collected from four adult Arabian stallions (one ejaculate per week for 10 consecutive weeks) were either processed directly without centrifugation (no centrifugation; NC) or subjected to centrifugation on the gel-free fraction. Centrifugation protocols were divid...
Sales F, Ferreira-Silva J, Vieira J, Basto S, Filho P, Rocha J, Moura MT, Oliveira M.Post-diluents could potentially increase semen cryotolerance, but remain poorly explored in horses. The aim was to evaluate the efficiency of post-diluents on frozen-thawed semen viability of two stallions (S1-S2). The cryopreserved semen was thawed at 50°C for 40 seconds. Semen motility and acrosin activity (AA) were determined during the thermo-resistance test (TRT). Progressive motility of S2 semen decreased after 60 and 90 minutes of TRT (TRT60 and TRT90) on the control compared to both post-diluents. The total motility of both S1 and S2 decreased on TRT60 and TRT90 semen control versus b...
Li B, He X, Zhao Y, Bai D, Bou G, Zhang X, Su S, Dao L, Liu R, Wang Y, Manglai D.P-element induced wimpy testis-interacting RNAs (piRNAs) are essential for testicular development and spermatogenesis in mammals. Comparative analyses of the molecular mechanisms of spermatogenesis among different organisms are therefore dependent on accurate characterizations of piRNAs. At present, little is known of piRNAs in non-model organisms. Here, we characterize piRNAs in the Mongolian horse, a hardy breed that reproduces under extreme circumstances. A thorough understanding of spermatogenesis and reproduction in this breed may provide insights for the improvement of fecundity and repr...
Blommaert D, Sergeant N, Delehedde M, Jouy N, Mitchell V, Franck T, Donnay I, Lejeune JP, Serteyn D.A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentra...
Alamaary MS, Haron AW, Ali M, Hiew MWH, Adamu L, Peter ID.Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equa...
Gonzalez-Castro RA, Carnevale EM.We determined if microfluidic sorting (MF) of frozen-thawed stallion sperm improves sperm population characteristics and results in embryo development after intracytoplasmic sperm injection (ICSI). The efficiency and efficacy of MF sperm separation was evaluated by comparing pre- and post-separation sperm population variables. Procedural comparisons were performed after sorting with MF, single-layer colloidal centrifugation (SLC) or swim-up (SU), and cleavage and embryo development were evaluated after ICSI using MF-sorted sperm. In Experiment 1, when compared to the original sperm sample, MF ...
Swegen A, Smith ND, Gibb Z, Curry BJ, Aitken RJ.Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibi...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Janečka JE, Davis BW, Ghosh S, Paria N, Das PJ, Orlando L, Schubert M, Nielsen MK, Stout TAE, Brashear W, Li G, Johnson CD, Metz RP, Zadjali AMA....Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transp...
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...
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...
Steger K, Balhorn R.Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint...
Ellerbrock RE, Honorato J, Curcio BR, Stewart JL, Souza JAT, Love CC, Lima FS, Canisso IF.Urospermia is a common ejaculatory dysfunction of stallions. Current practice suggests that urine contaminated semen should not be used for cryopreservation. The aim of this study was to determine effects of urine contamination on semen freezing. Sixty-five ejaculates from eight stallions were divided into no urine (CONT), low (20% urine, LOW), and high (50% urine, HIGH) samples. Semen was extended with a commercial cooling extender, cushion-centrifuged, resuspended to 200 million/mL in a commercial egg-yolk based extender, and cryopreserved in liquid nitrogen. A subset of ejaculates (n = ...
Pozor M, Conley AJ, Roser JF, Nolin M, Zambrano GL, Runyon SP, Kelleman AA, Macpherson ML.Recently, anti-Müllerian hormone (AMH) was validated as a reliable marker of testicular damage caused by various chemotherapy drugs in humans and in mice. In horses, the reference values of AMH concentrations in normal stallions, during different seasons of a year, have been recently reported. However, this hormone was not evaluated in subfertile or infertile stallions with testicular damage. Therefore, the objective of this study was to investigate the effects of experimentally induced testicular degeneration on the concentration of AMH in stallions. Severe but transient testicular degenerat...
Demyda-Peyrás S, Bottrel M, Acha D, Ortiz I, Hidalgo M, Carrasco JJ, Gómez-Arrones V, Gósalvez J, Dorado J.The aim of this study was to evaluate the effect of different cooling rates on post-thaw quality of cryopreserved donkey spermatozoa. Eighteen ejaculates from six adult Andalusian donkeys (three ejaculates per donkey) were collected using an artificial vagina. Pooled semen samples (two ejaculates per pool) were divided into three aliquots, and frozen in Gent freezing extender using three different cryopreservation protocols (P): P1 (conventional slow freezing, as control): semen pre-cooled in an Equitainer for 2 h and frozen in liquid nitrogen (LN) vapour; P2 (controlled pre-freeze cooling r...
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...
Suliman Y, Becker F, Wimmers K.Poor fertility of breeding stallions is a recognised problem in the equine industry. The aim of the present study was to detect molecular pathways using two groups of stallions that differed in pregnancy rates as well as in the proportion of normal and motile spermatozoa. RNA was isolated from spermatozoa of each stallion and microarray data were analysed to obtain a list of genes for which transcript abundance differed between the groups (P ≤0.05, fold change ≥1.2). In all, there were 437 differentially expressed (DE) genes between the two groups (P ≤ 0.05, fold change ≥1.2). Next, th...
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...
Sudheer Kumar C, Swamy MJ.The major protein of horse seminal plasma, HSP-1/2, exhibits membranolytic and chaperone-like activities and plays a crucial role in regulating sperm capacitation. L-Carnitine is a small polar molecule present in high concentrations in mammalian seminal plasma. The present results demonstrate that L-carnitine binds to HSP-1/2 and increases its thermal stability, enhances cooperativity of its chemical unfolding and decreases both chaperone-like and membranolytic activities of this protein. The HSP-1/2-L-carnitine complex exhibits anti-oxidative behaviour by inhibiting the production of hydroxyl...
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,...
Robles M, Dubois C, Gautier C, Dahirel M, Guenon I, Bouraima-Lelong H, Viguié C, Wimel L, Couturier-Tarrade A, Chavatte-Palmer P.Primiparous mares are known to produce smaller foals than multiparous mares. This difference seems to be partly explained by the reduced exchange surface and volume of the placental villi in primiparous compared to multiparous placentas. The effect of maternal parity on foals' post-natal growth, metabolism and sexual maturation, however, has been given little consideration. The objectives of this work were to analyse placental biometry and structure at term, growth of foals and yearlings, their metabolism and testicular maturation at one year of age. Twenty multiparous mares (M), aged over 6 y...
Pozor MA, Muehlhaus J, King A, Macpherson ML, Troedsson MH, Bailey CS.The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PT...
Prasinou P, De Amicis I, Fusaro I, Bucci R, Cavallini D, Parrillo S, Caputo M, Gramenzi A, Carluccio A.Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid cont...
Gacem S, Catalán J, Yánez-Ortiz I, Soler C, Miró J.The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates f...
Zong E, Fan G.Generally speaking, the majority of F1 hybrids between the horse and donkey are sterile and do conform to the hypothesis of gametogenic breakdown. The results of our own research, nevertheless, lead us to conclude that there is variability in degree of sterility and fertility for both F1 and B1 individuals, with a gradual advance from sterility to fertility being seen. This is the subject we will discuss from the point of view of oestrus, ovulation, mating, pregnancy, spermatogenesis, endocrine activity, isozymes and karyotypic analysis in this paper.
Varner DD.The conventional approach to evaluation of stallion semen dates back several decades, and includes evaluation of spermatozoal concentration, semen volume, spermatozoon morphological characteristics, and spermatozoal motility patterns initially and following in-vitro storage. While an analysis performed in this manner does have predictive value, incorporation of some more newly developed techniques may improve the predictive value of the examination. This communication addresses some newer tests that can be applied today for evaluation of semen, as well some tests that may be available in the c...
Stout TA, Colenbrander B.There are a number of situations in which it is desirable to suppress part or all of the reproductive endocrine system in a horse, notably the competing animal whose tractability during training, or performance during competition, is compromised by the expression of sexual or aggressive behavior. The current therapeutic approaches to reproductive endocrine suppression include gonadectomy and progestagen administration, where the former carries surgical risks and entails irreversible loss of breeding potential, and effective progestagen therapy requires frequent administration for extended peri...
Costa GMJ, Avelar GF, Lacerda SMSN, Figueiredo AFA, Tavares AO, Rezende-Neto JV, Martins FGP, França LR.The establishment of proper conditions for spermatogonial stem cells (SSCs) cryopreservation and storage represents an important biotechnological approach for the preservation of the genetic stock of valuable animals. This study demonstrates the effects of different cryopreservation protocols on the survival rates and phenotypic expression of SSCs in horses. The cells were enzymatically isolated from testes of eight adult horses. After enrichment and characterization of germ cells in the suspension, the feasibility of several cryopreservation protocols were evaluated. Three different cryomedia...
Pukazhenthi BS, Johnson A, Guthrie HD, Songsasen N, Padilla LR, Wolfe BA, Coutinho da Silva M, Alvarenga MA, Wildt DE.Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4°C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4°...
Li B, He X, Zhao Y, Bai D, Li D, Zhou Z, Manglai D.Numerous studies have shown that microRNAs (miRNAs) are essential for testicular development and spermatogenesis. In order to further characterise these physiological processes, three immature and three mature testes of the Mongolian horse were collected and six libraries were established. Using small RNA sequencing technology, 531 mature miRNAs were identified, including 46 novel miRNAs without previously ascribed functions. Among the 531 miRNAs, 421 were expressed in both immature and mature libraries, 65 miRNAs were found solely in immature testis libraries and 45 miRNAs were found solely i...
Pozor MA, Macpherson ML, McDonnell SM, Nollin M, Roser JF, Love C, Runyon S, Thomas BF, Troedsson MH.The objective of this study was to determine whether an indenopyridine derivative RTI-4587-073(l) was a good candidate for male contraception in horses. We hypothesized that a single administration of RTI-4587-073(l) causes significant suppression of testicular function in stallions without affecting sexual behavior. Three Miniature horse stallions received a single dose of 12.5 mg/kg RTI-4587-073(l) orally (group "treated"), whereas three other Miniature horse stallions received placebo only (group "control"). Semen was collected and evaluated from all stallions twice a week for three baselin...
Gadella BM, Rathi R, Brouwers JF, Stout TA, Colenbrander B.During sexual reproduction, the sperm and oocyte must fuse before the production of a diploid zygote can proceed. In mammals such as equids, fusion depends critically on complex changes in the plasma membrane of the sperm and, not surprisingly, this membrane differs markedly from that of somatic cells. After leaving the testes, sperm cease to synthesize plasma membrane lipids or proteins, and vesicle-mediated transport stops. When the sperm reaches the female reproductive tract, it is activated by so-called capacitation factors that initiate a delicate reorientation and modification of molecul...
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...
Foote RH.Several inherited conditions associated with testicular defects, abnormal spermatogenesis and morphologically abnormal sperm have been found. These usually are controlled by single gene pairs. A notable exception is testicular size, with heritability in young bulls ranging from .42 to .88. Testicular size directly affects sperm output potential. The major contributor to variation in semen quality is the environment. Environmental effects may be temporary or permanent. Permanent effects occurring during prenatal and prepubertal periods and temporary or permanent factors acting after spermatogen...
Pirhonen A, Linnala-Kankkunen A, Mäenpää PH.Protamines isolated from ejaculated human, stallion, bull, boar, and ram spermatozoa were subjected to phosphoserine conversion reaction and protein sequencing. Phosphoserines were detected as S-ethylcysteines. Endogenously phosphorylated protamines have previously been found only in ejaculated human sperm. In this study, we demonstrate that ejaculated sperm from other species also contain protamines phosphorylated at serine residues. In P1-protamines, the endogenously phosphorylated serines were located at the N-terminal region in all species studied, whereas in major forms of human and stall...
Heninger NL, Staub C, Blanchard TL, Johnson L, Varner DD, Forrest DW.Apoptosis in testicular germ cells has been demonstrated in many mammalian species. However, little is known about the stallion (Equus caballus) and rates of apoptosis during spermatogenesis. Morphological and biochemical features of apoptosis reported in other species were used to confirm that the TdT-mediated dUTP Nick end labeling (TUNEL) assay is an acceptable method for identification and quantification of apoptotic germ cells in histological tissue sections from stallion testis. Seminiferous tubules from eight stallions with normal testis size and semen quality were evaluated according t...
Tardif S, Brady HA, Breazeale KR, Bi M, Thompson LD, Bruemmer JE, Bailey LB, Hardy DM.Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acroso...
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...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design. Half of the ejaculate was analyzed as a fresh ...
Gross MK, Toscano DG, Toscano WA.Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by...
Calvete JJ, Mann K, Schäfer W, Sanz L, Reinert M, Nessau S, Raida M, Töpfer-Petersen E.We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (...
Jung HJ, Song H, Yoon MJ.Deleted in azoospermia-like (DAZL) is used as a germ cell marker in several species, including mice, rats, pigs, rhesus monkeys, bulls, and humans. Our objectives with this study were to investigate DAZL expression in stallion germ cells by using immunofluorescence, immunocytochemistry, and western blotting, and to determine the effects of reproductive stage and breeding season on the DAZL-positive cell population in seminiferous tubule cross sections. Testes were obtained during routine castration procedures at a large animal clinic and routine field service castration. The reproductive stage...
Love CC, Kenney RM.A variety of testicular insults can induce changes in the structure of spermatozoal chromatin, resulting in spermatozoal DNA that is more susceptible to acid-induced denaturation. The degree of change in the DNA can be measured using the sperm chromatin structure assay (SCSA). The SCSA measures the relative amounts of single- and double-stranded DNA after staining with the metachromatic dye, acridine orange. Here we used a stallion model (n = 4) to study the effects of scrotal heat stress on spermatozoal DNA. This model was created by insulating stallion testes for 48 h and collecting sperm da...
Lanneau M, Loir M.Using standardized methods for protein extraction and analysis, the testes of rams, bulls, goats, boars, stallions, rats, cats, hedgehogs, European mink and ferrets were examined for basic spermatid nucleoproteins by electrophoresis. The results suggest that differences exist in the total number of these proteins as well as in the number and amount of the cross-linked cystein-containing proteins. These differences appear to be more family-specific than species-specific.
Johnson L.Spermatocytogenesis plays a pivotal role in regulation of spermatogenesis; however, its details remain relatively obscure in nonrodent species. The equine testis contains approximately 100% more spermatogonia in summer than in winter and appears to be a good model to identify the flexible components of spermatocytogenesis that cause seasonal changes in daily sperm production. Testes were taken from horses in the winter (n = 47) and in summer (n = 43). Tissues were fixed by glutaraldehyde-perfusion and submission in osmium, embedded in Epon or methacrylate, sectioned at 0.5 micron or 5 microns,...
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...
Peña FJ, Ortega Ferrusola C, Martín Muñoz P.Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simul...
Kikuchi M, Takao Y, Tokuda N, Ohnami Y, Orino K, Watanabe K.Total 78 semen samples were obtained from 27 Thoroughbred stallions (aged 6 to 27 years), and were subjected to quantification of lactoferrin (Lf) in seminal plasma and examination of the seminal properties. The seminal plasma Lf concentration varied from 21 to 689 microg/ml, with a mean value of 244 +/- 151 microg/ml (S.D.). The seminal plasma Lf concentration and total seminal plasma Lf positively correlated with the sperm concentration (r=0.5938, P<0.001) and with the total sperm number (r=0.6959, P<0.001), respectively. There was no correlation between seminal plasma Lf and sperm mot...
Giesecke K, Hamann H, Stock KF, Klewitz J, Martinsson G, Distl O, Sieme H.The research of fertility in humans and other mammals has strongly advanced in the recent years. The examination of molecular mechanisms influencing horse fertility is relatively recent. We chose the angiotensin converting enzyme (ACE), the sperm autoantigenic protein 17 (SP17) and the follicle stimulating hormone (FSHB) as candidates for determining stallion fertility and to analyze associations of intragenic single nucleotide polymorphisms (SNPs), flanking microsatellites and candidate-gene linked haplotypes with the pregnancy rate per oestrus (PRO) in 179 Hanoverian stallions. Fertility tra...
Thompson DL, Pickett BW, Squires EL, Amann RP.Factors affecting testicular measurements in situ and the relationships among the measurements and various reproductive characteristics were studied using data from 48 stallions. Mean values during the breeding season are provided for scrotal width, widths and lengths of individual testes, combined weight of testicular parenchyma, daily sperm production and daily sperm output. Testicular measurements were highly repeatable from day to day and for repeated measurements on a given day; technician provided the largest source of variation in the measurements of a given stallion. Age significantly ...
Grignard E, Morin J, Vernet P, Drevet JR.We report here on the cloning of cDNAs coding bovine and equine orthologs of mouse epididymis-restricted and sperm-bound glutathione peroxidase 5 (GPX5), a selenium-independent member of the multigenic GPX family in mammals. The complete sequence of bovine GPX5 as well as a partial sequence of the equine GPX5 were characterized, conceptually translated and aligned with other known mammalian GPX5 proteins. Using Northern blotting assays, we show that the level of expression of GPX5 is high in bovine but low in equine and that in both species the regionalization of GPX5 expression in epididymis ...
