Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
Hoogewijs M, Morrell J, Van Soom A, Govaere J, Johannisson A, Piepers S, De Schauwer C, De Kruif A, De Vliegher S.The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E s...
Edmond AJ, Brinsko SP, Love CC, Blanchard TL, Teague SR, Varner DD.Centrifugal fractionation of semen is commonly done to improve quality of human semen in assisted-reproduction laboratories, allowing sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, promoting their retention above defined density media, with structurally normal sperm passing through the media following centrifugation. Three experiments were conducted to evaluate the effects of density-medium type, centrifuge-tube size, sperm number, and density-medium volume (column height) on stallion sperm quality and recovery rate in sperm pelle...
Harland MM, Stewart AJ, Böse R.A comprehensive purchase examination is expected by American clients intent on importing a horse from a foreign country. American veterinarians may be involved in performing purchase examinations in foreign countries or, more often, interpreting findings from foreign veterinarians for their American clients. Exportation and importation requirements for horses and semen vary from country to country. Detailed knowledge of the requirements by all involved veterinarians is essential for efficient and successful international equine travel.
Morillo-RodrÃguez A, MacÃas-GarcÃa B, Tapia JA, Ortega-Ferrusola C, Peña FJ.Ejaculates from six pure Spanish stallions were split, and one subsample frozen in a commercial extender supplemented with the lipid soluble antioxidant butylated hydroxytoluene (BHT), while the other subsample served as control. After at least 4 weeks of storage, samples were thawed and post-thaw sperm quality analysed: sperm motility and kinematics using a CASA system, membrane and acrosome integrity and mitochondrial membrane potential using flow cytometry. The outcome of cryopreservation varied significantly among stallions. However, the supplementation with 1 mm BHT had no significant eff...
Clulow JR, Buss H, Evans G, Sieme H, Rath D, Morris LH, Maxwell WM.Sex-sorted, frozen-thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex-sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex-sorted frozen-thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82(®) and a modified lactose-ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex-sorted stallion spermatozoa. No significant differences between the two freez...
Papa FO, FelÃcio GB, Melo-Oña CM, Alvarenga MA, De Vita B, Trinque C, Puoli-Filho JN, Dell'Aqua JA.The objective of this study was to determine whether replacing the egg yolk with soybean lecithin in the Botu-Crio® cryodiluent would maintain the fertility of cryopreserved stallion sperm. Two experiments were performed to evaluate cell freezability. In experiment 1, sperm from 15 stallions were frozen in Botu-Crio® (BC) or Botu-Crio® which contained 45g/L soybean lecithin (BCLS45) in place of the egg yolk. In experiment 2, we compared different concentrations of soybean lecithin: 0, 10.0, 12.5, 15.0, 17.5 and 20.0g/L (BC, BCLS10, BCLS12.5, BCLS17.5 and BCLS20, respectively). In experiment...
Pillet E, Labbe C, Batellier F, Duchamp G, Beaumal V, Anton M, Desherces S, Schmitt E, Magistrini M.Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these ...
Scherzer J.Mares can be artificially inseminated with chilled or frozen semen to increase the revenue from their offspring. Embryo transfer can be used to produce more than one foal from a single mare per season. Recent advances in using equine follicle-stimulating hormone to induce superovulation in mares have stimulated research on preserving equine embryos. Equine embryos are usually collected on day 7 or 8 after ovulation, and younger (day 6.5) embryos are typically cryopreserved. Cryopreservation improves the ability of veterinary clinicians to preserve embryos for implantation in recipient mares an...
Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K.Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sper...
Monteiro GA, Papa FO, Zahn FS, Dellaqua JA, Melo CM, Maziero RR, Avanzi BR, Alvarenga MA, Guasti PN.The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides ...
Peña FJ, GarcÃa BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO.We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.
Morrell JM, Garcia BM, Pena FJ, Johannisson A.The purpose of this study was to determine if the quality of stored stallion semen doses could be enhanced by the scaled-up version of Single Layer Centrifugation using Androcoll-E-Large. Three semen doses from each of fifteen stallions were transported overnight to the Swedish University of Agricultural Sciences (SLU) for processing 24 h after semen collection. Sperm quality in the resulting SLC-selected samples was significantly improved compared to the uncentrifuged samples: mean progressive motility was increased by 8% on the day of processing (P < 0.001) and by 13% after 24 h cold storage...
RodrÃguez AM, Ferrusola CO, GarcÃa BM, Morrell JM, MartÃnez HR, Tapia JA, Peña FJ.Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p<0.05) and velocity (p<0.05), membrane integrity (increases ...
Pozor MA, Muehlhaus J, King A, Macpherson ML, Troedsson MH, Bailey CS.The objective was to investigate the effects of pentoxifylline (PTX) on testicular perfusion and sperm production in stallions. In a preliminary study, six mature Miniature horse stallions were given 0, 8.5, or 17.0 mg/kg of PTX orally, twice daily, for 3 d. Total Arterial Blood Flow Rate (TABFR) was higher (P < 0.05) in all treated versus control stallions during and after treatment. Two months later (during the fall and winter), the same stallions received either 0 or 17 mg/kg of PTX orally, twice daily for 60 d. Resistance and pulsatility indices (RI and PI, respectively) decreased in PT...
Pamornsakda T, Pojprasath T, Suwimonteerabutr J, Tharasanit T.Equine epididymal sperm are known to be severely sensitive to cryopreservation, in terms of sperm quality and pregnancy rate. The objective of this study was to examine the effects of cholesterol loaded cyclodextrins (CLCs) on the quality of stallion epididymal sperm during cryopreservation. In experiment I, sperm were treated with different concentrations of CLCs: (1) 0mg (control), (2) 1.5mg, (3) 3mg, and (4) 6 mg per 120 × 10(6) sperm. The sperm viability and amount of cholesterol were determined at 15, 30 and 45 min after CLC treatment using viability markers (Ethidium homodimer-1 and Cal...
Jobim MI, Trein C, Zirkler H, Gregory RM, Sieme H, Mattos RC.The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spe...
Canisso IF, Carvalho GR, Morel MD, Ker PG, Rodrigues AL, Silva EC, Coutinho Da Silva MA.As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Objective: To compare the seminal parameters of cryopreserved donkey semen in lactose-EDTA and lactose-yolk extenders and the fertility rates on horse mares. Methods: TRIAL 1: Sperm total and progressive motility, vigour (scale 0-5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was ...
Mari G, Castagnetti C, Rizzato G, Mislei B, Iacono E, Merlo B.Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simp...
Kareskoski M, Sankari S, Johannisson A, Kindahl H, Andersson M, Katila T.Semen jets in the stallion's ejaculate differ in both the biochemical composition of seminal plasma (SP) and in sperm concentration, forming sperm-rich fractions (HIGH) and sperm-poor fractions (LOW). This study examined (i) the association of the levels of selected components of SP [alkaline phosphatase (AP), acid phosphatase (ACP), Na(+), Cl(-), K(+), Ca, Mg and prostaglandin E(2)] with semen quality, sperm longevity and fertility; (ii) the effects of the presence of SP on sperm survival during cooled storage; (iii) the differences in the composition of SP between sperm-rich and sperm-poor e...
Salazar JL, Teague SR, Love CC, Brinsko SP, Blanchard TL, Varner DD.Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), ...
da Silva CM, MacÃas-GarcÃa B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G....Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well-known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at ...
Pojprasath T, Lohachit C, Techakumphu M, Stout T, Tharasanit T.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integ...
Mattos MC, Bastos MR, Guardieiro MM, Carvalho JO, Franco MM, Mourão GB, Barros CM, Sartori R.The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estr...
Hoffmann N, Oldenhof H, Morandini C, Rohn K, Sieme H.Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is expo...
Foster ML, Varner DD, Hinrichs K, Teague S, Lacaze K, Blanchard TL, Love CC.Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and a...
Contri A, De Amicis I, Molinari A, Faustini M, Gramenzi A, Robbe D, Carluccio A.In this study, the effect of dietary supplementation of organic selenium, vitamin E, and zinc on raw semen characteristics was evaluated. Ten stallions with normal fertility were divided into two groups: a control group (CG), in which standard diet was provided, and a treated group (TG), in which the standard diet was supplemented with 1500 mg of α-tocopherol acetate, 360 mg of zinc, and 2.5 mg of organic selenium on a daily basis. Semen parameters on fresh semen were evaluated three times in all stallions before antioxidant supplementation (T0) and 30 (T1), 60 (T2), and 90 (T3) d after suppl...
Cocchia N, Pasolini MP, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G, Mancini A.New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: ...
Heckenbichler S, Deichsel K, Peters P, Aurich C.Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm ...
Heise A, Thompson PN, Gerber D.Fresh and post-thaw parameters (motility, morphology and viability) of stallion epididymal spermatozoa that have been and have not been exposed to seminal plasma were evaluated, and directly compared to fresh and post-thaw parameters of ejaculated spermatozoa. Six sperm categories of each stallion (n=4) were evaluated for motility, morphology and viability. These categories were fresh ejaculated spermatozoa (Fr-E), fresh epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+), fresh epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP-), frozen-thawed eja...
Brinsko SP, Rowan KR, Varner DD, Blanchard TL.This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loa...
Varner DD, Blanchard TL, Meyers PJ, Meyers SA.A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, fr...
Moura TCM, Arruda LCP, Araújo Silva RAJ, Silva RPF, Oliveira AS, Tobal LFM, Batista AM, Carneiro GF, Guerra MMP.The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 10 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (-196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasm...
Johannisson A, Lundgren A, Humblot P, Morrell JM.The decrease in foaling rates after artificial insemination with cooled semen warrants the search for new predictors of fertility. The objectives were to investigate levels of naturally occurring reactive oxygen species (ROS) in cooled, stored stallion semen doses for artificial insemination (AI), and their relationship with parameters of semen quality and with pregnancy rate. Semen was collected from warmblood stallions (n=15) and used to prepare commercial semen doses for AI. Sperm quality was evaluated after cooled transport to the laboratory overnight. The results were correlated with obse...
Diaz-Jimenez M, Dorado J, Consuegra C, Ortiz I, Pereira B, Carrasco JJ, Gomez-Arrones V, Domingo A, Hidalgo M.The aim of this study was to assess the effect of different factors affecting vitrification success of donkey sperm: extender, sperm concentration, volume and storage vessel type. In Experiment 1, sucrose supplementations at 0.25 and 0.1 M were compared using two base extenders (containing or not egg-yolk); in Experiment 2, three sperm concentrations were assessed: 100, 200 or 300 million sperm/mL; and in Experiment 3, three different sperm volumes (100, 160 and 200 μL) and two different storage vessels (0.25 and 0.5 mL straws) were assessed. Sperm motility variables (CASA), plasma memb...
Heckenbichler S, Deichsel K, Peters P, Aurich C.Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm ...
Podico G, Ellerbrock RE, Curcio BR, Cheong SH, Lima FS, Canisso IF.Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine conta...
Vigolo V, Gautier C, Falomo ME, Aurich C.The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen-thawed semen from 15 stallions that were...
de Vasconcelos Franco JS, Faheem M, Chaveiro A, Moreira da Silva F.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...
Lindeberg H, Karjalainen H, Koskinen E, Katila T.A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw a...
Al-Khaldi K, Yimer N, Al-Bulushi S, Haron AW, Hiew M, Babji AS.The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using com...
Turner RM, Casas-Dolz R, Schlingmann KL, Hameed S.To determine whether a homologue of A-kinase anchor protein 4 (AKAP4) is present and functional as an AKAP in equine spermatozoa and examine the effect of semen cooling and cryopreservation on binding of equine AKAP4 to the regulatory (RII) subunit of protein kinase-A (PK-A). Methods: Ejaculated semen collected from 2 fertile stallions, 3 bulls, and 3 humans. Methods: Identification of an equine homologue of AKAP4 was investigated via DNA sequencing. Protein was extracted from the spermatozoa of each species for immunoblot analysis to identify AKAP4 and its precursor protein, pro-AKAP4; immuno...
Bucci D, Spinaci M, Mislei B, Gadani B, Rizzato G, Love CC, Tamanini C, Galeati G, Mari G.Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some...
Alghamdi AS, Troedsson MH, Xue JL, Crabo BG.To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. Methods: 7 stallions from which we collected > or = 3 ejaculates/stallion. Methods: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or...
Smits K, Hoogewijs M, Woelders H, Daels P, Van Soom A.Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity...
Bedford SJ, Gowdy HL, Hinrichs K.This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38 degrees C in either air or 5% CO2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-m...
van Heule M, Verstraete M, Blockx Z, De Blende P, Dini P, Daels P.Cooled storage of semen after thawing can expand the use of frozen semen, providing the possibility of thawing and evaluating the semen at the storage site and subsequently shipping the semen. Our objectives were (1) to examine the motility and viability of frozen-thawed semen after cooled storage and (2) to compare two cooled-storage protocols for frozen-thawed semen. The samples (n = 31) were either placed immediately in a passive cooling box for 8 or 24 hours (CB) or placed in a refrigerator at 4°C for 30 minutes and then transferred to a passive cooling box (REF). Total and progressive ...
Diaz-Jimenez M, Dorado J, Ortiz I, Consuegra C, Pereira B, Gonzalez-De Cara CA, Aguilera R, Mari G, Mislei B, Love CC, Hidalgo M.The aim of this study was to evaluate the effect of different concentrations of sucrose combined with bovine serum albumin (BSA), as non-permeable cryoprotectants, on donkey sperm parameters after cryopreservation, in comparison to a control extender containing glycerol. Semen from five Andalusian donkeys (n = 12) were centrifuged and resuspended with a commercial extender for equine sperm (Gent A, Minitube) adding 1% BSA and different concentrations (M, mol/l) of water-diluted sucrose: 0.05, 0.1, 0.25, 0.35 and 0.45. Thereafter, semen (n = 24) were diluted in the same base extender co...
Oldenhof H, Friedel K, Akhoondi M, Gojowsky M, Wolkers WF, Sieme H.Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures...
Güvenc K, Reilas T, Katila T.Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen co...
Huber D, Amsler E, Vidondo B, Kaeser R, Wespi B, Sieme H, Burger D.There exist differences in the reproductive behavior of stallions and mares under free-running and domestic in-hand breeding conditions. Contrary to artificial insemination programs, a stallion mates a mare multiple times per estrus under natural conditions. The objective of this study was to determine if multiple periovulatory artificial inseminations (MI), four times in two different time intervals instead of two, would result in increased pregnancy outcome or higher incidences of breeding induced endometritis. Methods: Eighty-two estrous mares were allocated randomly to one of three experim...
Arriola J, Foote RH.Because microfloral content of stallion semen tends to be high, and strains may be resistant to commonly used antibiotics, amikacin was tested with stallion semen and compared with bull semen. Nine ejaculates to stallion semen were incubated at 37 C in egg yolk-tris extender for 0, 2, 4, 6, 8 and 10 h in the presence of amikacin concentrations of 0, 50, 100, 250, 500, 1,000 and 10,000 microgram/ml, with penicillin and penicillin-streptomycin as controls. Averaged over all incubations, spermatozoal motility was 44, 48, 49, 46, 45, 45 and 19%, for increasing concentrations of amikacin, compared ...
Delerue M, Breuil MF, Duquesne F, Bayon-Auboyer MH, Amenna-Bernard N, Petry S.Taylorella equigenitalis can be transmitted during artificial insemination. This report describes clinical T. equigenitalis transmission by cryopreserved stallion semen. T. equigenitalis isolates from a mare's vaginal discharge and semen from the same batch of the cryopreserved semen used for the insemination gave identical API ZYM, antibiotic susceptibility, and multilocus sequence typing results (ST-46); furthermore, the multilocus sequence typing lineage ST-46 is known to circulate in the country of semen collection. These results support the need for strict contagious equine metritis scre...
Clulow JR, Maxwell WM, Evans G, Morris LH.Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. Methods: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after tha...
Ponthier J, Teague SR, Franck TY, de la Rebière G, Serteyn DD, Brinsko SP, Love CC, Blanchard TL, Varner DD, Deleuze SC.Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO and its concentration is associated with decreased sperm motility. Recently, MPO concentration in post-thaw semen was shown to be associated with the presence of non-sperm cells (NSC). The objective of this study was to evaluate the effect of a single-layer colloidal centrifugation before cryopreservation on NSC and MPO concentrations in equine semen. The experimental design consisted of freezing semen with or without previous centrifugation t...
Roach J, Schnobrich M, Ellerbrock R, Feijo L, Bradecamp E, Alvarenga MA, Kline K, Canisso I.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...
Gil MC, Ferrusola CO, Anel-López L, Ortiz-Rodriguez JM, Alvarez M, de Paz P, Anel L, Peña FJ.Spermatozoa undergo apoptotic changes during the cryopreservation process. These changes, recently termed spermptosis, resemble the cryopreservation induced delayed onset of cell death observed after thawing of somatic cells. Due to its importance in cryobiology, methods to easily identify spermptotic cells are warranted. In this study, a well-validated method for identification of spermatozoa with caspase 3 activity was compared with use of the combination of Hoechst 33342 (H-42) and ethidium homodimer (Eth-1). Live, dead and apoptotic spermatozoa assessed with each method were compared using...
