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.
Bugno-Poniewierska M, Kij-Mitka B, Podstawski Z, Długosz B, Lasek O, Mrowiec P, Tischner M.The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, u...
Orsolini MF, Meyers SA, Dini P.As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towa...
Orsolini MF, Meyers SA, Dini P.As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.
Bresnahan DR, Lupole RE, Stilz CR, Carnevale EM.Zona pellucida (ZP) proteins are important for fertilization and sperm binding and are closely associated with cumulus cells. Communication between cumulus and oocytes is facilitated by intracellular membrane channels composed of connexins. The extent aging impacts potential differences in fertilization and reductions in fertility is not well understood. This study characterized age-related differences in transcript abundance of ZP proteins and connexins in cells from ovarian follicles. Additionally, differences in sperm binding to oocytes from old and young mares was evaluated. For experiment...
Gmel AI, Burger D, Neuditschko M.The use of frozen-thawed semen is an important reproduction tool to preserve the biodiversity of small, native horse breeds such as the Franches-Montagnes (FM). However, not all stallions produce cryotolerant semen with a progressive motility after thaw ≥ 35%. To improve our understanding of the genetic background of male fertility traits in both fresh and frozen-thawed semen, we performed genome-wide association studies (GWAS) on gel-free volume, sperm cell concentration, total sperm count, and progressive motility in fresh and frozen-thawed semen from 109 FM stallions using 335,494 genome-...
Serafini R, Varner DD, Love CC.Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to compare two testicular sample acquisition techniques (needle aspirate-N and tissue wedge-T) and results when using flow cytometry and histology procedures. Testicular cell types were stained with acridine orange, and nine regions (R2 to R10) were identified and enumerated following acquisition by either N or T. Testes were also grouped and analyzed by size and sexual maturity (Small [immature] compared with Large [mature]) and used...
Yánez-Ortiz I, Catalán J, Mateo-Otero Y, Dordas-Perpinyà M, Gacem S, Yeste N, Bassols A, Yeste M, Miró J.Jenny shows a large endometrial reaction after semen influx to the uterus with a large amount of polymorphonuclear neutrophils (PMN) migrating into the uterine lumen. PMN act as a sperm selection mechanism through phagocytosis and NETosis (DNA extrudes and, together with proteins, trap spermatozoa). While a reduced percentage of spermatozoa are phagocytosed by PMN, most are found to be attached to neutrophil extracellular traps (NETs). This selection process together with sperm metabolism produces a large amount of reactive oxygen species (ROS) that influence the reproductive success. The pres...
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...
Scarlet D, Handschuh S, Reichart U, Podico G, Ellerbrock RE, Demyda-Peyrás S, Canisso IF, Walter I, Aurich C.It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy wer...
Wiebke M, Hensel B, Nitsche-Melkus E, Jung M, Schulze M.This review is part of the Festschrift in honor of Dr. Duane Garner and provides an overview of current techniques for cooled storage of semen from livestock animals. The first part describes the current state of the art of liquid semen preservation in boars, bulls, and stallions, including the diluents, use of additives, processing, temperature, and cooling of semen. The species-specific physiology and varying extents of cold shock sensitivity are taken into consideration. In addition, factors influencing the quality of cooled-stored semen are discussed. Methods, trends, and the most recent a...
Fernández-Hernández P, Marinaro F, Sánchez-Calabuig MJ, García-Marín LJ, Bragado MJ, González-Fernández L, Macías-García B.Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida . Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle ( = 4) or a recent ovulation ( = 4); the samples were kept at -80°...
Kútvölgyi G, Brabender K, Andersson M, Javkhlan A, Nagy S, Páble T, Egerszegi I, Hidas A, Soós I, Kovács A.The case of an 8-year-old, sexually active but infertile Przewalski's stallion (Equus ferus przewalskii) was studied. Besides the infertility, the stallion also showed permanent problems with its body condition, being obviously weaker than all the other group members. The horse was kept in a separate place for two years with 12 mares in its harem group (six mares had foals earlier); however, none of the mares covered got pregnant. Andrological and cytogenetic investigations revealed underdeveloped testes, arrested spermatogenesis, azoospermia, and XY/XXY/X0 mosaicism. We classify the case as a...
Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS.The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using com...
Mateo-Otero Y, Zambrano F, Catalán J, Sánchez R, Yeste M, Miro J, Fernandez-Fuertes B.In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. Objective: To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in respon...
Gacem S, Valverde A, Catalán J, Yánez Ortiz I, Soler C, Miró J.This study aimed to characterize the sperm kinematic values with high frames per second, to define the subpopulation structure of a horse and a donkey and compare them. A total of 57 fresh semen ejaculates (26 Spanish and 16 Arabian horse breeds and 10 donkeys) were collected and subsequently analyzed for kinematic parameters using the Computer-aided sperm motility analysis ISAS®v1.2 system and using a Spermtrack® 10-μm depth counting chamber. Sequences were recorded at 250 frames per second, and eight kinematic parameters were automatically evaluated. All kinematic parameters showed signif...
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...
Bazzano M, Laus F, Spaterna A, Marchegiani A.Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from differ...
Olğaç KT, Akçay E.In this study, the effects of polyamines, Spermine and Spermidine, on long-term preservation and post-thaw spermatological parameters were evaluated. Moreover, determination of the most suitable polyamine and its dose that can be added to standard extenders were aimed. Four adult Arabian stallions were used in the study. Five ejaculates were collected from each of four stallions via artificial vagina two days interval. Each ejaculate was divided into 13 aliquots. INRA96 (95,5%), egg yolk (2%), and glycerol (2,5%) were used as a control extender. Extenders of experimental groups were prepared w...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, Silva-Rodríguez A, Gil MC, Ortega-Ferrusola C, Peña FJ.Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a spli...
Magalhaes HB, Canuto LEF, Zhao F, Li M, Li X, Ji C, Canisso IF.This study aimed to assess the sexual behavior of jacks mounting jennies in estrus and determine the daily sperm output (DSO) and spermatogenic efficiency using two equations to calculate testicular volume (TV). Eight sexually rested mature jacks, aging 5 to 10 years old, had semen collected once a day for 10 consecutive days using jennies in good standing estrus for mounting. Sexual behavior and semen parameters were assessed during each collection. Testicular measurements of height, width, and length were taken immediately before the first semen collection, and these measurements were used t...
Silva DEC, Penitente-Filho JM, Souza Netto DL, Waddington B, de Oliveira RR, Guimarães JD.This study aimed to describe the testicular growth curve in Mangalarga Marchador stallions fitting nonlinear models to data of total scrotal width (TSW) and testicular volume (TV). A cross-sectional study was conducted with 120 stallions, aging from 1.75 to 21.93 years. The parameters of nonlinear models (Brody, Gompertz, Hill, Logistic I and II, Meloun I and II, Michaelis-Menten, Mitscherlich, and von-Bertalanffy) were estimated by Gauss-Newton iterative process. Goodness of fit was evaluated by the corrected Akaike (AICc) and Bayesian (BIC) information criteria, adjusted R, error sum of squa...
D'Anza E, Albarella S, Galdiero G, Tafuri S, Del Prete C, Cocchia N, Ciani F, Mastellone V, Pasolini MP, Carotenuto D, Selvaggi M, Ciotola F....The reproductive performances of livestock play an essential role in the economic management of the farm. The improvement of semen quantity and quality through the use of food supplements that lack substances which are forbidden in animal feeding, or that may have detrimental effects, is an important goal. Maca (Lepidium meyenii) is a plant that has been used for centuries in the Andes for nutrition and fertility enhancement in humans and animals. The aim of this study was to evaluate the effects of food supplementation of stallions with maca during the breeding season on spermatozoa parameter...
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 ...
Gimeno BF, Bariani MV, Laiz-Quiroga L, Martínez-León E, Von-Meyeren M, Rey O, Mutto AÁ, Osycka-Salut CE.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we e...
Papin J, Stuhtmann G, Martinsson G, Sieme H, Lundeheim N, Ntallaris T, Morrell JM.Removal of seminal plasma facilitates stallion sperm survival during storage, but washing may damage sperm chromatin. Therefore, sperm quality was compared in samples following single-layer centrifugation (SLC) or sperm washing and controls (extension only) in two extenders, INRA82 and INRA96. Ejaculates from six stallions were split among six treatments: SLC, sperm washing, and controls, in INRA82 and INRA96. Sperm motility and acrosome status were evaluated at 0, 24, 48, 72 and 96 hours; morphology at 0, 24, 48, 72 hours and chromatin integrity at 0 and 96 hours, with storage at 6°C. Sperm ...
Cavalero TMS, Segabinazzi LGTM, Scheeren VFDC, Freitas-Dell'Aqua CP, Papa FO.The present study compared the quality of sperm collected by artificial vagina or pharmacologically induced ejaculation from a 10-year-old thoroughbred stallion with seminal vesiculitis. The pharmacological protocol involved intravenous administration of detomidine (0.01 mg/kg) and oxytocin (20 IU) and successfully induced ejaculation in all attempts of semen collection. Sperm motility, plasma membrane and acrosome integrity (PMAI), reactive oxygen species (ROS) levels, polymorphonuclear neutrophil (PMN) percentage, and bacterial profiles of fresh and cooled semen (5°C for 24 hr) were eval...
Ponthier J, Rigaux G, Parrilla-Hernandez S, Egyptien S, Gatez C, Carrasco Leroy C, Deleuze S.This report describes an option to modulate the testicular function of wild horses and field methods to assess it. Non-surgical castration of a captive wild Przewalski's stallion with anti-gonadotropin-releasing hormone (GnRH) immunization was performed by sub-cutaneous injection of two doses of 450 μg (3 ml) of GnRH conjugated to diphtheria toxin, further repeated every 6 months. Semen quality was assessed after collection by electro-ejaculation under general anesthesia. Endocrine and behavioral consequences were studied during a 2-year follow-up period. The procedure of electro-ejaculation ...
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...
Crespo F, Wilson R, Díaz-Jimenez M, Consuegra C, Dorado J, Barrado BG, Gosálvez J, Smit RL, Hidalgo M, Johnston S.Horses are long-day seasonal breeding animals, however, with modern stallion reproductive management it is important for collection of semen during periods that are not part of the traditional breeding season. This study was conducted to examine variation in the seminal characteristics of individual stallions in Avila, Spain during 1 year with a particular emphasis on sperm DNA fragmentation. Semen was collected twice per season from a total of 20 stallions. There was a marked seasonal effect on all seminal characteristics, with the greatest on progressive motility, % membrane integrity and le...
Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips G, Silva A, Tapia JA, Gil MC, Redondo E, Masot J, Ortega-Ferrusola C, Peña FJ.Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter f...
Hoffmann B, Landeck A.To gain further information on gonadal function of the stallion, concentrations of testicular steroids in blood plasma (bpl) and seminal plasma (spl) and their distribution in the ejaculate were determined. Blood and semen samples from a total of 11 stallions were collected from November to July. Estrone (E1), estrone sulfate (E1S), estradiol-17beta (E2beta) and testosterone (T) were determined in bpl and spl, and in addition androstenedione (A), dehydroepiandrosterone (DHEA) and 5alpha-dihydrotestosterone (5alpha-DHT) were measured in spl. At certain points of time, aliquots of an ejaculate w...
Pereyra-Bonnet F, Fernández-Martín R, Olivera R, Jarazo J, Vichera G, Gibbons A, Salamone D.Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated ...
Contri A, De Amicis I, Molinari A, Faustini M, Gramenzi A, Robbe D, Carluccio A.In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after suppl...
Claes A, Ball BA, Almeida J, Corbin CJ, Conley AJ.Anti-Müllerian hormone (AMH), a homodimeric glycoprotein, is secreted early in fetal life when it exerts a crucial function in sexual differentiation. The secretion of AMH in male humans persists after birth and is characterized by high prepubertal concentrations followed by a significant decrease at the onset of puberty. The expression of AMH in the normal and cryptorchid equine testis is well characterized but data regarding circulating AMH concentrations are lacking. The objectives of this study were to determine serum AMH concentrations in neonatal colts and fillies, prepubertal colts, an...
Kareskoski M, Katila T.Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, and ejaculated as several consecutive fluid fractions. The composition of seminal plasma and the effects on sperm longevity vary between fractions and individual stallions. This review focuses on the sequence of ejaculation, constituents of seminal plasma and their potential use as fertility markers as well as the influence of seminal plasma on spermatozoa during storage.
Nicander L, Malmqvist M.Principal cells in the initial segment of the epididymis in horses, cattle, pigs, sheep, dogs, cats, and rabbits have an abundant, partly rough, endoplasmic reticulum and a large Golgi complex. Small vacuoles with opaque content seem to be formed by the Golgi complex and move to the cell apex, where they empty their contents into the lumen by a merocrine mechanism.
Oldenhof H, Wolkers WF, Sieme H.In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation allows for long-term storage of insemination doses and secures reproduction at a desired time point. In order to cryopreserve semen, it needs to be carefully processed to preserve its vital functions after thawing. In this chapter, we describe the processes involved in cryopreservation of bull, stallion, and boar sperm. These include preparation of diluents, dilution of sperm in primary and freezing extender, slow cooling from room temperature to 5 °C, packaging of insemina...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 1...
Janett F, Thun R, Niederer K, Burger D, Hässig M.The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawe...
Jung H, Roser JF, Yoon M.Spermatogonial stem cells (SSCs) continuously undergo self-renewal and differentiation to sustain spermatogenesis throughout adulthood in males. In stallions, SSCs may be used for the production of progeny from geldings after cryopreservation and therapy for infertile and subfertile stallions. Undifferentiated cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans and rats. The main purposes of this study are to determine the following: 1) changes in the expression pattern of UTF1 at various reproductive stages of stallions, 2) subpopulations of sp...
Mateo-Otero Y, Zambrano F, Catalán J, Sánchez R, Yeste M, Miro J, Fernandez-Fuertes B.In several mammalian species, acute endometritis driven by the recruitment of polymorphonuclear cells (PMN) occurs in response to semen. These PMNs release DNA to form neutrophil extracellular traps (NETs) in cattle, horse and human, leading to sperm entrapment. While there is no evidence of this phenomenon occurring in donkeys, artificial insemination (AI) with frozen-thawed semen, which results in very poor pregnancy rates, leads to a large PMN recruitment to the uterus. Objective: To investigate whether donkey semen can trigger NET release (NETosis) and if excessive NETosis occurs in respon...
Turner RM, McDonnell SM.Significant amounts of alkaline phosphatase (AP) activity have been found in semen plasma from numerous species. In species in which the majority of semen plasma AP (SPAP) activity originates from the epididymis and testicle, SPAP activity can be used clinically as a marker to differentiate testicular origin azoospermia or oligospermia from ejaculatory failure. Information on SPAP activity in stallions to date has been limited. In this study, a standard clinical chemistry analyzer was used to determine AP activity in pre-ejaculatory fluid and ejaculates from groups of normal stallions. Additio...
Livolant F.The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chroma...
Martin Muñoz P, Ortega Ferrusola C, Vizuete G, Plaza Dávila M, Rodriguez Martinez H, Peña FJ.Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard tech...
Hejmej A, Bilińska B.Evidence collected over the years has demonstrated that cryptorchidism is associated with a defect in spermatogenesis and, as a consequence, with either reduced fertility or infertility. However, the effect of cryptorchidism on Leydig cell function is less clear. The aim of our study therefore was to investigate the regulation of steroid hormone biosynthesis and, additionally, intercellular communication in the cryptorchid equine testes. Methods: Testes of mature bilaterally cryptorchid horse and healthy stallions were used for this study. The expression of luteinising hormone receptor (LHR), ...
Talluri TR, Mal G, Ravi SK.To investigate various biochemical components of seminal plasma in Marwari stallions and Poitou Jacks and to find out their correlation with that of the seminal characteristics. Methods: In this study, semen was collected from six Marwari stallions and six Poitou jacks aged from 4 to 6 years and with known fertility status. The semen collection from the stallions were collected during the breeding season, i.e., between the months of April and June. From the collected semen ejaculates, we estimated the values of some biochemical components, viz., total protein content, total lipid content, and ...
Pearl CA, Mason H, Roser JF.In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular...
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...
Yoon MJ, Roser JF.Leydig cells located in the interstitial space of the testicular parenchyma produce testosterone which plays a critical role in the maintenance and restoration of spermatogenesis in many species, including horses. For normal spermatogenesis, maintaining Leydig cells is critical to provide an optimal and constant level of testosterone. Recently, an anti-apoptotic effect of IGF-I in testicular cells in rats has been reported, but a similar effect of IGF-I on equine Leydig cells remains to be elucidated. If IGF-I also protects stallion testicular cells from undergoing apoptosis, then IGF-I may ha...
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...
Kavak A, Lundeheim N, Aidnik M, Einarsson S.The standard procedure for assessing the breeding potential of a stallion includes the parameter total number of spermatozoa classified as morphologically normal. This study investigated sperm morphology of fresh semen in randomly chosen Estonian (E, n = 8) and Tori (T, n = 7) breed stallions with proven fertility. Two ejaculates were examined from each stallion. An aliquot from each ejaculate was fixed in 1 mL formol-saline immediately after collection and examined with phase-contrast microscope at a magnification 1000x for all types of morphological abnormalities. Furthermore smears were pre...
Rode K, Sieme H, Richterich P, Brehm R.The formation of the blood-testis barrier (BTB) is defined as occurring with the first appearance of spermatocytes at around puberty and is vital for normal spermatogenesis. This barrier between two adjacent Sertoli cells (SCs) consists of a cell junctional protein complex, which includes tight junctions (TJs), adherens junctions, and gap junctions. In many mammalian species, BTB composition has already been investigated, whereas little is known about the equine BTB. In the present study, immunohistochemistry and qualitative Western Blot analysis were used to assess the expression and distribu...
Johnson L, Thompson DL.Testes and blood samples were obtained from 201 stallions aged 6 months to 20 years in either December-January (nonbreeding season) or June-July (breeding season) to study the effect of age and season on reproductive parameters. Seasonal differences in the Sertoli cell population of adult (4-20 years old) horses were characterized by a 36% larger number of Sertoli cells in the breeding season than in the nonbreeding season. Seasonal elevation in the Sertoli cell population was associated with an increase in testicular weight and daily sperm production per testis (DSP/testis). Concentrations of...
Calvete JJ, Raida M, Gentzel M, Urbanke C, Sanz L, Töpfer-Petersen E.In the bovine, seminal plasma heparin-binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin-like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin- and phosphorylcholine-binding proteins of stallion and boar seminal plasma. Horse seminal plasma proteins HSP-1 and HSP-2, and boar protein pB1, belong to the same family as the bull heparin- and phosphorylcholine-binding proteins BSP-A1/2, BSP-A3, and BSP-30K. We have determined the amino acid sequence and posttrans...
Giesecke K, Hamann H, Stock KF, Woehlke A, Sieme H, Distl O.Stallion fertility is an economically important trait because the use of artificial insemination is increasing in the horse industry and superior sires are used more intensely. Molecular genetic markers may be useful as early indicators for a stallion's fertility and genetic improvement programmes. The testis-specific SPATA1 protein is involved in shaping the sperm head during spermatogenesis. Thus, the spermatogenesis associated 1 (SPATA1) gene was chosen as candidate for stallion fertility, and we analysed intragenic single nucleotide polymorphisms (SNPs) as genetic markers for the least squ...
Almeida J, Conley AJ, Ball BA.Cryptorchidism affects 2-8% of male horses and the affected testis undergoes a disruption of normal spermatogenesis. The underlying molecular changes are poorly understood in the cryptorchid equine testis. Objective: Compare the expression of anti-Müllerian hormone (AMH), anti-Müllerian hormone receptor (AMHR2), androgen receptor (AR), cyclin kinase inhibitor (CDKN1B), connexin 43 (Cx43), 3β hydroxysteroid dehydrogenase/Δ(5) -Δ(4) - isomerase (3βHSD), P450c17 hydroxylase/lyase (P450c17) and cytochrome P450 aromatase (P450arom) in the undescended testis of cryptorchid stallions with that ...
Moraes CR, Runcan EE, Blawut B, Coutinho da Silva MA.The iSperm is a newly released semen analysis tool from Aidmics Biotechnology Co. LTD, which allows an iPad Mini to be transformed into a handheld microscope with objective semen analysis software for equine available through the Apple Store (version 4.5.2). The aim of this study was to compare iSperm values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. Two ejaculates from each of five Standardbred stallions were used to compare sperm motility (computer-assisted semen anal...
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...
Muñoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Peña FJ.Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the prese...
Johnson L, Blanchard TL, Varner DD, Scrutchfield WL.Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells) and germ cells (spermatogonia, spermatocytes, and spermatids). Activities of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic materia...
