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Topic:Equid Semen
Equid semen refers to the reproductive fluid produced by male horses, which contains sperm cells necessary for fertilization. The quality and viability of equid semen are important for successful breeding programs and can be influenced by various factors, including genetics, age, diet, and environmental conditions. Key parameters used to assess semen quality include volume, concentration, motility, and morphology of sperm cells. Techniques for collecting, evaluating, and preserving equid semen, such as cryopreservation, are critical for artificial insemination practices. This page compiles peer-reviewed research studies and scholarly articles that explore the physiology, assessment, and management of equid semen in the context of equine reproduction.
Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells. The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell po...
Cryopreservation of Peruvian Paso horse spermatozoa: dimethylacetamide preserved an optimal sperm function compared to dimethyl sulfoxide, ethylene glycol and glycerol. The objective was to evaluate the effect of different cryoprotectant agents in the cryopreservation of Peruvian Paso horse semen. Twenty semen samples were collected from five Peruvian Paso horse stallions. Each sample was divided into 12 parts to form the groups: dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (GLY), at 3%, 4% and 5%. Samples were frozen using a rate-controlled freezer. Sperm parameters evaluated were motility and viability/acrosomal status. After thawing, progressive motility in DMA group was higher (p < .05) than in DMSO, EG and GL...
Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa. 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...
Genome-wide association study for semen quality traits in German Warmblood stallions. We performed a genome-wide association study for semen quality traits in 139 German Warmblood stallions. Stallions were genotyped using the Illumina equine SNP50 Beadchip. Traits analysed were de-regressed estimated breeding values (EBVs) for gel-free volume, sperm concentration, total number of sperm, progressive motility and the total number of progressively motile sperm. The GWAS revealed 29 SNPs on 12 different chromosomes as genome-wide significantly associated with semen quality traits. For ten genomic regions we could retrieve candidate genes influencing stallion fertility. Among the ca...
Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm. The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Cytosine methylation of sperm DNA in horse semen after cryopreservation. Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ej...
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 ...
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...
Heritability of semen traits in German Warmblood stallions. The objectives of the present study were to evaluate genetic parameters for semen quality traits of 241 fertile German Warmblood stallions regularly employed in artificial insemination (AI). Stallions were owned by the National Studs Celle and Warendorf in Germany. Semen traits analyzed were gel-free volume, sperm concentration, total number of sperm, progressive motility and total number of progressively motile sperm. Semen protocols from a total of 63,972 ejaculates were collected between the years 2001 and 2014 for the present analysis. A multivariate linear animal model was employed for es...
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...
Fluorescence investigations on choline phospholipid binding and chemical unfolding of HSP-1/2, a major protein of horse seminal plasma. Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
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...
Our current understanding of the pathophysiology of equine endometritis with an emphasis on breeding-induced endometritis. Equine endometritis is characterized by the expression of pro- and anti- inflammatory cytokines and an influx of polymorphonuclear neutrophils (PMNs) into the uterus. Mares resistant to persistent endometritis clear the inflammation within 24-36 h after exposure to microorganisms or semen. These mares have a rapid increase of pro-inflammatory cytokines and an upregulation of inflammatory modulating cytokines within 6h after exposure to inflammatory challenge. In conjunction with effective uterine contractions, these events are believed to be responsible for the transient nature of the inflamma...
Freeze-dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays. During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-...
Milk, caseinate and lactoferrin addition to equine semen cooling extenders. Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml-1 , C4) C2 + Lf 500 μg ml-1 and C5) C2 + Lf 1000 μg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motili...
Diagnosis and effects of urine contamination in cooled-extended stallion semen. Urospermia is known to affect semen quality in many mammals, including stallions. Determinations of semen pH and creatinine and urea concentrations have been used to diagnose urine contamination in raw stallion semen. Unfortunately, practitioners suspecting urine contamination in cooled-shipped samples have no proven means to confirm the presence of urine. Therefore, the objectives of this study were (1) to assess the effects of urine contamination on sperm motility of extended fresh and cooled-stored stallion semen, (2) to evaluate the usefulness of semen color, odor, pH, and creatinine and u...
Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm. The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G...
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...
Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death. 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...
Pregnancy outcomes using stallion epididymal sperm stored at 5 °C for 24 or 48 hours before harvest. The cryopreservation of epididymal sperm can be useful in a variety of circumstances for ensuring genetic preservation of a valued stallion. Although early studies have reported pregnancy rates significantly lower than those achieved with cryopreserved ejaculated sperm, two recent studies report over 60% one-cycle pregnancy rates with epididymal sperm stored for 24 hours at 5 °C before harvest and cryopreservation. The aims of this study were to: (1) attempt to replicate the one-cycle pregnancy rate of over 60% using epididymal sperm cooled and stored within the epididymis for 24 hours before...
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...
Sperm Membrane Behaviour during Cooling and Cryopreservation. Native sperm is only marginally stable after collection. Cryopreservation of semen facilitates transport and storage for later use in artificial reproduction technologies, but cryopreservation processing may result in cellular damage compromising sperm function. Membranes are thought to be the primary site of cryopreservation injury. Therefore, insights into the effects of cooling, ice formation and protective agents on sperm membranes may help to rationally design cryopreservation protocols. In this review, we describe membrane phase behaviour of sperm at supra- and subzero temperatures. In a...
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...
Characterization of alkaline phosphatase activity in seminal plasma and in fresh and frozen-thawed stallion spermatozoa. Alkaline phosphatase (AP) has been studied in several situations to elucidate its role in reproductive biology of the male from different mammalian species; at present, its role in horse sperm physiology is not clear. The aim of the present work was to measure AP activity in seminal plasma and sperm extracts from freshly ejaculated as well as in frozen-thawed stallion spermatozoa and to verify whether relationship exists between AP activity and sperm quality parameters. Our data on 40 freshly ejaculated samples from 10 different stallions demonstrate that the main source of AP activity is semi...
A comparative study of two cooling protocols on stallion sperm cryosurvival. There are many protocols for horse sperm cryopreservation, but results are inconsistent; sperm survival after freeze-thawing is usually poor; in consequence, fertility is low. The objective of this work was to see whether slow cooling before freezing to minus 3 °C instead of +5 °C, the traditional target temperature, could improve horse sperm cryosurvival, capability to carry out capacitation and the acrosome reaction induced by progesterone. Spermatozoa from five stallions were packaged in straws and slowly cooled to +5 °C. Half of the straws were frozen directly and the other half was ...
