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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.
Mitochondrial oxygen consumption is a unique indicator of stallion spermatozoal health and varies with cryopreservation media. Mitochondrial oxygen consumption is a sensitive indicator of spermatozoal health in the context of cryopreservation. We investigated oxygen consumption of equine sperm mitochondria during incubation in four commercially available sperm cryopreservation extenders: modified INRA 96, BotuCrio, EZ Freezin-"LE" and "MFR5", in addition to several other parameters including motility, reactive oxygen species (ROS) production and viability. All experimental endpoints, with the exception of average path velocity, were affected significantly by freezing extender type after freezing and thawing. Sperm in ...
The effects of urine concentration, and cushion centrifugation to remove urine, on the quality of cool-stored stallion sperm. Urine-contaminated stallion semen is a clinical problem due to a variety of causes. The effect of the level of urine contamination on the longevity of sperm quality has not been evaluated. The aim of this study was to determine the effects of urine concentration level (0%, 10%, 20%, 30%, and 40%) and cushioned centrifugation and resuspension of the sperm pellet in fresh extender, on measures of sperm quality, immediately after semen collection (T0), after 1 hour of storage at room temperature (T1), and after 24 hours of cooled storage (T24). In general, most sperm quality measures declined w...
New flow cytometry approaches in equine andrology. Flow cytometry is currently recognized as a robust tool for the evaluation of sperm quality and function. However, within equine reproduction, this technique has not reached the sophistication of other areas of biology and medicine. In recent years, more sophisticated flow cytometers have been introduced in andrology laboratories, and the number of tests that can be potentially used in the evaluation of sperm physiology has increased accordingly. In this review, recent advances in the evaluation of stallion spermatozoa will be discussed. These new techniques in flow cytometry are able to simul...
Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility. Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these ...
Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm. The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were fro...
Lipidomics of equine sperm and seminal plasma: Identification of amphiphilic (O-acyl)-ω-hydroxy-fatty acids. Using a nontargeted lipidomics analysis of equine sperm and seminal plasma, we were able to characterize a diverse array of individual lipids including ethanolamine and choline ether lipids and seminolipids essential to membrane raft function. We also detected, for the first time in sperm, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths, which were localized to the head and not the tail of sperm. The only previous identification of OAHFAs has been in meibomian glands and their sebaceous secretions. The identities of these lipid amphiphiles were valida...
Development of a new fertility prediction model for stallion semen, including flow cytometry. Several laboratories routinely use flow cytometry to evaluate stallion semen quality. However, objective and practical tools for the on-field interpretation of data concerning fertilizing potential are scarce. A panel of nine tests, evaluating a large number of compartments or functions of the spermatozoa: motility, morphology, viability, mitochondrial activity, oxidation level, acrosome integrity, DNA integrity, "organization" of the plasma membrane, and hypoosmotic resistance, was applied to a population of 43 stallions, 33 of which showing widely differing fertilities (19%-84% pregnancy rat...
Effects of α-tocopherol and freezing rates on the quality and heterologous in vitro fertilization capacity of stallion sperm after cryopreservation. The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C...
The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions. Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of au...
Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen. This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the cen...
Effects of a long-day light programme on the motility and membrane integrity of cooled-stored and cyropreserved semen in Shetland pony stallions. Increasing day length in spring stimulates reproductive functions in horses. In this study, we have analysed the effect of artificial long days on the quality of cooled-stored and cryopreserved semen in Shetland stallions. Stallions of the treatment group (AL, n = 8) were exposed to 16 h light and 8h darkness from 15th December to 20th March while control stallions (CON, n = 7) were kept under natural photoperiod. Semen was collected once weekly and processed for cooled-storage and cryopreservation once per month. Total and progressive motility and percentage of membrane intact spermatozoa wer...
Effect of clinically-related factors on in vitro blastocyst development after equine ICSI. Equine intracytoplasmic sperm injection (ICSI) is being used clinically for foal production, but little information is available on factors affecting the efficiency of this procedure. We examined factors that may influence blastocyst development when ICSI is performed clinically, i.e., on oocytes recovered from live mares by transvaginal ultrasound-guided follicle aspiration (TVA), using sperm from the stallion of the client's choice. In a clinical setting, there may be a delay from the time of TVA to isolation of oocytes from the aspirated fluid. In a preliminary study, oocytes from fluid hel...
Analysis of breed effects on semen traits in light horse, warmblood, and draught horse breeds. In the present study, systematic effects on semen quality traits were investigated in 381 stallions representing 22 breeds. All stallions were used for AI either at the Lower Saxon National Stud Celle or the North Rhine-Westphalian National Stud Warendorf. A total of 71,078 fresh semen reports of the years 2001 to 2014 were edited for analysis of gel-free volume, sperm concentration, total number of sperm, progressive motility, and total number of progressively motile sperm. Breed differences were studied for warmblood and light horse breeds of both national studs (model I) and for warmblood b...
The cholesterol transporter ABCA1 is expressed in stallion spermatozoa and reproductive tract tissues. In the present study, we assessed the presence of the ATP-binding-cassette (ABC) transporter molecules ABCA1 in spermatozoa of adult stallions and in testicular and epididymal tissue of prepubertal and adult stallions. For this purpose, semen samples from six fertile Shetland pony stallions aged 4 to 19 years were collected. Semen was collected from each stallion on three consecutive days. Ejaculates were analyzed immediately after collection, and only ejaculates meeting minimal requirements for fertile stallions were further evaluated. ABCA1 immunosignal was localized after staining of semen ...
Ejaculate Characteristics Depend on Social Environment in the Horse (Equus caballus). Sperm competition theory predicts semen characteristics to be affected by the social environment. We used the polygamous horse (Equus caballus) to experimentally study within-subject plasticity in response to different social environments. Stallions were sequentially exposed, over a period of 8 weeks each, to other stallions and then singly to mares, or vice versa (in adjacent boxes separated by grills). Ejaculates were collected to determine semen characteristics. Highest sperm numbers were found in stallions that were first exposed to other stallions and then to mares, while lowest sperm num...
A long-day light program accelerates seasonal coat changes but is without effect on semen and metabolic parameters in Shetland pony stallions. Horses are seasonal breeders, and robust breeds may exhibit a winter hypometabolism when kept under semiferal conditions. In this study, we analyzed the effects of artificial long days on rectal temperature, heart rate, heart rate variability, hematology, coat changes, semen parameters, and plasma testosterone concentrations in Shetland stallions stabled overnight and assigned to a control group (CON, n = 9) kept under natural photoperiod, and a treatment group exposed to a long-day light program from 15 December to 20 March (AL, n = 9). During the 8-month study, rectal temperature, heart rate...
Coenzyme Q10 and α-Tocopherol Prevent the Lipid Peroxidation of Cooled Equine Semen. Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with...
Draft Genome Sequence of Taylorella equigenitalis Strain ERC_G2224 Isolated from the Semen of a Lipizzaner Stallion in South Africa. Taylorella equigenitalis is the causative agent of contagious equine metritis (CEM), a sexually transmitted infection of horses. We report here the genome sequence of T. equigenitalis strain ERC_G2224, isolated in 2015 from a semen sample collected in 1996 from a Lipizzaner stallion in South Africa.
Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess w...
Characteristics of frozen epididymal spermatozoa from stallions that died 12 to 36 hours after colic surgery. Equine spermatozoa from the cauda epididymis were previously collected and frozen, and the fertility was assessed. Most studies were performed on healthy stallions that had undergone routine castration or on the epididymis collected at the abattoir, but there are no studies on the quality of epididymal semen in subjects which have died from colic or which underwent intensive care. The present study was designed to verify whether a severe illness could affect epididymal semen quality and freezability in the stallion. Therefore, epididymal semen characteristics during the freezing process in sta...
The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses. The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., av...
Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen. Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a ...
Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion sperm. Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm reco...
Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy. Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i....
Effects of ground semen collection on weight bearing on hindquarters, libido, and semen parameters in stallions. Collection of semen on the ground from the standing stallion represents an alternative method to dummy mount semen collection and is of increasing popularity for sport stallions, males suffering from health problems, or in studs without a dummy or suitable mare at disposal. Our aim was to collect and compare spermatological and physiological data associated with traditional and ground semen collection. Twelve of 23 Franches-Montagnes stallions were selected to carry out semen collection on a dummy and while standing in a crossed experimental protocol. Semen quantity and quality parameters, wei...
Increased cortisol release and transport stress do not influence semen quality and testosterone release in pony stallions. The use of breeding stallions for equestrian competitions requires that fertility is not negatively affected by competition or transport to the competition site. In this study, effects of cortisol release induced by road transport (600 km), adrenocorticotropic hormone (ACTH) administration (3 × 0.5 mg synthetic ACTH) and placebo treatment on semen quality and testosterone release were investigated in Shetland stallions (N = 13) using a crossover design. Saliva for cortisol and blood for testosterone analysis were collected for 10 weeks after treatments. Semen was collected daily for 5 days di...
Dexamethasone acutely regulates endocrine parameters in stallions and subsequently affects gene expression in testicular germ cells. Testicular steroidogenesis and spermatogenesis are negatively impacted by stress-related hormones such as glucocorticoids. The effects of two injections of a therapeutic dose of dexamethasone (a synthetic glucocorticoid, 0.1mg/kg; i.v.) given 24h apart to each of three stallions were investigated and compared to three saline-injected control stallions. Dexamethasone decreased circulating concentrations of cortisol by 50% at 24h after the initial injection. Serum testosterone decreased by a maximum of 94% from 4 to 20h after the initial injection of dexamethasone. Semen parameters of the dexame...
Cryopreservation of semen from domestic livestock. In modern livestock breeding, cryopreserved semen is routinely used for artificial insemination. Sperm cryopreservation secures future reproduction, and insemination doses can be easily shipped. Processing of semen for cryopreservation can be done with minimal efforts and relatively low costs. In this chapter we describe the entire cryopreservation process for stallion and bull sperm including dilution of sperm in primary and freezing extender, cooling and packaging in straws, freezing in liquid nitrogen vapor, cryogenic storage, and thawing. Special emphasis is given on preparation of commonl...
The “dilution effect” in stallion sperm. Dilution of semen to less than 20 × 10(6) sperm/mL has been reported to decrease sperm quality in multiple species, a phenomenon known as the semen "dilution effect." Critical evaluation of stallion semen diluted to these concentrations, however, has not been reported. This study evaluated sperm motion characteristics (percent total motility [TMOT], percent progressive motility [PMOT], curvilinear velocity [μm/s], and percent straightness) and plasma membrane integrity (percent plasma membrane intact [PMI]) in semen samples diluted to 2.5 × 10(6) sperm/mL with the addition of 0%, 7.5%, ...
Use of hypometabolic TRIS extenders and high cooling rate refrigeration for cryopreservation of stallion sperm: presence and sensitivity of 5′ AMP-activated protein kinase (AMPK). This study evaluated the effect of the use of hypometabolic TRIS extenders in the presence or the absence of AMPK activators as well as the utilization of high cooling rates in the refrigeration step on the freezability of stallion sperm. Twelve ejaculates were cryopreserved using Botucrio® as a control extender and a basic TRIS extender (HM-0) separately supplemented with 10 mM metformin, 2mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), 2 mM Adenosine monophosphate (AMP), 40 μM compound C AMPK inhibitor or 2 mM AMP+40 μM compound C. Our results showed that the utilization ...
