Freezing techniques in horses involve the controlled application of low temperatures to preserve equine biological samples, tissues, or cells for research and clinical purposes. These techniques are employed in various contexts, including the preservation of semen for artificial insemination, the storage of embryos for breeding programs, and the conservation of genetic material. The process typically involves the use of cryoprotectants to prevent ice crystal formation, which can damage cellular structures. Research in this area focuses on optimizing freezing protocols to enhance viability and functionality post-thaw. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and outcomes of freezing techniques in equine science.
Miró J, MartÃnez-Rodero I, Yeste M, Catalán J.Cryopreservation is currently the only strategy for long-term conservation of equine sperm. To get optimal post-thaw sperm survival, carefully following each step of the freezing protocol is crucial. First, one needs to obtain and exhaustively analyze an ejaculate of good sperm quality. Then, the seminal plasma is removed by centrifugation, and the resulting pellet is resuspended in a certain volume of the freezing medium to reach the right sperm concentration. Finally, sperm samples are packaged into 0.5-mL straws, cooled, and frozen using an automatic, controlled-rate freezer. Once the tempe...
McDonald S, Harley JJ, Hockenhull J.Free-ranging native Dartmoor and Exmoor ponies have not only held strong cultural and environmental significance for thousands of years within their respective national parks, but their environmental benefits and naturally selected characteristics have also been acknowledged and harnessed for conservation grazing and rewilding programmes. Despite a wealth of literature regarding the welfare of sports, leisure and working horses, there is little information concerning the welfare of free-ranging and extensively grazing ponies. The present study compared the welfare of native Exmoor and Dartmoor...
Long AE, Pitta D, Hennessy M, Indugu N, Vecchiarelli B, Luethy D, Aceto H, Hurcombe S.Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different ...
Schoenecker KA, King SRB, Hennig JD, Cole MJ, Scasta JD, Beck JL.There are two species of free-roaming feral equids in North America: horses (Equus caballus) and donkeys or "burros" (E. asinus). Both species were introduced as domestic animals to North America in the early 1500s and currently inhabit rangelands across the western United States, Canada, and all continents except Antarctica. Despite their global distribution, little is known about their fine scale spatial ecology. Contemporary research tools to assess space use include global positioning system (GPS) tracking collars, but older models were problematic due to stiff collar belting causing poor ...
Lavado RA, Lewis J, Montgomery JB.Laminitis is a severely debilitating and life-threatening condition that occurs as a consequence of different primary triggering factors. Continuous digital hypothermia (CDH) is recommended in horses at risk of, or diagnosed with, acute laminitis due to its several physiological and biochemical alterations that may be positive for the prevention and early treatment of the condition, representing a low risk of adverse effects. Modulation of the inflammatory response, profound vasoconstriction, and prevention of tissue damage are the most notable protective effects of cryotherapy on the lamellae...
El-Shalofy A, Gautier C, Khan Y, Aurich J, Aurich C.This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ -150 °C) or on dry ice (≤ -78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intac...
Contreras MJ, Arias ME, Fuentes F, Muñoz E, Bernecic N, Fair S, Felmer R.Cryopreservation of stallion semen does not achieve the post-thaw quality or fertility results observed in other species like cattle. There are many reasons for this, but the membrane composition and intracellular changes in stallion sperm predispose them to low resistance to the cooling, freezing, and subsequent thawing process. Damage to the sperm results from different processes activated during cryopreservation, including oxidative stress, apoptosis, and structural modifications in the sperm membrane that increase the deleterious effect on sperm. In addition, significant individual variabi...
de Moura TCM, Arruda LCP, Cahú TB, Bezerra RS, Carneiro GF, Guerra MMP.Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were ...
Jhamb D, Talluri TR, Sharma S, Juneja R, Nirwan SS, Yadav D, Pargi KK, Tanwar A, Kumar P, Kumar R, Mehta SC, Parashar M, Gaur M.Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing...
Umair M, Beitsma M, de Ruijter-Villani M, Deelen C, Herrera C, Stout TAE, Claes A.The cryotolerance of equine blastocysts larger than 300 μm can be improved by aspirating blastocoele fluid prior to vitrification; however, it is not known whether blastocoele aspiration also enables successful slow-freezing. The aim of this study was therefore to determine whether slow-freezing of expanded equine embryos following blastocoele collapse was more or less damaging than vitrification. Grade 1 blastocysts recovered on day 7 or 8 after ovulation were measured (>300-550 μm, n = 14 and > 550 μm, n = 19) and blastocoele fluid was aspirated prior to slow-freezing in 10...
Hatami M, Qasemi-Panahi B, Daghigh Kia H, Moghaddam G, Janmohammadi H.Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, β-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β-carotene. Subse...
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...
Rosa MVD, Rosa M, Botteon PTL.This study aimed to evaluate the use of equine amniotic membrane (EAM), frozen indirectly using liquid nitrogen and stored between -10° and -24°C, in the treatment of equine skin lesions. Six healthy female horses, aged 3-10 years, were included in this study. EAM was collected from previously evaluated healthy parturient mares. Wounds were surgically created at the distal ends of the forelimbs. One limb was chosen for treatment, and the contralateral limb was chosen as the control. Pain sensitivity, presence of granulation tissue, secretions, and bleeding after debridement during cleaning w...
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
Freitas NPP, Silva BDP, Bezerra MRL, Pescini LYG, Olinda RG, Salgueiro CCM, Nunes JF, Martins JAM, Neto SG, Martins LT.This study investigated platelet-rich plasma (PRP) and adipose stem cell-conditioned medium (ASC-CM) use as a strategy to accelerate tissue healing. Platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) were quantified in fresh and freeze-dried PRP and ASC-CM, and a stability test was performed in the freeze-dried samples (90 and 180 days of storage). A cell proliferation test was performed using equine mesenchymal stem cell culture in reconstituted PRP gel mesh after freeze-drying. In vivo PRP, ASC-CM applications, or their association were performed in induced w...
Morse-Wolfe B, Bleach E, Kershaw C.Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three f...
Aguiar CS, Barros CHSC, Machado WM, Allaman IB, Leite AO, Barbosa LP, Snoeck PPDN.The aim of this study was to evaluate the association of different concentrations of Trolox and the addition of a fixed concentration of DHA in the freezing of semen of Mangalarga Marchador stallions. To that end, 16 ejaculates were frozen in the following extenders: E1) BotuCrio (BC; Control); E2) BC + 50 ngml DHA + 30 µM Trolox (BCDHA30T); E3) BC + 50 ngml DHA + 40 µM Trolox (BCDHA40T); E4) BC + 50 ngml DHA + 50 µM Trolox (BCDHA50T). All the tested extenders were similar in preserving different kinematic parameters, cell functional integrity, compacted DNA, and high and intermediate mitoc...
Peña FJ, Gibb Z.The growing understanding of the mechanisms regulating redox homeostasis in the stallion spermatozoa, together with its interactions with energetic metabolism, is providing new clues applicable to the improvement of sperm conservation in horses. Based on this knowledge, new extenders, adapted to the biology of the stallion spermatozoa, are expected to be developed in the near future. The preservation of semen either by refrigeration or cryopreservation is a principal component of most animal breeding industries. Although this procedure has been successful in many species, in others, substantia...
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 ...
Gray SM, Gutierrez-Nibeyro SD, Horn GP, McCoy AM, Schaeffer DJ, Stewart M.To assess the effect of repeated freezing and thawing on the suture pull-out strength in arytenoid and cricoid cartilages subjected to the laryngoplasty (LP) procedure. Methods: Ex vivo experimental study. Methods: Ten grossly normal equine cadaveric larynges. Methods: Bilateral LP constructs were created using a standard LP technique. One hemilarynx was randomly allocated to the single freeze and thaw group and the other allocated to the repeated freeze and thaw (3 complete cycles) group. The suture ends of each LP construct were attached to a load frame and subjected to monotonic loading unt...
Ortiz I, Dorado J, Pereira B, Diaz-Jimenez M, Consuegra C, Gosalvez J, Hidalgo M.The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dispersion test. CC-DF rates between vitrified and control COCs were statistically compared by Student's t-test. The rates of CC-DF from control COCs were lower than in vitrified COCs...
Catalán J, Yánez-Ortiz I, Tvarijonaviciute A, González-Aróstegui LG, Rubio CP, Barranco I, Yeste M, Miró J.The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to h...
Nogueira BG, Pereira RR, Bitencourt JL, Milan B, Reis WVA, Junior MV, Acácio BR, Sampaio BFB, I L Souza M.The objective of the experiment was to evaluate the effect of the addition of different concentrations of the antioxidants Coenzyme Q10 (CoQ10) and melatonin to equine semen freezing diluent, alone or in combination, during the cryopreservation process. Twenty ejaculates (n = 5 stallions) were divided in groups: Control (C, without the addition of antioxidants), melatonin 0.75 mM (MEL1), melatonin 1.5 mM (MEL2), CoQ10 40 μg/mL (Q1), CoQ10 200 μg/mL (Q2), and CoQ10 40 μg/mL+ 0.75 mM melatonin (Q1 +MEL1). Q1 and Q2 groups demonstrated intact plasma membrane and high mitochondrial me...
Contreras MJ, Arias ME, Silva M, Cabrera P, Felmer R.Cryopreservation of stallion semen is less efficient than other species such as bovine. This is mainly because of the greater susceptibility of stallion sperm to the freezing damage that generates oxidative stress and plasma membrane injury, resulting in DNA fragmentation and cell death. These data suggest the need to develop new strategies of sperm cryopreservation that can improve the efficiency of this technique in stallions by reducing or preventing membrane damage and cell death. The present study aimed to evaluate the effect of adding membrane stabilizers to the freezing medium and asses...
Fanelli D, Tesi M, Monaco D, Diaz-Jimenez M, Camillo F, Rota A, Panzani D.In the literature, the very low pregnancy rates after artificial insemination (AI) with frozen semen in donkeys were improved in one study after re-extension of the frozen-thawed semen in autologous seminal plasma. The aims of our study were (1) to describe in vitro post-thaw parameters of donkey jackass semen after re-extension in seminal plasma (SP) or in INRA96 and (2) to compare pregnancy rates in jennies bred with frozen-thawed semen using two different AI protocols. Semen collected from two Amiata donkey stallions, known to be fertile, was frozen in INRA96 supplemented with 2% egg yolk, ...
Nikitkina EV, Dementieva NV, Shcherbakov YS, Atroshchenko MM, Kudinov AA, Samoylov OI, Pozovnikova MV, Dysin AP, Krutikova AA, Musidray AA....The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds. Methods: Semen samples were collected from the stallions of 23 horse breeds. The following semen characteristics were examined: progressive motility (PM), p...
Yánez-Ortiz I, Catalán J, RodrÃguez-Gil JE, Miró J, Yeste M.Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. In some farm animals, the use of cryopreserved sperm has so many benefits for which relevance has become more evident in recent decades. Values for post-thaw sperm quality, however, are variable among species and within individuals of the same species. There is no standardized methodology for each of the stages of the cryopreservation procedure (andrological examination, semen collection, dilution, centrifugation, resuspension of the pellet with the freezing medium, p...
Parks JE, Lynch DV.Composition and thermotropic phase behavior of sperm membrane lipids from species ranging in sensitivity to cold shock were determined. Lipids from whole sperm and sperm plasma membrane were fractionated into neutral lipid, glycolipid, and phospholipid fractions. Compositional analyses were completed for free sterols, phospholipids and phospholipid-bound fatty acids. Phase transition temperatures were determined for phospholipid and glycolipid fractions using differential scanning calorimetry. Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ra...
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...
Sieme H, Oldenhof H, Wolkers WF.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...
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...
Loomis PR, Graham JK.One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen ...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Roth SP, Glauche SM, Plenge A, Erbe I, Heller S, Burk J.Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficia...
Peña FJ, O'Flaherty C, Ortiz RodrÃguez JM, MartÃn Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C.Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine resid...
Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A, Kurosaka K, Kobayashi Y, Murata T, Obata Y, Yokoyama K.Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells diffe...
Yeste M, Estrada E, Rocha LG, MarÃn H, RodrÃguez-Gil JE, Miró J.Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) an...
Aparicio IM, Martin Muñoz P, Salido GM, Peña FJ, Tapia JA.Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of au...
Canisso IF, Panzani D, Miró J, Ellerbrock RE.Donkeys are nonseasonal, polyestrous, territorial, and nonharem breeders. Although there are many similarities between horses and donkeys, there are also reproductive features that differ, from the longer cervix in the jenny to spermatogenic efficiency in the jack. Mules display reproductive cyclic activity but are rarely fertile. Frozen donkey semen has high pregnancy rates in mares, but lower rates in jennies. This article reviews key aspects of donkey and mule reproductive physiology, reproductive medicine, and assisted reproductive techniques that are useful for practitioners offering assi...
Ball BA, Vo AT, Baumber J.To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
Moore AI, Squires EL, Graham JK.Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sper...
Vanin AF, Serezhenkov VA, Mikoyan VD, Genkin MV.The parameters of EPR signal from dinitrosyl-iron complexes (DNIC) with bovine serum albumin (BSA), horse hemoglobin (Hb), and apometallothionein (apo-Mt) of horse kidney incorporating one (BSA, Hb) or two thiol-containing ligands (apo-Mt) were compared. The EPR signal from DNIC-BSA was characterized by the rhombic symmetry of g tensor at room temperature of signal recording (ambient temperature) or at 77K in the solution frozen in the presence of glycerol. In freezing of the solution in the absence of glycerin, under the exposure of DNIC-BSA to negatively charged sodium dodecyl sulfate (SDS) ...
Aurich C, Schreiner B, Ille N, Alvarenga M, Scarlet D.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...
Ortega Ferrusola C, González Fernández L, Salazar Sandoval C, MacÃas GarcÃa B, RodrÃguez MartÃnez H, Tapia JA, Peña FJ.In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active cas...
McClain AK, McCarrel TM.Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our ob...
Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M.Mammalian adult stem cells show, in vitro, extensive differentiative ability and may represent a versatile tool for tissue regenerative purposes, even after long-term storage. Multipotent stem cells isolated from horse blood have been shown to possess the capacity to differentiate into diverse mesenchymal lineages although their full characterization is still at an early stage. The aim of this study was to examine the effects of cryopreservation on stemness characteristics of adult equine mesenchymal stem cells isolated from peripheral blood (ePB-MSC). Each sample of ePB-MSC was analyzed immed...
Darr CR, Cortopassi GA, Datta S, Varner DD, Meyers SA.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 ...
Souris AC, Kaczensky P, Julliard R, Walzer C.The Przewalski's horse (Equus ferus przewalskii) became extinct in the wild in the 1960s, but survived as a species due to captive breeding. There have been several initiatives to re-introduce the species in central Asia, but until now only two projects in Mongolia establish free-ranging populations. Data on basic ecology and behavior of the species prior to extinction is largely lacking and a good documentation of the re-introduction process is essential. Between 13 May and 2 September 2003 we documented the time budget-, group synchrony and body score development of a newly released Przewals...
Moore AI, Squires EL, Graham JK.Seminal plasma is generally removed from equine spermatozoa prior to cryopreservation. Two experiments were designed to determine if adding seminal plasma back to spermatozoa, prior to cryopreservation, would benefit the spermatozoa. Experiment 1 determined if different concentrations of seminal plasma affected post-thaw sperm motility, viability and acrosomal integrity of frozen/thawed stallion spermatozoa. Semen was washed through 15% Percoll to remove seminal plasma and spermatozoa resuspended to 350 x 10(6)sperm/mL in a clear Hepes buffered diluent containing either 0, 5, 10, 20, 40 or 80%...
Papas M, Catalán J, Fernandez-Fuertes B, Arroyo L, Bassols A, Miró J, Yeste M.While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. T...
Brinsko SP, Varner DD, Love CC, Blanchard TL, Day BC, Wilson ME.Eight stallions were used in 2 x 2 crossover study to determine if feeding a nutriceutical rich in docosahexaenoic acid (DHA) would improve semen quality. Stallions were randomly assigned to one of two treatment groups (n = 4 per group). Stallions were fed their normal diet (control) or their normal diet top-dressed with 250 g of a DHA-enriched nutriceutical. Feeding trials lasted for 14 week, after which a 14-week washout period was allowed and the treatment groups were reversed for another 14 week feeding trial. Feeding the nutriceutical resulted in a three-fold increase in semen DHA levels ...
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...
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...
Oldenhof H, Gojowsky M, Wang S, Henke S, Yu C, Rohn K, Wolkers WF, Sieme H.The aim of this study was to determine how different membrane-permeable and -impermeable cryoprotective agents modulate tolerance of stallion sperm to osmotic stress and stabilize membranes during cryopreservation. Special emphasis was on hydroxyl ethylene starch (HES), which exposes cells to minimal osmotic stress due to its large molecular weight. Percentages of motile sperm post-thaw were found to be similar when glycerol, sucrose, and HES were used at their optimal concentrations. Percentages of plasma membrane intact sperm after return to isotonic medium were highest for HES. Fourier tran...
Martin de Bustamante M, Plummer C, MacNicol J, Gomez D.Sample storage conditions are an important factor in fecal microbiota analyses in general. The objective of this study was to investigate the effect of sample storage at room temperature on the equine fecal microbiota composition. Fecal samples were collected from 11 healthy horses. Each sample was divided into 7 sealed aliquots. One aliquot was immediately frozen at -80 °C; the remaining aliquots were stored at room temperature (21 to 22 °C) with one transferred to the freezer at each of the following time points: 6, 12, 24, 48, 72 and 96 h. The Illumina MiSeq sequencer was used for high-th...
Batellier F, Vidament M, Fauquant J, Duchamp G, Arnaud G, Yvon JM, Magistrini M.In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in ord...
