Topic:Sperm
Equine sperm refers to the male reproductive cells produced by stallions, essential for the process of fertilization and successful breeding in horses. The study of equine sperm encompasses various aspects, including morphology, motility, viability, and genetic integrity. These parameters are critical for assessing stallion fertility and improving breeding outcomes. Research in this field often focuses on understanding the factors that influence sperm quality, such as age, nutrition, and environmental conditions. Additionally, advancements in assisted reproductive technologies, such as artificial insemination and cryopreservation, rely heavily on the detailed study of sperm characteristics. This page compiles peer-reviewed research studies and scholarly articles that explore the biology, evaluation, and technological applications related to equine sperm.
Aerobic bacterial flora of semen and stallion reproductive tract and its relation to fertility under field conditions. This study was initiated in order to investigate the bacterial flora of the stallion genital tract by taking consecutive samples from normal stallions in regular use. The objective was to determine whether any growth of potential pathogens, particularly P. aeruginosa and K. pneumoniae, in fresh semen and urethra was associated with the presence of inflammatory cells in the semen and whether bacterial growth had any effect on sperm morphology and pregnancy results. Sixteen stallions, only used for A.I., housed at 3 different commercial stud farms, were used. A wide variety of microorganisms was...
Equine CRISP-3: primary structure and expression in the male genital tract. Although originally described in the male rodent genital tract, cysteine-rich secretory proteins (CRISPs) are expressed in a variety of mammalian tissue and cell types. The proteins of the male genital tract have been observed associated to spermatozoa and are believed to play a role in mammalian fertilization. Here we describe the identification and primary structure of the first equine member of the CRISP family. Equine CRISP-3 is transcribed and expressed in the stallion salivary gland, in the ampulla and the seminal vesicle. It displays all 16 conserved cysteine residues and shows 82% homo...
Progesterone-induced acrosome reaction in stallion spermatozoa is mediated by a plasma membrane progesterone receptor. The aim of the present study was to investigate whether the induction of stallion sperm acrosome reaction (AR) by progesterone is mediated by binding of progesterone to a receptor on the sperm plasma membrane or to an intracellular progesterone receptor. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Alternatively, an indirect immunofluorescence technique employing a monoclonal antibody (C-262) against human intracellu...
Prostasome-like particles in stallion semen. Human semen contains membranous vesicles called prostasomes. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin, and Ca2+. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. No prostasome-like vesicles have been described in horse semen up to now. We have demonstrated the presence of prostasome-like vesicles in the equine semen and characterized them as to size, morphology, and lipid composition; we have found that they are similar to human prostasomes in many respects. We prop...
Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida. The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a...
Treatment of equine oocytes with A23187 after intracytoplasmic sperm injection. In vitro matured horse oocytes with a first polar body (n = 68) were each injected with a single spermatozoon and divided into 2 groups: Group 1 oocytes were treated with 10 microM calcium ionophore A23187 for 5 min while Group 2 oocytes received no activation treatment. After culture in vitro for 2 days, significantly more oocytes treated with A23187 (5/24, 21%) cleaved than oocytes without activation treatment (2/44, 5%, P<0.05). All 7 cleaved zygotes from both treatment groups were transferred to recipient mares but no pregnancies resulted.
Assessing the fertility potential of equine semen samples using the reducible dyes methylene green and resazurin. The objective of this study was to determine if spermatozoal reduction of the dyes methylene green to colorless and resazurin to pink or colorless was associated with the fertility potential of an equine semen sample. Fifty samples from 38 stallions were evaluated for the number of spermatozoa per milliliter and number of motile sperm per milliliter. Methylene green (20 micrograms/mL of semen) or resazurin (85 micrograms/mL of semen) was added to 3-mL aliquots of semen. Semen samples were identified as having low fertility potential (< 200 x 10(6) total cells/mL and or = 200 x 10(6) total ...
Intracytoplasmic sperm injection of in vitro-matured equine oocytes. Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and process...
Extraction and quantification of acrosin, beta-N-acetylglucosaminidase, and arylsulfatase-A from equine ejaculated spermatozoa. Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in ...
Isolation and characterization of a protein with homology to angiotensin converting enzyme from the periacrosomal plasma membrane of equine spermatozoa. The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68-70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related to 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ...
[Separation techniques ro achieve vital and reproduction competent equine spermatozoa populations–a survey]. Equine ejaculates are significantly characterized by widely varying parameters especially in those of practical relevance for equine Al. Therefore it is of interest for practical purposes to get subpopulations of concentrated, vital, and competent spermatozoa from the origin ejaculates. Special preparation of the donor stallions will stabilize sperm output. Fractionated semen collection from stallions supplies sperm enriched seminal fractions very useful to work with further in semen preservation. Most important to achieve a concentrated sperm subpopulation are semen manipulations post ejacula...
Effect of milk fractions on survival of equine spermatozoa. Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts...
Hydrolysis of extracellular adenine nucleotides by equine epidydimal spermatozoa. Ectoenzymic activities capable of hydrolyzing ATP sequentially to adenosine are present on equine epidydimal spermatozoa membranes. Kinetic parameters for ATPase, ADPase and 5'-nucleotidase were obtained by analysis of progress reactions curve when ATP, ADP and AMP were supplied as initial substrates. These values are not different from those found when the substrates were supplied from the preceding reactions. Feed-forward inhibition on 5'-nucleotidase by ATP/ADP was taken into account to fit simulated data to the experimental results. None of the substrates supplied by the preceding reaction...
Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa. Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin...
Assessment of viability and mitochondrial function of equine spermatozoa using double staining and flow cytometry. An objective double-staining method was developed to evaluate viability and mitochondrial function of stallion spermatozoa using flow cytometry. Sperm viability was assessed by propidium iodide (PI) exclusion, and mitochondrial function was measured by the intensity of rhodamine 123 (R123) fluorescence. Flow cytometry estimates of sperm viability measured by PI were equivalent (P > 0.05) to estimates made using Hoechst 33258 stain and fluorescent microscopy (% dead: 25 +/- 2.4 vs 21.5 +/- 3.5). The use of both PI and R123 was validated by addition of various proportions of freeze-shocked (m...
Immunohistochemical localization in the stallion genital tract, and topography on spermatozoa of seminal plasma protein SSP-7, a member of the spermadhesin protein family. SSP-7 is a protein originally isolated from stallion seminal plasma. It has extensive amino acid sequence homology with boar spermadhesin AWN, and, like its porcine counterpart, SSP-7 displays zona pellucida-binding activity. Strikingly, however, immunohistochemical studies presented here show that the stallion and the boar spermadhesin homologues are secreted at different places of the male genital tract. Furthermore, indirect immunofluorescence shows that the topography of SSP-7 on the surface of stallion spermatozoa is restricted to the equatorial segment, whereas boar AWN epitopes cover th...
Sperm head morphometry analysis of ejaculate and dismount stallion semen samples. The evaluation of seminal characteristics is important in the clinical detection of stallion subfertility. Conventional semen evaluation includes subjective determination of sperm concentration, motility, and gross morphology. Due to the subjectivity and variability of the manual morphology assessment, computer automated sperm morphology analyses has been developed. Computer automated sperm morphology analysis was applied in the current study to determine if the morphometric measurements of sperm heads from collected and dismount samples of the same ejaculate were similar. If the post-ejaculat...
Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1. 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...
Intracytoplasmic sperm injection (ICSI) versus conventional IVF on abattoir-derived and in vitro-matured equine oocytes. Conventional IVF as well as several assisted microfertilization techniques have shown limited success in the horse. After recent positive results achieved with intracytoplasmic injection of a single spermatozoon (ICSI) in human IVF, we chose to try the method in the horse. We compared conventional IVF to ICSI by fertilization rates of oocytes with compact and expanded cumuli and by developmental potential of the resulting embryos. Cumulus-oocyte complexes (COCs) were obtained by aspirating the follicular fluid from the ovaries of slaughtered mares. Complexes showing complete cumulus investment...
Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC...
Antibody directed against plasma membrane components of equine spermatozoa inhibits adhesion of spermatozoa to oviduct epithelial cells in vitro. Before fertilization, equine spermatozoa adhere to oviduct epithelial cells (OEC) of the mare. The biochemical basis for this adhesion has not been determined. Our objective was to produce an antiserum to block this interaction. Ejaculated spermatozoa were subjected to nitrogen cavitation and spermatozoal plasma membranes enriched by sucrose density gradient centrifugation; membrane enrichment was confirmed by comparative alkaline phosphatase analysis, electron microscopy, and one- and two-dimensional PAGE. Periacrosomal plasma membrane was used as an immunogen for the production of an antiser...
Pregnancies from imipramine and xylazine-induced ex copula ejaculation in a disabled stallion. Breeding or semen collection was attempted using: natural cover, manual stimulation, artificial vagina, pharmacologic induction of ejaculation, and electroejaculation. Sperm cells were recovered from the ductus deferens and epididymides post mortem. Only semen collected ex copula by imipramine and xylazine treatment resulted in conceptions (4/5). This is the first report of pregnancies in horses from ex copula semen collection.
Subjecting horse spermatozoa to hypoosmotic incubation: effects of ouabain. Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na (+)K (+), ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenney's medium (T...
Intrauterine fluid accumulation in oestrous mares. Intrauterine fluid (IUF) was collected using a tampon from mid-oestrous mares (n = 57) with and without ultrasonically detectable accumulations of free intraluminal fluid. Bacteria were cultured and neutrophils counted from all samples (n = 57). Total protein concentration, trypsin-inhibitor capacity (TIC), and plasmin, beta-glucuronidase (B-Gase) and N-acetyl-beta-D-glucosaminidase (NAGase) activities were determined in 27 IUF samples. The motility of spermatozoa in the presence of IUF, IUF extended with Kenney's medium (1:1) and Kenney's medium alone was analysed in 9 samples using a Hamilto...
Primary structure of stallion seminal plasma protein HSP-7, a zona-pellucida-binding protein of the spermadhesin family. The primary-structure of HSP-7, a 14-kDa protein isolated from stallion seminal plasma, has been determined, HSP-7 belongs to the spermadhesin protein family, shares 98% sequence identity with the boar seminal plasma protein AWN, and, like its boar homolog, displays zona-pellucida-binding activity. Despite these conserved structural and functional features, the equine and porcine spermadhesins differ in their topography on spermatozoa.
Seasonal rhythm of semen characteristics of a Brazilian breed (“Mangalarga”) stallion. An attempt has been made to define semen seasonality in a horse in the Southern Hemisphere. Repeated measurements of three variables in the semen were made for 36 months (Jan/90-Dec/92) in a 21-year old "Mangalarga" stallion living under natural photoperiod and temperature conditions in a farm situated in São José do Rio Pardo, São Paulo, Brazil (latitude 21 degrees) 36'S; longitude 46 degrees 53' W). The horse fed on natural pasture and a nutritionally balanced feed twice a day (11:00 and 17:00 h). Water and mineral supplement were available ad libitum. Semen was collected almost daily by ...
Role of carbohydrates in the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells in vitro. To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...