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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.
Advances in Stallion Semen Cryopreservation. The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation a...
Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Cl) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47...
Intramuscular Transplantation of Allogeneic Mesenchymal Stromal Cells Derived from Equine Umbilical Cord. Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. Results: For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs dilute...
Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program. There is a clinical demand for cryopreservation of both in vivo-recovered and in vitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both in vivo-recovered and IVP blastocysts. In vivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10 hours to the laboratory before vitrificat...
Reproductive stage-dependent effects of additional cryoprotectant agents for the cryopreservation of stallion germ cells. The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell po...
Cryopreservation of Peruvian Paso horse spermatozoa: dimethylacetamide preserved an optimal sperm function compared to dimethyl sulfoxide, ethylene glycol and glycerol. The objective was to evaluate the effect of different cryoprotectant agents in the cryopreservation of Peruvian Paso horse semen. Twenty semen samples were collected from five Peruvian Paso horse stallions. Each sample was divided into 12 parts to form the groups: dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol (GLY), at 3%, 4% and 5%. Samples were frozen using a rate-controlled freezer. Sperm parameters evaluated were motility and viability/acrosomal status. After thawing, progressive motility in DMA group was higher (p < .05) than in DMSO, EG and GL...
Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media. Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol(®) FRS (HTS-FRS), CryoSt...
Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa. Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the prese...
Field fertility of liquid stored and cryopreserved flow cytometrically sex-sorted stallion sperm. The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Equine Mesenchymal Stromal Cells from Different Sources Efficiently Differentiate into Hepatocyte-Like Cells. Adult equine hepatocytes have proven challenging to culture long term in vitro as they rapidly lose their morphology and functionality, thus limiting studies on liver function and response to disease. In this study, we describe for the first time the differentiation of equine mesenchymal stromal cells (MSC) from a variety of sources into functional hepatocyte-like cells (HLC). First, we differentiated equine umbilical cord blood (UCB)-derived MSC into HLC and found that these cells exhibited a distinct polygonal morphology, stored glycogen as visualized by periodic acid Schiff's reagent staini...
Cytosine methylation of sperm DNA in horse semen after cryopreservation. 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...
Mitochondrial oxygen consumption is a unique indicator of stallion spermatozoal health and varies with cryopreservation media. 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 ...
CD47 expression in cryopreserved equine cutaneous masses and normal skin. We investigated CD47 expression in cryopreserved sections of equine cutaneous masses and normal skin. CD47 is a cell surface protein expressed on many cell types and overexpressed in some tumors. Interaction of CD47 and signal regulatory protein-alpha (SIRPα) inhibits phagocytosis by macrophages. Formalin-fixed tissues from horses prospectively enrolled in the study were used to establish a histologic diagnosis. Immunohistochemical assays were performed on cryopreserved tissues using anti-CD47 antibodies or IgG control antibodies. CD47 was not expressed on equine normal skin but positivity to...
Effect of various concentrations of butylated hydroxyanisole and butylated hydroxytoluene on freezing capacity of Turkman stallion sperm. The present study aimed to determine the effect of different concentrations of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on post-thaw stallion sperm quality. The ejaculates collected from four healthy mature Turkmen stallions were pooled and divided into eight aliquots. The samples were diluted with extenders containing different concentrations (0.5, 1 or 2mM/mL) of BHA or BHT. The positive control (PC) samples were diluted with extender containing 0.5% ethanol (v/v) whereas; the negative control (NC) samples were diluted with basic extender only. Semen samples were fro...
Effects of α-tocopherol and freezing rates on the quality and heterologous in vitro fertilization capacity of stallion sperm after cryopreservation. The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C...
The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions. 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...
Micromanipulation of equine blastocysts to allow vitrification. Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embr...
Comparison of cushioned centrifugation and SpermFilter filtration on longevity and morphology of cooled-stored equine semen. This study compares two methods for seminal plasma removal by evaluating sperm recovery rates, and motility and morphology of cooled-stored semen. Ejaculates were divided into three groups: control, filtration and cushioned centrifugation. Semen was extended to 25 million sperm/ml using a skim-milk-based extender and stored at 5°C for all groups. Sperm motility (total motility (%TM) and progressive motility (%PM)) was determined at 0, 24, 48 and 72 hours by a computer-assisted sperm analyser. Sperm morphology was assessed using differential interference microscopy. Overall, %TM of the cen...
Effects of a long-day light programme on the motility and membrane integrity of cooled-stored and cyropreserved semen in Shetland pony stallions. Increasing day length in spring stimulates reproductive functions in horses. In this study, we have analysed the effect of artificial long days on the quality of cooled-stored and cryopreserved semen in Shetland stallions. Stallions of the treatment group (AL, n = 8) were exposed to 16 h light and 8h darkness from 15th December to 20th March while control stallions (CON, n = 7) were kept under natural photoperiod. Semen was collected once weekly and processed for cooled-storage and cryopreservation once per month. Total and progressive motility and percentage of membrane intact spermatozoa wer...
Milk, caseinate and lactoferrin addition to equine semen cooling extenders. Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 μg ml-1 , C4) C2 + Lf 500 μg ml-1 and C5) C2 + Lf 1000 μg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motili...
Substitution of egg yolk by a cyclodextrin-cholesterol complex allows a reduction of the glycerol concentration into the freezing medium of equine sperm. The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G...
Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation. Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryo...
Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95% and DMSO at 10 or 5. Equine superficial digital flexor tendon injury is a well-accepted model of human tendon injury and is routinely treated with local injections of autologous mesenchymal stem cells (MSCs). Identification of a clinically safe medium for short-term cryopreservation of MSCs prior to cell implantation would streamline laboratory and clinical procedures for autologous regenerative therapies. Veterinary experience with short-term (MSCs prepared after the injury has occurred) cryopreserved MSCs in naturally occurring injury in the horse will be of value to human practitioners. Methods: Equine bone mar...
Effect of Solid Medium During Cooled Storage on Stallion Sperm Parameters. Solid storage medium prevents cellular sedimentation, reduces metabolic demand via limiting movement, and avoids the modification of an extender composition in the sedimentary microenvironment. It has been proven to prolong spermatozoa viability in mammalians. Objective: This experiment aims to evaluate the effect of cool storage in solid phase extender on stallion sperms. Methods: Semen was collected from 10 Crioulo stallions (n=30) and submitted to treatments: control group (semen extender) and groups with gelatin addition in different concentrations (semen extender + 1%, 2% and 3%). Seminal...
Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification. Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experim...
Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death. Oxidative stress has been linked to sperm death and the accelerated senescence of cryopreserved spermatozoa. However, the molecular mechanisms behind this phenomenon remain poorly understood. Reactive oxygen species (ROS) are considered relevant signaling molecules for sperm function, only becoming detrimental when ROS homeostasis is lost. We hereby hypothesize that a major component of the alteration of ROS homeostasis in cryopreserved spermatozoa is the exhaustion of intrinsic antioxidant defense mechanisms. To test this hypothesis, semen from seven stallions was frozen using a standard tech...
Pregnancy outcomes using stallion epididymal sperm stored at 5 °C for 24 or 48 hours before harvest. The cryopreservation of epididymal sperm can be useful in a variety of circumstances for ensuring genetic preservation of a valued stallion. Although early studies have reported pregnancy rates significantly lower than those achieved with cryopreserved ejaculated sperm, two recent studies report over 60% one-cycle pregnancy rates with epididymal sperm stored for 24 hours at 5 °C before harvest and cryopreservation. The aims of this study were to: (1) attempt to replicate the one-cycle pregnancy rate of over 60% using epididymal sperm cooled and stored within the epididymis for 24 hours before...
Coenzyme Q10 and α-Tocopherol Prevent the Lipid Peroxidation of Cooled Equine Semen. Biotechnology applied for equine semen increases the levels of reactive oxygen species and reduces the natural antioxidant defence, by both dilution and removal of seminal plasma. Therefore, the aims of this study were to evaluate the effect of adding coenzyme Q10 (CoQ10) and α-tocopherol (α-TOH) to the cooling extender, singly or in combination, on sperm parameters, and their effectiveness in preventing lipid peroxidation (LPO) of equine semen during cooling at 5°C for 72 h. Ten adult stallions of proven fertility were used, using two ejaculates each, subjecting them to the treatments with...
Cryopreservation of stallion spermatozoa using different cryoprotectants and combinations of cryoprotectants. The present study investigates the effects of five cryoprotectants (CPAs) and cryoprotectant combinations on the post-thaw total motility, progressive motility, viability, mitochondrial membrane potential and acrosome integrity in stallion spermatozoa. In Experiment 1, the objective was to compare the impact of different concentrations (2.5%, 3.5% and 5%) of a single CPA, including glycerol (Gly), ethylene glycol (EG), dimethyl sulphoxide (DMSO), methyl formamide (MF), and dimethylformamide (DMF) for stallion spermatozoa cryopreservation. In Experiment 2, two or more CPAs were used to assess w...
Sperm Membrane Behaviour during Cooling and Cryopreservation. 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...
