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Topic:Cryopreservation
Cryopreservation in horses involves the process of cooling and storing biological samples at very low temperatures to preserve their viability for future use. This technique is primarily applied to equine gametes, embryos, and genetic material, such as semen and oocytes. The process aims to halt all biological activity, thereby maintaining the integrity of the samples over extended periods. Cryopreservation is utilized in equine breeding programs to enhance genetic diversity, support conservation efforts, and facilitate international genetic exchange. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, challenges, and applications of cryopreservation in equine reproduction and genetics.
Iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation. Density gradient centrifugation can be used for selection of sperm of superior quality and removal of seminal plasma for use in artificial insemination. In this study, the use of two-layer iodixanol density gradient centrifugation was evaluated for processing of stallion semen. The protocol includes centrifugation through a 16% iodixanol top layer of 1.090 g mL(-1) and collection of motile and intact sperm on a 30% iodixanol bottom layer of 1.165 g mL(-1). Sperm recovery and effects on sperm quality were determined during cold storage as well as after cryopreservation and compared with ordinar...
Cryopreservation of equine embryos: current state-of-the-art. During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the follo...
Mesenchymal stromal cell cryopreservation. The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins...
Applicability of a new cell culture device for cooled-storage of stallion semen. A new device for storage and shipping of cell cultures--the Petaka G3 cell management device--was tested for its applicability for cooled-storage of equine semen. Semen from three stallions was processed with EquiPro extender either without antibiotics (three ejaculates per stallion) or with gentamicin (250 mg/l; three ejaculates per stallion). Semen was either stored at five (anaerobic conditions) or 15 °C (aerobic conditions) in syringes or cell culture devices. Total and progressive motility, as well as membrane integrity of spermatozoa, were evaluated from days 1 to 7 after collection wit...
Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa. Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4...
Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity. There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin i...
Membrane phase behavior during cooling of stallion sperm and its correlation with freezability. 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...
Post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. The objective was to verify the relationship between equine semen cryopreservation and changes related to increased lipid peroxidation. Also, addition of autologous or homologous seminal plasma from a stallion with a good freezing response to post-thawed sperm was tested to determine whether it would confer protection. Frozen-thawed sperm were evaluated and allocated into three groups: without plasma addition, and supplemented with either homologous or autologous seminal plasma. All groups were evaluated at 0, 60 and 120 min after incubation at 37 °C. Cryopreservation did not increase plasma ...
Induction of pluripotency in adult equine fibroblasts without c-MYC. Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining ...
Dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. 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...
Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells. Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digesti...
Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Glycerol is, to date, the most widely used cryoprotectant to freeze stallion spermatozoa at concentrations between 2% and 5%. Cryoprotectant toxicity has been claimed to be the single most limiting factor for the success of cryopreservation. In order to evaluate the toxic effects of the concentrations of glycerol used in practice, stallion spermatozoa were incubated in Biggers Whitten and Whittingham (BWW) media supplemented with 0%, 0.5%, 1.5%, 2.5%, 3.5%, and 5% glycerol. In two additional experiments, a hyposmotic (75 mOsm/kg) and a hyperosmotic (900 mOsm/kg) control media were included. Sp...
Autophagy and apoptosis have a role in the survival or death of stallion spermatozoa during conservation in refrigeration. Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Seminal freezing in pure breed andalusian horse: difference in individual stallions and correlation between pre and post-freezing sperm parameters. The aim of this study was the optimization of the sperm freezing protocols for the Pure Breed Andalusian Horse (AH) stallions. The study was performed in 84 ejaculates from 14 stallions (6 ejaculates per stallion). We examined the effect of individual stallion, centrifugal force and centrifugation extender on post-thaw sperm quality. Neither centrifugal force nor centrifugal extender had any significant effect on post-centrifugation or post-thawing sperm quality. Stallion was the principal source of variation in our experiments, showing individual significant differences (p < 0.05) in all p...
Sperm selection using single layer centrifugation prior to cryopreservation can increase thawed sperm quality in stallions. 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...
Consequences of butylated hydroxytoluene in the freezing extender on post-thaw characteristics of stallion spermatozoa in vitro. 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...
Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation. 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...
Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen. 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...
Liposomes as an alternative to egg yolk in stallion freezing extender. 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 ...
Artificial insemination and embryo transfer in mares. 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...
Cryopreservation and fertility of ejaculated and epididymal stallion sperm. 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 ...
Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols? 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.
Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker. The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in t...
Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro. Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ± 1 capsules separated media in the "donor" chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the "recipient" chamber. Concentrations of CPA, ...
Freezing stallion semen with the new Cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability. 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 ...
A cryogenic clamping technique that facilitates ultimate tensile strength determinations in tendons and ligaments. To describe the use of a cryogenic clamp of novel design for tensile strength testing of tendinous and ligamentous tissues with inherently high tensile strength. Methods: Inexpensive, easily machined steel clamps were manufactured to facilitate rapid insertion into a standard wedge-screw grip apparatus installed on a testing system with a control system attached. The deep digital flexor tendon (DDFT) of six horses was trimmed to a uniform dumbbell shape and secured in clamps using partial submersion in liquid nitrogen for approximately 45 seconds and immediately tested. Approximate time betwee...
Effects of cholesterol-loaded cyclodextrins on the quality of frozen-thawed equine epididymal sperm. 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...
Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix. Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Methods: Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes...
Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares. 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 ...
Laser-assisted vitrification of large equine embryos. The major difficulty in providing the benefits of embryo cryopreservation for equine agriculture is the mismatch between the optimal embryo age for collection from the mare (7-8 days after ovulation was detected) and the optimal age for freezing under current methods (6.5 days after ovulation). To overcome this limitation, we tested a method to enhance penetration of cryopreservative across the capsule and trophoblast of day 7 and 8 embryos combined with rapid freezing by vitrification. Six small embryos (<300 μm in diameter) were collected on day 6-7 after ovulation and twelve larger embr...
