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.
Comparison of the longevity of motility of stallion spermatozoa incubated at 38 degrees C in different capacitating media and containers. This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38 degrees C in either air or 5% CO2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-m...
Squamous cell carcinoma of the urethral process in a horse with hemospermia and self-mutilation behavior. A 14-year-old Arabian stallion was examined because of acute hemospermia. The stallion was used in an artificial breeding program and had a 6-year history of low-grade hemospermia and a 4-year history of self-mutilation behavior. During previous examinations, minor irritation of the urethral process was identified as the source of the bleeding. Physical examination revealed a mucosal ulceration in the distal portion of the urethra. Histologic examination of a biopsy specimen from this area revealed low-grade squamous cell carcinoma. The urethral process was excised, and the hemospermia resolve...
Effect of antioxidants on the motility and viability of cooled stallion spermatozoa. The aim of the present study was to determine whether antioxidants in semen extenders help to maintain the motility and viability of stallion spermatozoa incubated for 48 h at 5 degrees C. Semen samples were collected from ten stallions and washed to remove the seminal plasma. Five antioxidant treatments (control, xanthurenic acid, glutathione, taurine and hypotaurine) were prepared in each of three different semen extenders (skimmed milk, skimmed milk + egg yolk, and cream gel extenders). The spermatozoa were suspended in 15 treatments (three extenders x five treatments). Sub-samples from eac...
Oviductal insemination of mares. A technique was developed for oviductal insemination of mares, in which a small number of motile spermatozoa are deposited directly into the oviduct. Pregnancy rates in mares inseminated by traditional intrauterine artificial insemination were compared with rates in mares inseminated by oviductal insemination. Fifteen mares were inseminated with 5 x 10(8) progressively motile spermatozoa by intrauterine artificial insemination, and 14 mares were inseminated with 5 x 10(4) progressively motile spermatozoa by oviductal insemination. Pregnancy rates in mares inseminated by intrauterine artificial...
Transport of spermatozoa in the reproductive tracts of mares. A scintigraphic method was developed to study sperm migration in the reproductive tracts of mares. Mares (n=5) and stallions (n=2) were used to test various steps of the procedure and three other mares and a stallion were used to study sperm transportation. A radiolabelling solution was prepared from 99mTc (Technetium-99m) and hexamethyl propylene amine oxime. The highest labelling of spermatozoa (57-72%) was obtained by incubation of the spermatozoa with the radiolabelling solution for 20 min at 20 degrees C. Radioactivity outside the spermatozoa was removed by centrifugation and by two subse...
Effect of cryopreservation and oviductal cell conditioned media on Ca2+ flux of equine spermatozoa. Movement of Ca2+ into spermatozoa is a critically important event for capacitation and the acrosome reaction. In the present study, the nature of Ca2+ movement in fresh equine spermatozoa was established and the effects of oviductal cell conditioned medium (OCM) and cryopreservation on Ca2+ flux were investigated. The ability of fresh and cryopreserved stallion spermatozoa to regulate Ca2+ concentration over time was evaluated in Ca2+ -free PBS. Intracellular Ca2+ concentrations were higher in cryopreserved spermatozoa than in fresh spermatozoa. However, extracellular Ca2+ concentrations were ...
Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing. The aim of the present study was to investigate the cryoprotectant properties of cholesterol after incorporation into the plasma membranes of equine spermatozoa. A cholesterol-methyl-beta-cyclodextrin complex was used to alter sperm plasma membrane cholesterol content. Ejaculates from six stallions were centrifuged in a non-fat skimmed milk glucose-sucrose extender (MK) or a modified Tyrode's medium (TALP). The sperm pellets were resuspended in the appropriate extender with or without added cholesterol (0.125 mmol cholesterol-methyl-beta-cyclodextrin complex l(-1)) and incubated at 24 degrees ...
Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes. The use of intracytoplasmic sperm injection (ICSI) for in vitro fertilization of equine oocytes and the developmental potential of these oocytes after transfer to the Fallopian tubes of synchronized mares were examined. Oocytes were aspirated from mature follicles 39 h after injection of a GnRH analogue and transported 190 km at 39 degrees C. Semen from a fertile and an infertile stallion was frozen and prepared for injection. Successfully injected oocytes were transferred surgically into the ampulla of the Fallopian tube either: (i) 4-8 h after semen injection; or (ii) after 24-48 h culture b...
Zona pellucida-sperm binding assay for equine oocytes. The binding of a spermatozoon to the zona pellucida is the first step in fertilization. The number of spermatozoa bound to a zona pellucida may reflect the functional status of both the oocyte and spermatozoa. The aim of the present study was to determine whether the stage of maturation of the equine oocyte affects the capacity of the zona pellucida to bind with spermatozoa. Cumulus-oocyte complexes (COCs) were collected from the ovaries of mares from abattoirs or were obtained in vivo by ultrasound-guided follicular aspiration. Oocytes were inseminated directly after collection or after 30 h ...
Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions. In the present study, the interactions among stallions, semen extenders and cooling treatments before stallion semen samples were frozen were studied. In Expt 1, the effects of four cooling treatments and three semen extenders were investigated (11 stallions x four split ejaculates), whereas in Expt 2, the effects of two semen extenders, two egg yolk concentrations and two glycerol concentrations were investigated (six stallions x five split ejaculates). Sperm motility after thawing was evaluated. In Expt 1, the extender x cooling treatment interaction was significant. Centrifugation and addit...
Exposure of progesterone receptors on the plasma membranes of stallion spermatozoa as a parameter for prediction of fertility. Subfertility in stallions is attributed to the inability of spermatozoa to undergo the acrosome reaction in response to progesterone. In the present study, it was assessed whether there is a correlation between stallion fertility, defined on the basis of first cycle foaling rate and first cycle 'non-return rate', and the proportion of spermatozoa with exposed progesterone receptors on their plasma membranes. Semen from Dutch Warmblood (n=10) and Friesian (n=4) stallions was analysed. Progesterone 3-(o-carboxymethyl) oxime-BSA coupled with fluorescein isothiocyanate was used as a progesterone r...
Relationship between sperm nuclear protamine free -SH status and susceptibility to DNA denaturation. Data from the sperm chromatin structure assay (SCSA), a flow cytometric measurement of susceptibility of sperm nuclear DNA to denaturation, show strong correlation with the fertility potential of bulls, boars, men and stallions. Previous studies showed a strong relationship between stallion spermatozoa with denatured DNA and the presence of DNA strand breaks. In the present study, the relationship between stallion sperm DNA denaturation and the redox status of -SH groups on the cysteine residues of sperm nuclear protamines that are thought to stabilize chromatin was investigated. Semen samples...
Which insemination results in fertilization when several are performed before ovulation? The aim of the present study was to determine which artificial insemination results in fertilization when mares are inseminated several times before ovulation. Mares in oestrus were inseminated over 62 cycles with fresh semen at 48 h intervals from when a follicle > or =30 mm in diameter was detected until ovulation. The number of inseminations was limited to three. Three fertile stallions were used and a different stallion was used for each artificial insemination. The order of the three stallions was changed for each cycle. Embryos were collected between day 10 and day 12 after ovulation and...
Localization and cellular distribution of a unique hyaluronidase in stallion spermatozoa during epididymidal transit. Three protein bands with hyaluronidase activity and molecular masses of 87, 48 and 43 kDa were isolated from purified equine sperm plasma membranes. Indirect immunofluorescence was used to assess sperm labelling patterns using a polyclonal antibody to sperm hyaluronidase. In ejaculated spermatozoa, surface-associated hyaluronidase was localized to the posterior head region of 98 +/- 2% of spermatozoa (n=10). Epididymides were isolated from mature stallions (n=5) and divided into caput, corpus and cauda epididymides in separate Petri dishes. The epididymidal tubules were dissected and washed us...
Effects of follicular fluid or progesterone on in vitro maturation of equine oocytes before intracytoplasmic sperm injection with non-sorted and sex-sorted spermatozoa. In Expt 1, compact cumulus oocyte complexes (COCs) were matured in: (i) control medium (Hepes-buffered TCM-199 with 10% oestrous cow serum (OCS) + oestradiol, LH and FSH); (ii) Hepes-buffered TCM-199 with 20% follicular fluid; or (iii) control medium containing 250 ng progesterone ml(-1). Mature oocytes were collected by transvaginal aspiration as a positive control for the in vitro maturation (IVM) treatments. Oocytes were fertilized by ICSI and cultured in Menezo's B2 + 5% fetal calf serum (FCS). There were no significant differences among IVM treatments. In Expt 2, oocytes with expanded COC...
The structural morphology and epithelial association of spermatozoa at the uterotubal junction: a descriptive study of equine spermatozoa in situ using scanning electron microscopy. Uterotubal junction tissues (n=12) recovered from young nulliparous mares 4 h after insemination were examined for spermatozoa in situ using scanning electron microscopy. Mares were inseminated before ovulation with semen containing 66-85% morphologically abnormal spermatozoa and approximately 1 x 10(9) progressively motile spermatozoa. Spermatozoa were found in all the tissues and displayed a variety of relationships to the epithelium, depending on regional location, spatial constraints and epithelial surface characteristics. Spermatozoa were most abundant in the epithelial folds on the uteri...
Preservation of stallion sperm quality by native phosphocaseinate: a direct or indirect effect? Milk-based diluents are generally considered efficient for survival of stallion spermatozoa in vitro. However, milk is a complex and variable medium and native phosphocaseinate is a milk component that is more efficient for preservation of sperm motility and fertility, although the mechanisms involved in this protection have not yet been elucidated. The aim of the present study was to characterize the interactions between native phosphocaseinate and equine spermatozoa. No binding between sperm membranes and native phosphocaseinate was observed using indirect immunofluorescent staining or elect...
Production of live foals from sperm-injected oocytes harvested from pregnant mares. In vitro fertilization in horses has been less successful than anticipated owing to: (i) the inability to collect large numbers of good quality oocytes; (ii) alterations in the zona pellucida that occur during in vitro maturation of equine oocytes; and (iii) inadequate preparation of equine sperm cells. In addition, studies in humans, mice and cattle have indicated that high concentrations of glucose in culture media may inhibit embryonic development in vitro and this may also be a problem for development of equine embryos in vitro. The aims of the present study were: (i) to achieve fertilizat...
Measurements of reproductive function in stallions treated with trimethoprim-sulfamethoxazole and pyrimethamine. To evaluate the effects of trimethoprim-sulfamethoxazole and pyrimethamine treatment on various measures of reproductive function in healthy pony stallions. Methods: Randomized complete block study. Methods: 12 healthy, mature pony stallions. Methods: Stallions were assigned to treatment and control groups balanced for age and various characteristics of reproductive function. The treated group received trimethoprim-sulfamethoxazole and pyrimethamine for 90 days during summer and fall; the control group was not treated. Semen characteristics, sexual behavior, testicular volume, and sperm produc...
CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa. Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, ...
A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion sperm and is associated with spermatozoal function. Sperm hyaluronidase has been implicated in sperm penetration of the extracellular matrix of the cumulus oophorus and may play a crucial role in gamete interaction and fertility in mammals. The objectives of this study were to characterize the enzyme activity of equine sperm hyaluronidase and to investigate its cellular distribution. Zymography of stallion sperm plasma membrane extracts was used to identify hyaluronidase activity in protein bands. Affinity-purified polyclonal IgG raised against equine sperm hyaluronidase was used to label fresh and capacitated stallion sperm, followed by indire...
Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro. In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured in serum-free medium. The hardened ZP is refractory to sperm penetration. Fetuin, a component of fetal calf serum, inhibits ZPH and allows normal fertilization rates in oocytes cultured in the absence of serum. We evaluated whether fetuin is present in horse serum and follicular fluid (FF) and whether fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increas...
In vitro interactions of cryopreserved stallion spermatozoa and oviduct (uterine tube) epithelial cells or their secretory products. Formation of a spermatozoa ('sperm') reservoir in the mare is thought to occur through lectin-mediated sperm attachment to the oviductal epithelium. Once attached, prefertilization sperm survival is supported by oviductal factors. Cryopreservation of stallion sperm decreases the number of sperm attaching to oviduct epithelial cells (OEC) and the length of time these sperm survive. Quantification of in vitro interactions between sperm and OEC in a co-culture system may provide an assay for functional integrity of cryopreserved or fresh sperm samples. Additionally, superior additives for in vitr...
Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture. To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. Methods: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. Methods: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozo...
A 105- to 94-kilodalton protein in the epididymal fluids of domestic mammals is angiotensin I-converting enzyme (ACE); evidence that sperm are the source of this ACE. SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was...
Scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. 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...
Preliminary observations in in vitro development of equine embryo after ICSI. The objective of this study was to perform intracytoplasmic sperm injection (ICSI) on in vitro matured equine oocytes and to improve in vitro embryonic development on Vero cells after activation of the microinjected oocytes with calcium ionophore. After maturation (23 or 40 h, 38.5 degrees C, 5% CO2), the cumulus-oocyte complexes were denuded, centrifuged and all oocytes exhibiting the first polar body were microinjected. ICSI was performed using fresh semen from three fertile stallions. Microinjected oocytes were activated with calcium ionophore A23187 (10 min, 10 microM) and cultured individ...
Immunohistochemical localization of the spermadhesin AWN-1 in the equine male genital tract. Spermadhesins are proteins with various functions in sperm capacitation and zona pellucida binding. In this study the cellular localization of the spermadhesin AWN-1 has been examined in the equine male genital tract. Results obtained by immunohistochemical methods reveal that in the horse AWN-1 is synthesized in spermatogonia, in the rete testis, the ductus epididymidis and the seminal vesicles. These findings indicate that the cellular origin of spermadhesins is species-specific.
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...