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.
Lima-Neto JF, Fernandes CB, Alvarenga MA, Golim MA, Landim-Alvarenga FC.This experiment aimed to study equine fibroblasts in culture analyzing and the cell cycle and viability of cells pre- and post-freezing. Skin fragments were obtained from 6 horses and cultured in DMEM high glucose + 10% FCS in 5% CO(2) until the beginning of confluence. Two passages were performed before freezing. Cells subjected to serum starvation (0.5% FCS) were analyzed for viability and cell cycle at 24, 48, 72, 96, 120, 144 and 168 h of culture. For the confluent groups, cells were analyzed at the moment they achieved confluence. Cellular viability was assisted with Hoescht 33342 and pro...
Ponthier J, Franck T, Detilleux J, Mottart E, Serteyn D, Deleuze S.Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different s...
Tharasanit T, Colleoni S, Galli C, Colenbrander B, Stout TA.Vitrifying oocytes is a potentially valuable means of preserving the female germ line, but significantly compromises oocyte developmental competence. This study examined the hypothesis that the cumulus complex protects the oocyte during vitrification. Vitrified-warmed immature cumulus oocyte complexes (COCs) were labelled with a plasma membrane impermeant DNA marker (ethidium homodimer-1) to examine the percentage and location of dead cumulus cells, and to investigate the effect of the proportion of dead cells (+1,+2 or +3) on the success of in vitro maturation (IVM). Further, oocytes were lab...
Ortega-Ferrusola C, Macías García B, Suárez Rama V, Gallardo-Bolaños JM, González-Fernández L, Tapia JA, Rodríguez-Martinez H, Peña FJ.In an attempt to improve the information obtained after computer-assisted sperm analysis (CASA), data from five stallions (three ejaculates from each) were analysed before (fresh, extended semen) and after cryopreservation using traditional statistics as well as a cluster analysis. The data matrix consisted of 13 987 observations of individual spermatozoa for fresh, extended semen, and 8305 for frozen-thawed samples. As expected, freezing and thawing resulted in a marked decrease of CASA-derived variables of sperm kinematics. All sperm velocities were significantly lower in frozen-thawed sampl...
Tablin F, Walker NJ, Hogle SE, Pratt SM, Norris JW.To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. Methods: 6 clinically normal adult horses. Methods: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. R...
Ortega-Ferrusola C, García BM, Gallardo-Bolaños JM, González-Fernández L, Rodríguez-Martinez H, Tapia JA, Peña FJ.In an attempt to identify valuable markers for potential freezeability of the equine spermatozoa, three ejaculates were collected from five Andalusian stallions and frozen using a standard protocol. Before freezing, three apoptotic cell markers were studied by flow cytometry (early changes in sperm membranes, mitochondrial membrane potential and caspase activity). Post-thaw, spermatozoa were again evaluated for these parameters. Sperm kinematics using CASA were also studied before and after freezing and thawing. Receiving operating system curves were used to evaluate the relative value of the ...
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...
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...
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...
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...
Karasiewicz J, Andrzej-Modlinski J.Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. ...
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...
Scherzer J, Fayrer-Hosken RA, Ray L, Hurley DJ, Heusner GL.Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstratio...
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 ...
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 ...
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...
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. ...
Crews LJ, Waelchli RO, Huang CX, Canny MJ, McCully ME, Betteridge KJ.To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concep...
Kardak A, Leibo SP, Devireddy R.The rate at which equine and macaque ovarian tissue sections are first cooled from +25 degrees C to +4 degrees C has a significant effect on the measured water transport when the tissues are subsequently frozen in 0.85 M solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG. Sections of fr...
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...
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...
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...
Matsukawa K, Akagi S, Adachi N, Sato F, Hasegawa T, Takahashi S.In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates...
Hill DE, Forbes L, Gajadhar AA, Gamble HR.Many aspects of the biology and epidemiology of Trichinella infection in the horse are poorly understood, including survival of Trichinella spp in horse muscle. In this study, we have assessed the freeze tolerance of T. spiralis in horse meat stored at 5, -5, and -18 degrees C for 1 day to 24 weeks. Results demonstrate a steady reduction in the number of live ML recovered from the cold stored meat samples. On Day 1, recovery of live larvae had been reduced by 18.6%, 50.1%, and 37.2%, and by 4 weeks, recovery of larvae had been reduced by 65.4%, 66.5%, and 96.2% in samples stored at 5, -5, and ...
Carnevale EM.Vitrification can be used successfully to cryopreserve equine embryos. Embryos for vitrification should be collected from donor mares' uteri when they are 300 mm or less in diameter, however,and at the morula or early blastocyst stage of development. No special equipment is required for vitrification; the straw containing the embryo is exposed to vapor for 1 minute before plunging it into liquid nitrogen. Warming of the straw requires no special equipment,and the embryo can be transferred directly from the straw into a recipient's uterus. Vitrification has been repeatedly successful when the p...
Kútvölgyi G, Stefler J, Kovács A.A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs....
Czlonkowska M, Boyle MS, Allen WR.Fourteen horse embryos recovered non-surgically on Days 6-8 after ovulation (Day 0) were cooled slowly to - 35 degrees C (7 embryos) or - 40 degrees C (7 embryos) and stored in liquid nitrogen (- 196 degrees C) for 4-98 days. Surgical transfer of the thawed embryos to unmated recipient mares that had ovulated - 2 to + 1 days with respect to the embryo donors resulted initially in the establishment of 4 conceptuses. However, only one mare maintained her pregnancy to term.
Wilsher S, Rigali F, Kovacsy S, Allen WT.Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill. Objective: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Methods: In vivo experiment. Methods: Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits cont...
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...
Guasti PN, Monteiro GA, Maziero RR, Carmo MT, Dell'Aqua JA, Crespilho AM, Rifai EA, Papa FO.The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being h...
Craig AM, Blythe LL, Rowe KE, Lassen ED, Barrington R, Walker KC.Recent evidence concerning the pathogenesis of equine degenerative myeloencephalopathy indicated that low blood alpha-tocopherol values are a factor in the disease process. Variables that could be introduced by a veterinarian procuring, transporting, or storing samples were evaluated for effects on alpha-tocopherol concentration in equine blood. These variables included temperature; light; exposure to the rubber stopper of the evacuated blood collection tube; hemolysis; duration of freezing time, with and without nitrogen blanketing; and repeated freeze/thaw cycles. It was found that hemolysis...
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...
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...
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...
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...
Harris RC, Snow DH, Katz A, Sahlin K.Muscle biopsies taken after exercise, in comparison to those at rest, contain increased amounts of blood and this is a particular problem in studies of the horse. The inclusion of blood in muscle will introduce an upward bias in values of pH measured in muscle homogenates. In an attempt to control this, muscle biopsy samples of the middle gluteal from Thoroughbred horses were freeze-dried and dissected free of blood before determination of pH. Following exercise, muscle pH measured after freeze-drying was similar to that measured in homogenates prepared from frozen samples. In contrast, freeze...
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...
Pasch L, Schmidt A, King W.The use of autologous blood processing tools including platelet-rich plasma (PRP) devices is increasingly widespread in veterinary medicine. In equine reproduction, a number of studies have explored the effects of intrauterine infusion of PRP on persistent mating-induced endometritis. Artificial insemination with thawed frozen semen incites an intrauterine inflammatory response and we sought to extend the applications of intrauterine PRP to normal mares being inseminated with frozen semen. We investigated a subset of our normal breeding population to observe the clinical effects of prebreeding...
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...
Shepard KN, Haffner JC, Neal DL, Grubbs ST, Pearce GL.Plasma adrenocorticotropic hormone (ACTH) concentration is used in the diagnosis of pituitary pars intermedia dysfunction (PPID) in horses. We enrolled 10 horses, 5 PPID-positive and 5 PPID-negative, in our study, September 20-22, 2016. On day 0, 5 mL of whole blood was collected into each of 6 EDTA tubes and immediately placed in a refrigerator at 7°C. One tube was centrifuged within 15 min of collection, followed by centrifugation of one tube from each horse at 4, 8, 12, 24, and 36 h following collection. At each time, centrifuged plasma was pipetted into 1.5-mL polypropylene tubes and stor...
Morris LH, Tiplady C, Allen WR.To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Hidalgo M.Lipids and proteins can be used for sperm vitrification to preserve the integrity of sperm membranes or to increase the viscosity of the medium. This study evaluated the effect of low-density lipoproteins (LDL) and milk serum proteins (Pronexcell) for stallion sperm vitrification. Hippex extender (Barex Biochemical Products, The Netherlands), plus 1% of bovine serum albumin and 100 mM of trehalose, was used as control for sperm vitrification. In experiment 1, different concentrations of LDL (L1 = 0.25, L2 = 0.5, L3 = 1%) and in experiment 2 of Pronexcell (P1 = 1, P2 = 5, P3 = 10%)...
Pereira BC, Ortiz I, Dorado J, Consuegra C, Diaz-Jimenez M, Demyda-Peyras S, Gosalvez J, Hidalgo M.DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liqui...
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...
Young CA, Squires EL, Seidel GE, Kato H, McCue PM.Larger grade 1 or 2 (1 = excellent,.... 4 = degenerate) equine embryos that ranged in diameter from 300 to 680 microm and were recovered from mares on Day 7 or 8 after ovulation, were randomly assigned to 3 widely divergent cryopreservation treatments. Treatment 1 consisted of cooling from -6 degrees C to -35 degrees C at 0.5 degrees C per min followed by plunging into liquid nitrogen, with a one-step addition and a 4-step removal of 1.0 M glycerol. Treatment 2 (step-down equilibration) consisted of a 2-step addition of glycerol to 4.0 M followed by a decrease to 2.0 M prior to freezing, with ...
Gastal GDA, Aguiar FLN, Ishak GM, Cavinder CA, Willard ST, Ryan PL, Feugang JM, Gastal EL.Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC) and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7 days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes; (ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of apoptosis for Bax and...
Avanzi BR, Ramos Rdos S, Araujo GH, Fioratti EG, Trinca LA, Dell'Aqua JA, Melo E Oña CM, Zahn FS, Martin I, Alvarenga MA, Papa FO.The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n = 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were in...
Ferreira JC, Meira C, Papa FO, Landin e Alvarenga FC, Alvarenga MA, Buratini J.Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were remov...
Matthews GL, Keegan KG, Graham HL.To determine effects of tendon grip technique on in vitro surface strain measurements of equine deep digital flexor tendon (DDFT) when loaded in tension. Methods: 12 hind limb DDFT from 8 adult horses (mean age, 9.8 years [range, 4.5 to 17 years]; mean body weight, 472 kg [range, 450 to 509 kg]), with no clinical evidence of hind limb lameness. Methods: After calibration, liquid mercury strain gauges were sutured to plantar surfaces of the tendons at distal (position 1), middle (position 2), and proximal (position 3) metatarsal regions. Each tendon was affixed to a materials testing machine (d...
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, ...
Morillo Rodriguez A, Balao da Silva C, Macías-García B, Gallardo Bolaños JM, Tapia JA, Aparicio IM, Ortega-Ferrusola C, Peña FJ.A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL-1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL-2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 we...
Desjardins MR, Hurtig MB, Palmer NC.Two 10 mm thick osteochondral grafts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. The grafts were frozen in a 7.5% DMSO solution and stored in liquid nitrogen. The horses were anesthetized again on day 14 and the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifle. A fresh graft was transferred from the right hock to the left stifle. To control for the effects of surgery, another fresh graft was transferred from the right stifle to the left stifle. The result was two grafts in...
Ortiz I, Dorado J, Morrell JM, Diaz-Jimenez MA, Pereira B, Consuegra C, Hidalgo M.The aim of this study was to compare the post-thaw distribution of motile sperm subpopulations, following simple or colloid centrifugation. A new analysis was used to evaluate the available number of sperm from each subpopulation after each centrifugation protocol. Frozen/thawed semen samples were divided into the following after-thawing treatments: uncentrifuged control (UDC), sperm washing (SW) and two colloid centrifugation procedures (Equipure, SLC-E, and Androcoll, SLC-A). Percentage of total and progressive motility (TM and PM), as well as sperm motility kinematics, distribution of motil...
