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Topic:Semen Analysis
Semen analysis involves the evaluation of semen quality to assess reproductive potential and health in horses. This process typically includes the examination of several parameters such as sperm concentration, motility, morphology, and volume. Semen analysis is used in breeding programs to determine the fertility of stallions and to identify any reproductive issues. The collected data can assist in making informed decisions about breeding management and interventions. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, findings, and implications of semen analysis in equine reproduction.
Single layer centrifugation of stallion spermatozoa improves sperm quality compared with sperm washing. This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods. Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-b...
Seasonal functional relevance of sperm characteristics in equine spermatozoa. A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus). Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September-December, January-March, late March-May, and June-July. Parameters monitored included sperm concentration, sperm motility, sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential. Overall, seminal parameters monitored are affected mostly by time period, followed by animal and last...
Alkaline and acid phosphatase, β-glucuronidase and electrolyte levels in fractionated stallion ejaculates. Seminal plasma (SP) is a mixture of contents from the testes, epididymides and accessory sex glands. The sperm concentration is highest in the first few jets, or fractions, of the ejaculate, and the composition of SP varies between these fractions because accessory gland secretions are released in a specific order. The aim of this study was to compare the levels of Na, Cl, K, Mg, Ca, inorganic phosphate (Pi) and the enzymes alkaline phosphatase (AP), acid phosphatase (ACP) and β-glucuronidase (BG) in the different fractions of the ejaculate and in different stallions. All semen collections we...
Evaluation of shape variability of stallion sperm heads by means of image analysis and Fourier descriptors. This study quantified and evaluated the variability of sperm head shape for 10 different stallions. Sperm head shape characteristics including sperm head length to width ratio, position of the center of gravity, curvature, and degree of roundness were assessed and analysed from images using elliptic Fourier descriptors and inverse Fourier transformation. The first four principal components accounted for 88.46-92.33% of the total variance and provided a good summary of the overall data. In the case of the ejaculate with defective sperm heads the components accounted for 97.35-98.21% of variatio...
Immediate and delayed (after cooling) effects of centrifugation on equine sperm. The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25x10(6) cells/mL, and subjected for 10min to (1) no centrifugation (NC) or (2) centrifugation at 400xg, (3) 900xg, or (4) 4500xg. Before and after centrifugation (Day 0), and after 24h of cooling (Day 1), sperm motility ...
Sexual behavior and ejaculate characteristics in Pêga donkeys (Equus asinus) mounting estrous horse mares (Equus caballus). The objectives were to (i) characterize sexual behavior of donkey stallions (jacks; Equus asinus) during on-farm semen collection using estrous horse mares (mares; Equus caballus); (ii) compare behavior of young (less experienced) versus older (more experienced) jacks; (iii) determine whether semen suitable for artificial insemination (AI) could be collected using mares; and (iv) determine the suitability of using mares in field collection of semen from jacks. Six Pêga jacks (3.5 to 16 yr old), previously conditioned to breed mares, were used. Mount mares were confirmed in estrus by a teaser ...
Purification and partial characterization of proteinase inhibitors of equine seminal plasma. The aims of the study were: 1/ to isolate and identify equine seminal plasma proteinase inhibitors, 2/ to evaluate their inhibitory potential, and 3/ to test a correlation between protein concentration in seminal plasma supernatant (obtained after precipitation with 36% ammonium sulfate) and stallion sexual maturity. Seminal plasma proteins obtained from six stallions were chromatographed in a Superose 12 (FPLC system) column followed by C(18) HPLC reverse-phase. Inhibition of trypsin amidase activity was evaluated in the collected fractions. Active proteins with a molecular mass of 6.3-7.0 KD...
Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily. 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...
Change in Morphology of Spermatozoa from Dismount Semen during the Breeding Season in Thoroughbred Stallions in Japan. To clarify the physiological changes of sperm morphology in active Thoroughbred stallions during the breeding season, we examined the dismount semen collected from the penile urethra immediately after service. The spermatozoa were analyzed for relationships between the morphology and the stallion's age or the number of services. Seasonal variation was apparent in the rate of the sperm tail abnormalities, spermatozoa with cytoplasmic droplets, appearance of medusa cells, and sperm head length. Area and width of the sperm head correlated negatively with age (P<0.05). The rate of appearance of...
Evaluation of the presence of equine viral herpesvirus 1 (EHV-1) and equine viral herpesvirus 4 (EHV-4) DNA in stallion semen using polymerase chain reaction (PCR). In the horse, the risk of excretion of two major equine pathogens (equine herpesvirus types 1 (EHV-1) and 4 (EHV-4)) in semen is unknown. The objective of our study was to assess the possible risks for the horizontal transmission of equine rhinopneumonitis herpesviruses via the semen and the effect of the viruses on stallion fertility. Samples of stallion semen (n=390) were gathered from several different sources. Examination of the semen involved the detection of viral DNA using specific PCR. The mean fertility of the stallions whose sperm tested positive for viral DNA and the mean fertility ...
Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound. Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm...
Can programmed cell death be induced in post-ejaculatory bull and stallion spermatozoa? Apoptosis is common during spermatogenesis. Here, it was tested whether apoptosis could be induced in sperm after ejaculation. There were several lines of evidence to indicate that sperm are resistant to induction of apoptosis. First, incubation of bull sperm at temperatures characteristic of normothermia (38.5 degrees C) or heat shock (40 and 41 degrees C) for 4h did not increase the proportion of sperm positive for the TUNEL reaction. There was also no reduction in mitochondrial polarity caused by exposure to 40 or 41 degrees C. Incubation at 38.5 degrees C (least-squares mean+/-SEM=4.0+/-1....
Semen characteristics in a sub-fertile Arabian stallion with idiopathic teratospermia. A 5-year-old Arabian stallion was managed for breeding with fresh/extended semen during a period of 8 months with a resulting per cycle pregnancy rate of 26.3%. The stallion was in good health and no abnormalities of the reproductive tract were observed. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, that sperm production was normal and that semen quality was extremely poor. The most prevalent sperm defects were abnormal heads and mid-pieces. Most abnormal heads were microcephalic and/or tapered and consi...
Quality and fertilizing ability in vivo of sex-sorted stallion spermatozoa. Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex-sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14-propidium iodide), mitochondrial function (JC-1) and acrosomal status (fluorescein isothiocyanate Pisum sativ...
Suppression of testicular function and sexual behavior by vaccination against GnRH (Equity) in the adult stallion. The aim of this study was to evaluate the effects of an anti-GnRH vaccine on testosterone concentration, antibody titer, scrotal width, semen quality and sexual behavior in the stallion. Adverse reactions to the vaccine were also determined. Eight clinically healthy sexually experienced stallions aged between 6 and 15 years from the National Stud in Avenches (Switzerland) were used. Five stallions were immunized 3 times at an interval of 4 and 8 weeks, respectively, with 200 microg of a GnRH-protein conjugate (Equity, CSL Limited, Australia) intramuscularly in the neck and 3 control animals re...
Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test. Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present....
Spermatozoa morphology during the breeding season in Thoroughbred stallions in Japan. The morphology of spermatozoa of modern Thoroughbred stallions in Japan was investigated during the breeding season. A total of 299 semen samples were collected from the penises of 16 stallions immediately after service. The rate of abnormalities in sperm heads and tails, spermatozoa with cytoplasmic droplets and slides with medusa cells to total observed slides in each stallion were 3.9 +/- 2.1%, 11.5 +/- 5.9%, 2.4 +/- 2.6% and 20.1%, respectively. The values for the area, length, width and aspect ratio of the stallion sperm head were 12.54 +/- 1.34 microm(2), 5.93 +/- 0.40 microm, 2.69 +/- 0...
Species-specific interaction of seminal plasma on sperm-neutrophil binding. Bovine semen is naturally deposited in the vagina and spermatozoa migrate through the cervix into the uterus leaving the bulk of seminal plasma (SP) behind. In equine, both spermatozoa and SP are deposited directly in the uterus and SP reduces sperm binding to neutrophils and prevents the formation of DNA-based neutrophil extracellular traps (NETs). We investigated the role of bovine SP on sperm-neutrophil binding using the four most common bovine semen extenders. Contrary to equine, bovine spermatozoa removed from SP had low binding to neutrophils for up to 3h, but as little as 10% SP increas...
Morphology and head morphometric characters of sperm in Thai native crossbred stallions. One of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions. Methods: Semen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Stand...
Centrifugation on a single layer of colloid selects improved quality spermatozoa from frozen-thawed stallion semen. The present study attempted to select the subpopulation of stallion spermatozoa that best survived a conventional freezing and thawing procedure, using centrifugation of post-thawed semen samples through a single layer of a glycidoxypropyltrimethoxysilane-coated silica colloid with a species-specific formulation (Androcoll-E). Sperm motility, sperm chromatin structure, membrane integrity and mitochondrial membrane potential were studied in filtered and non-filtered spermatozoa. Single-layer centrifugation (SLC) using Androcoll-E significantly improved all the sperm parameters studied, implying...
Factors influencing the “sortability” of stallion spermatozoa into X- and Y-chromosome bearing populations. Intrinsic differences between stallions exist for semen traits such as motility, morphology fertility and the ability of spermatozoa to survive cryopreservation processes. Ejaculates from 11 stallions were used to test the differences between stallions when selecting X- and Y-chromosome bearing spermatozoa using a modified flow cytometer. Data on orientation and viability of spermatozoa were collected during sex-sorting, and motility characteristics of sex-sorted and non-sorted (control) spermatozoa were assessed before and after cryopreservation. An index was created to rank each stallion in ...
Factors impacting equine sperm recovery rate and quality following cushioned centrifugation. Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear...
Seminal traits, suitability for semen preservation and fertility in the native Portuguese horse breeds Puro Sangue Lusitano and Sorraia: Implications for stallion classification and assisted reproduction. The Puro Sangue Lusitano (PSL) is the major national breed of horse in Portugal, but no studies exist on its seminal characteristics, or on the possibility of conserving semen for future use. The aim of this study was to evaluate semen parameters, fertility and the aptness to semen preservation in Lusitano Stallions. In order to compare characteristics defined by a single or by multiple semen collections per stallion 152 ejaculates obtained from 152 Lusitano stallions presented at an annual breeding soundness examination as well as data related to 371 ejaculates obtained from 9 PSL were analyz...
When to refer an infertile mare to a theriogenologist. Most equine infertility cases can be solved with a methodical, thorough physical and reproductive examination and appropriate diagnostic laboratory aids. Repeated examinations may be needed in some cases to identify subtle anatomical abnormalities or irregularities between hormonal and physiological relationships of the reproductive tract. For pregnancy to occur, hormonal signaling must be exquisitely synchronized with physical changes of the reproductive tract and deposition of fertile semen in the uterus. Asynchrony of these events, infection, inflammation, previous trauma to the reproductiv...
Sperm production and sperm morphology of Swedish Warmblood stallions. The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5-8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology wa...
Influence of long-term treatment with equine somatotropin (EquiGen) on gonadal function in stallions with poor semen quality. The aim of the present study was to investigate the spermatogenic and Leydig cell activity in stallions with impaired semen quality after treatment with equine somatotropin. Experiments were performed using 18 adult clinically healthy stallions with poor semen quality which did not pass breeding soundness evaluation. The animals were randomly divided into a treatment (n = 9) and a control (n = 9) group. Over a period of 90 days, nine stallions received a daily intramuscular injection of 10 mg recombinant equine somatotropin (EquiGen, BresaGen Limited, Adelaide, Australia) and 9 control animals...
Developments in stallion semen evaluation. 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...
Oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse. Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and f...
Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity. 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.
