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.
Alghamdi AS, Lovaas BJ, Bird SL, Lamb GC, Rendahl AK, Taube PC, Foster DN.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...
Macías García B, González Fernández L, Morrell JM, Ortega Ferrusola C, Tapia JA, Rodriguez Martínez H, Peña FJ.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). After freezing and thawing, four sperm subpopulations were identified, listed as FT1 to FT4. While subpopulations FT1 and FT2 were characterized by low sperm velocity, high velocities characterized the ones called FT3 and FT4. The single-layer centrifugation (SLC)-handle...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically norma...
Macías García B, Morrell JM, Ortega-Ferrusola C, González-Fernández L, Tapia JA, Rodriguez-Martínez H, Peña FJ.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...
Clulow JR, Evans G, Morris LH, Maxwell WM.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 ...
Palm F, Walter I, Budik S, Kolodziejek J, Nowotny N, Aurich C.After artificial insemination or mating an inflammatory response is induced by spermatozoa and components of the inseminate or ejaculate. In order to investigate the inflammatory reaction of the endometrium to different semen extenders, phosphate buffered saline (PBS), seminal plasma (SP), skim milk-based extender (SM) or egg yolk semen extender (EY) was inoculated into the uterus of oestrous mares (n=8) during four consecutive cycles in alternating order. Twelve hours after treatment, a uterine lavage was performed and an endometrial biopsy was taken. An additional biopsy was taken in the oes...
Miller CD.This manuscript is a review of current protocols, advantages, and disadvantages of breeding mares with frozen-thawed equine semen. Issues affecting pregnancy rates are discussed, including proper mare selection, induction of ovulation, insemination dose, timing of insemination (single-dose versus multiple-dose insemination), methods of insemination (transrectal-guided deep-horn versus hysteroscopic insemination), and post-insemination mare management procedures. In a retrospective analysis of breeding records, a single-dose of frozen-thawed semen was inseminated within 6h post-ovulation; the p...
Gamboa S, Machado-Faria M, Ramalho-Santos J.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...
De Botton D, Janett F, Burger D, Imboden I, Kähn W, Thun R.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...
Varner DD.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...
Sieme H, Harrison RA, Petrunkina AM.Success in cryopreserving stallion semen has been very variable. Several different freezing regimes have been published. However, because extenders and procedures used in each regime have differed, direct comparison of these techniques has been very difficult, and controlled studies comparing different techniques have not been reported. A number of different factors affect sperm cryosurvival. In this article we review briefly current cryopreservation procedures for stallion semen, and then in more detail cryobiological determinants of sperm function, and mechanisms of cryoinjury and cryoprotec...
Ball BA.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...
Kareskoski M, Katila T.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.
Aurich C.The majority of horse registries approve the use of artificial insemination, and horse breeding has widely taken benefit from the use of cooled-stored semen. New insights into cooled-semen technology open possibilities to reduce problems such as impaired semen quality after cooled-storage in individual stallions. The stallion itself has major impacts on quality and fertility of cooled-stored semen. Dietary supplementation of antioxidants and polyunsaturated fatty acids improves semen quality in a variety of species, but only few studies on this topic exist in the horse. Proper semen collection...
Brum AM, Sabeur K, Ball BA.The objective was to evaluate apoptotic markers in ejaculated equine spermatozoa after separation by density-gradient centrifugation and after cryopreservation. Subpopulations of percoll-separated equine spermatozoa differed (P<0.05) in the percentage of live, caspase-activated spermatozoa (2.9+/-0.7% vs 14.2+/-6.4%; mean+/-S.E.M.), low mitochondrial membrane potential (MMP; 6.8+/-1.1 vs 23.8+/-3.7), altered plasma membrane permeability (1.3+/-0.2 vs 3.0+/-0.5), DNA fragmentation (2.0+/-1.3 vs 14.3+/-3.6), total motility (81.8+/-3.3 vs 35.1+/-5.4), and progressive motility (66.3+/-4.3 vs 24...
Vidament M, Vincent P, Martin FX, Magistrini M, Blesbois E.A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and...
Deichsel K, Palm F, Koblischke P, Budik S, Aurich C.Lipid peroxidation contributes to the damage of the sperm plasma membrane. In different species, dietary supplementation with antioxidants has been shown to improve semen quality. Therefore, we tested effects of dietary supplementation with antioxidants and l-carnitin on semen quality in Shetland pony stallions (n=6). Semen was collected twice a week over a time period of 16 weeks. From weeks 5 to 12, a special diet for stallions containing a variety of antioxidants (STALLION, Pavo Pferdenahrung GmbH, Goch, Germany; tocopherol 300 mg/day; ascorbic acid 300 mg/day; l-carnitin 4000 mg/day; folic...
Crowe CA, Ravenhill PJ, Hepburn RJ, Shepherd CH.Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. Objective: Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. Methods: Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 200...
Price S, Aurich J, Davies-Morel M, Aurich C.This study was undertaken to investigate the effects of storage of stallion semen in a defined milk protein extender at 5 and 15 degrees C under either anaerobic or aerobic conditions, with or without addition of the antibiotic gentamicin. Semen samples were collected from eight fertile stallions and stored for 96 h (day 0-4) and assessed daily for motility, velocity and membrane integrity (viability) using a CASA system. Samples for bacteriology assessment were taken on day 2 of storage. No significant (p > 0.05) differences in motility, velocity or viability were observed between treatmen...
Saragusty J, Gacitua H, Pettit MT, Arav A.Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were ...
Ortega-Ferrusola C, Sotillo-Galán Y, Varela-Fernández E, Gallardo-Bolaños JM, Muriel A, González-Fernández L, Tapia JA, Peña FJ.The kinematics of the appearance of apoptotic markers was studied by flow cytometry and immunoblot assays in equine spermatozoa subjected to freezing and thawing. Caspase activity, low mitochondrial membrane potential, and increases in sperm membrane permeability were observed in all of the phases of the cryopreservation procedure. Freezing and thawing caused an increase in membrane permeability and changes in the pattern of caspase activity; decreases in mitochondrial membrane potential were observed after centrifugation and cooling to 4 degrees C and after freezing and thawing. It is propose...
López-Fernández C, Crespo F, Arroyo F, Fernández JL, Arana P, Johnston SD, Gosálvez J.The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. ...
Clulow JR, Buss H, Sieme H, Rodger JA, Cawdell-Smith AJ, Evans G, Rath D, Morris LH, Maxwell WM.In the 2004/2005 breeding season, the fertility of sex-sorted (SS) and non-sorted (NS) frozen stallion spermatozoa from two Hannovarian stallions was compared. A hysteroscopic insemination technique [Morris, L.H., Tiplady, C., Allen, W.R., 2003a. Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen-thawed spermatozoa onto the uterotubal junction. Equine Vet. J. 35, 197-201] was used to deposit low doses (6, 13 or 25 x 10(6) frozen-thawed SS or NS spermatozoa) onto the utero-tubal junction at 32 or 38 h after the administration of Chorulon (2500...
Clulow JR, Mansfield LJ, Morris LH, Evans G, Maxwell WM.The effects of sperm freezing concentration (40 x 10(6)mL(-1) vs. 400 x 10(6)mL(-1)), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam box vs. programmable freezing machine) were evaluated in a 2 x 2 x 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 x 10(6)mL(-1) was higher than for spermatozoa frozen at 400 x 10(6)mL(-1). No significant differences were observed in the semen parameters assesse...
Dietz JP, Sertich PL, Boston RC, Benson CE.Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate (n=11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1h after collection) to evaluate sperm motility and microbial conce...
Zemanová J, Lukác N, Massányi P, Trandzík J, Burócziová M, Nad P, Capcarová M, Stawarz R, Skalická M, Toman R, Koréneková B, Jakabová D.In this study, the concentration of nickel in stallion, bull, ram, boar and fox semen, and its relation with spermatozoa quality was analyzed. The concentration of nickel in semen was 0.20 mg kg(-1) in stallion, 0.12 mg kg(-1) in bull, 0.31 mg kg(-1) in ram, 0.06 mg kg(-1) in boar and 0.36 mg kg(-1) in fox. Seminal nickel concentration was significantly higher (P < 0.05) in foxes than that in bulls and significantly higher (P < 0.01) in rams and foxes in comparison with boars. Evaluation of total pathological spermatozoa revealed the highest number in stallions followed by rams, bulls, boars a...
Cran DG.Many tens of thousands of calves resulting from artificial insemination (AI) have been born worldwide after XY sperm separation and commercial production is underway in several countries. Accuracy of sex selection is some 90% and can be achieved both in research facilities and at AI studs in rural locations. Most facilities sort X- sperm which have also be utilised for superovulation and embryo transfer projects and for in vitro fertilisation (IVF) as well as AI. Sort rates of some 15 x 10(6) sperm/h are currently achievable and are used for low dose insemination, generally at 2 x 10(6) frozen...
Morris GJ, Faszer K, Green JE, Draper D, Grout BW, Fonseca F.The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultra...
Schober D, Aurich C, Nohl H, Gille L.Cryopreservation of spermatozoa is of essential importance for artificial insemination and breeding programs in horses. Besides other factors, spermatozoal motility depends on mitochondrial energy metabolism. Based on changes of single mitochondrial functions it has been suggested that mitochondrial damage during cryopreservation could be a major reason for diminished post thaw semen quality. However, it is still unclear to which extent this influences the whole bioenergetic performance of mitochondria and whether this plays a role during routine cryopreservation procedures. Therefore, it was ...
Roasa LM, Choi YH, Love CC, Romo S, Varner DD, Hinrichs K.In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates wer...
Monteiro RA, Cunha RM, Guerra MMP, de Almeida VM, Peña-Alfaro CE, Silva SV.The aim of this study was to test equine semen cryopreservation techniques for the conservation of donkey germplasm. Ejaculates of three male donkeys were used (n = 18; six ejaculates per donkey; six repetitions), collected by the artificial vagina method. To remove the seminal plasma, the ejaculates were split and submitted to filtration or centrifugation methods. To assess the freezing method, each fraction was submitted to the automated system or the conventional system, and groups were formed: automated centrifuge, automated filtrate, conventional centrifuge and conventional filtrate. Af...
Ponthier J, Franck T, Parrilla-Hernandez S, Niesten A, de la Rebiere G, Serteyn D, Deleuze S.Myeloperoxidase (MPO) is a pro-oxidant enzyme associated with decreased motility in thawed equine semen. This study aimed to describe MPO concentration, activity and subunits in raw and thawed semen and to correlate these data with motilities in raw and thawed semen. Semen samples from five stallions were collected four times. Motilities were assessed in raw and thawed semen. MPO assays were performed in raw seminal plasma, raw sperm-rich pellet and thawed semen. Total and active MPO concentrations were, respectively, assayed by enzyme-linked immunosorbent assay and specific immunological extr...
Thompson DL, Pickett BW, Berndtson WE, Voss JL, Mett TM.Stallions were subjected to a gradually increasing photoperiod beginning on October 15, 1973. The maximum artificial daylength (16 hr) was imposed on February 8, 1974, and maintained until October 6, 1974. Two ejaculates were collected from each of five treated and four control stallions weekly, with an interval of 24 hr between die paired ejaculates on the first and alternate weeks, and an interval of 1 hr on the second and alternate weeks. During summer, stallions subjected to the artificial photoperiod produced less (P<.05) gel-free semen and gel per ejaculate and had more (P<.05) spermatoz...
Burger D, Meroni G, Thomas S, Sieme H.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...
Vizcarra JA, Ford JJ.The sperm mobility assay used in the present study measures the rate of sperm penetration in a biologically inert cell-separation solution (Accudenz). When a sample of sperm is overlaid in a cuvette containing Accudenz, sperm penetrate the solution and absorbance of the sample can be measured with a spectrophotometer. This assay has been successfully used to select chicken and turkey semen donors. We validated this assay for semen from boars and stallions. Absorbance was measured after overlaying fresh semen from each species in prefilled cuvettes for 1, 5, 10, 15, 20, and 40 min. There were n...
McDonnell SM, Hinze AL.Periodic spontaneous erection and penile movements known as masturbation (SEAM) occur normally at approximately 90 min intervals in awake equids. SEAM in horses has traditionally been misunderstood by many horsemen as aberrant behavior that should be eliminated. Accordingly, it is not uncommon for trainers of performance stallions or managers of breeding stallions to punish SEAM in an attempt to eliminate the behavior. Previous clinical observations and preliminary unsystematic trials had suggested that attempts to stop stallion SEAM may lead to an increase rather than a decrease in SEAM, and ...
Hoogewijs M, De Vliegher S, De Schauwer C, Govaere J, Smits K, Hoflack G, de Kruif A, Van Soom A.Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with compu...
Halo M, Tirpák F, Slanina T, Tokárová K, Massányi M, Dianová L, Mlyneková E, Greń A, Halo M, Massányi P.This study was aimed to determine the impact of different taurine and caffeine combinations on the motility, viability, and oxidative markers of chilled stallion spermatozoa. Each stallion semen sample was diluted in a ratio of 1:2, with various taurine and caffeine concentrations (2.5-7.5 mg/mL taurine + 0.625-1.25 mg/mL caffeine) dissolved in a conventional extender. The control samples (CON) were prepared by diluting ejaculate only using the conventional extender. The motility was analyzed using a CASA system at different time intervals (0, 6, 12, 24, and 30 h) and the viability was evaluat...
Veeramachaneni DN, Sawyer HR.Conventional light microscopic evaluation does not fully utilize potential indicators in seminal ejaculates for diagnosis of disorders of the reproductive tract. The technique of evaluation of all cellular components of semen, as described in this article, utilizing both light and transmission electron microscopy is a valuable diagnostic tool. Compare with other common biopsy procedures, use of semen as biopsy material is noninvasive, more representative than excisional biopsy, less expensive, and helps in the longitudinal evaluation after a therapeutic regimen.
Filho JS, Corcini CD, Santos FCC, Anciuti AN, Gatti NLS, Anastacio E, Mielke R, Nogueira CEW, Curcio BR, Varela AS. BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. Objective: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. Methods: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. R...
Brogna R, Fan J, Sieme H, Wolkers WF, Oldenhof H.Even though dried sperm is not viable, it can be used for fertilization as long as its chromatin remains intact. In this study, we investigated drying- and temperature-induced conformational changes of nucleic acids and stallion sperm chromatin. Sperm was diluted in preservation formulations with and without sugar/albumin and subjected to convective drying at elevated temperatures on glass substrates. Accumulation of reactive oxygen species was studied during storage at different temperatures, and the sperm chromatin structure assay was used to assess DNA damage. Fourier transform infrared spe...
Álvarez C, Gil L, González N, Olaciregui M, Luño V.The rise of assisted reproduction techniques in equine medicine has fostered investigations that seek to optimize methods to increase fertility rates. Since cryopreservation continues to give low values of viability in stallions, the handling and preservation of the sperm is of vital importance. This reduction of fertility makes it essential for farmers to find new options that ensure reliability in the use of these techniques. The main objective of this study was to assess the effect of INRA 96® (manufactured commercial extender for cooling of Equine semen) as an extender for cryopreservatio...
Bustamante-Filho IC, Rosa AP, Van der Linden LS, Pederzolli CD, Neves AP, Dutra-Filho CS, Jobim MI, Mattos RC.The use of stallion semen collected from cauda epididymis for AI has increased due to the new protocols available for cryopreservation. Preserving the genetic material from valuable males that suffer sudden death or other events that prematurely end the stallion's reproductive life is an important strategy for Stud breeding management. While protecting spermatozoa from oxidative stress and infectious agents, the epididymis promotes the enhancement of sperm cell morphology and changes in membrane protein profile, increasing its fertility potential. The epididymal fluid must be a balanced redox ...
Ferreira HN, Ferreira-Silva JC, Rocha JM, Farras MC, Calixto M, Moura MT, Alvarenga MA, Oliveira AL.Semen cryopreservation causes DNA damage, thus requiring continuous monitoring. Objective: To compare two assays for sperm DNA fragmentation (SDF) from stallions with contrasting semen freezability. Methods: Thirteen stallions were classified as good semen freezers (GSF) or bad semen freezers (BSF). Ejaculates were cryopreserved with three diluents. Semen was subject to SDF evaluation using the sperm chromatin structure assay (SCSA) and Halomax after thawing (0 h) and after a 4 h thermoresistance test. Results: On semen of BSF, analysis by SCSA was similar between evaluations, but Halomax show...
Weiss S, Janett F, Burger D, Hässig M, Thun R.The aim of the present study was to investigate the influence of various centrifugation methods on sperm loss and quality of frozen-thawed semen. From at a total of 8 Warmblood stallions of the National Stud Farm in Avenches, 3 ejaculates each were collected and seminal plasma was removed using 3 different centrifugation regimes. In method I (reference method) centrifugation occurred by a speed of 600 x g during 10 minutes. In method II 1000 x g was used during 2 minutes while in method III centrifugation was performed by 2000 x g during 2 minutes. After centrifugation 90%, of the supernatant ...
Prell MJ, McCue PM, Moffett PD, Graham JK.Breeding mares with cryopreserved semen requires specialized equipment for storage and thawing and more intensive mare management. The objectives of this study were (1) evaluate the longevity of frozen stallion semen once it had been thawed, extended, and maintained at 5°C for 48 hours in a passive cooling container, and (2) determine fertility potential of frozen semen that had been thawed, extended, and used to inseminate mares after 24 hours of cooled storage. Eight ejaculates were collected and aliquots were cooled in either INRA96 and CryoMax LE minus cryoprotectant at a concentration ...
Oldenhof H, Heutelbeck A, Blässe AK, Bollwein H, Martinsson G, Wolkers WF, Sieme H.The aim of this study was to evaluate inter-individual variability in osmotic properties of stallion spermatozoa and its correlation with cryosurvival. In addition, temperature dependency of hypo-osmotic tolerance and membrane fluidity were studied. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15-30 °C temperature range, whereas membrane stability was found to be decreased at 4 and 37 °C. Bull spermatozoa showed greater hypo-osmotic tolerance compared with stallion spermatozoa, especially at temperatures above 30 °C, which coincided with decreased membr...
Morrell JM, Timoney P, Klein C, Shuck K, Campos J, Troedsson M.Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, ...
Ortiz I, Felix M, Resende H, Ramírez-Agámez L, Love CC, Hinrichs K.To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods: Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. R...
Johannisson A, Al-Essawe EM, Al-Saffar AK, Karkehabadi S, Lima-Verde I, Wulf M, Aurich C, Morrell JM.The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding...
Andrade Junior LRP, Segabinazzi LGTM, Oliveira SN, Dell'Aqua JA, Papa FO.A high amount of blood and not the mere presence of blood in equine semen impacts fertility. The aim of this study was to develop an approach to rescue the fertility of stallions with high hemospermia levels. Semen from 15 stallions was divided into four experimental groups: (a) Control-pure raw semen, (b) WB50-50% (v/v) whole blood added into semen, (c) E1-WB50 extended in a 1:1 (v/v) ratio with milk-based extender and (d) E2-WB50 extended in a 2:1 ratio with milk-based extender. Sperm kinetics, plasma membrane integrity (PMI), lipid peroxidation (PER) and intracellular superoxide (O ) produc...
Caselles AB, Miro-Moran A, Morillo Rodriguez A, Gallardo Bolaños JM, Ortega-Ferrusola C, Salido GM, Peña FJ, Tapia JA, Aparicio IM.Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and ...
Segabinazzi LGTM, Scheeren VFDC, Freitas-Dell'Aqua CP, Papa FO, Alvarenga MA, Dell'Aqua JA, Canisso IF.The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with t...
Len JA, Beehan DP, Lyle SK, Eilts BE.It was hypothesized that optimal sperm recovery rate (RR) without damage to the sperm would be obtained after centrifugation without a cushion solution. Semen collected three times from six light breed stallions was extended to 25 × 10(6) sperm/mL and centrifuged at CON (noncentrifuged), 900NC (no-cushion), 900C (cushion), 1800NC, and 1800C × g for 10 minutes. Sperm concentration, motility (TM and PM), and intact plasma membranes (PLM) and acrosomes (ACR) pre- and postcentrifugation (D0) and after 24 hours (D1) of cooling were evaluated. The RR in the CON (100 ± 0.0), 900NC (93.7 ± 2.9), a...
Serafini R, Varner DD, Blanchard TL, Teague SR, LaCaze K, Love CC.The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP) or unexposed (SP) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP vs. SP) or freezing treatment (FS vs. FZ...
Desantis S, Ventriglia G, Zizza S, Nicassio M, Valentini L, Di Summa A, Lacalandra GM.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...
Bazzano M, Zhu C, Laus F, Giambattista AD, Laghi L.The application of the 'omics' studies in the field of animal reproduction has been aimed at identifying novel biomarkers of fertility since the last few years. When assessing reproductive efficiency in horses, breed should also be taken into account as it can influence semen quality and fertility. Considering the growing interest in metabolomic analysis to evaluate male fertility, we aimed to investigate the metabolomic profile of seminal plasma in two different horse breeds. Twelve healthy stallions, n.6 American Quarter Horse (AQH) and n.6 Italian Draft Horse (IDH) stallions, regularly used...
Loureiro KC, Lima-Verde IB, Johannisson A, Ntallaris T, Jager A, Štěpánek P, da Costa Mendonça M, Severino P, Morrell JM.Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes ...
Clulow JR, Evans G, Morris LH, Maxwell WM.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 ...
Batellier F, Magistrini M, Fauquant J, Palmer E.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...
