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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.
Alginate encapsulation of stallion sperm for increasing storage stability. The aim of this study was to establish an alginate encapsulation procedure for stallion sperm, and investigate if sperm encapsulation enhances longevity during cold storage and survival after cryopreservation. First, biocompatibility of the compounds needed for encapsulation was tested and factors determining capsule structure were identified. Sperm encapsulation was realized either by depositing droplets (20 µL) of sperm solution supplemented with barium or calcium chloride (10 mM) in alginate solution (0.25%, w/v), or by adding sperm-alginate droplets in solution containing barium or calc...
Use of Equine Sperm Cryopreservation Techniques as a Conservation Method of Donkey Germplasm. 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...
Relationship between sperm ubiquitination and equine semen freezability. This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very l...
Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro. In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three s...
The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation. The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O/mL. The semen samples were diluted (200 x 10 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by ...
Advances in sperm cryopreservation in farm animals: Cattle, horse, pig and sheep. 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...
Effect of platelet lysate on uterine response of mares susceptible to persistent mating-induced endometritis. Many mares are susceptible to persistent mating-induced endometritis (PMIE), an important cause of reduced fertility. Platelet lysate (PL) derives from freeze-thawing platelets after concentration, so that growth factors are released from the platelets. Among the advantages of PL compared to platelet-rich plasma (PRP), it can be frozen stored and allogenic use for PL might also be conceivable. Platelet-rich plasma beneficially reduced inflammatory response in PMIE mares when administered 24 h pre- or 4 h post-AI. The aim of this study was to test the effect of PL on inflammatory uterine resp...
Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm. In donkeys, the use of frozen-thawed sperm for artificial insemination (AI) leads to low fertility rates. Furthermore, donkey sperm produce a large amount of reactive oxygen species (ROS), and post-AI inflammation induces the formation of neutrophil extracellular traps (NETosis), which further generates many more ROS. These high ROS levels may induce lipid peroxidation in the sperm plasma membrane, thus affecting its integrity. Enzymatic and non-enzymatic antioxidants, mainly found in the seminal plasma (SP), are responsible for maintaining the redox balance. However, this fluid is removed pri...
Diluent Containing Dimethylformamide Added With Sucrose Improves In Vitro Quality After Freezing/Thawing Stallion Sperm. The aim of this study was to investigate the effects of sucrose on post-thawed equine semen quality. Semen samples (n = 24) were collected from six stallions. They were diluted (200 × 10 sperm/mL) in a freezing medium based on skimmed milk, egg yolk, dimethylformamide, and supplemented with sucrose at concentrations of 0 (Control), 25, 50, and 100 mM and in a commercial extender (BotuCrio). Subsequently, they were filled in straws (0.5 mL) and subjected to freezing and storage (-196°C). Immediately after thawing (37°C, 30 seconds), semen samples were evaluated for kinetics (CASA), plasm...
Characteristics of diluted-stored and post-thawed semen of Hutsul stallions. The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, u...
Cryotherapy Techniques: Best Protocols to Support the Foot in Health and Disease. Treatment of equine laminitis continues to be a challenge despite recent advancements in knowledge of the pathophysiology of laminitis. With more evidence supporting its use, distal limb hypothermia or cryotherapy has become a standard of care for both prevention of laminitis and treatment of the early stages of acute laminitis. Recent studies have demonstrated that cryotherapy reduces the severity of sepsis-related laminitis and hyperinsulinemic laminitis in experimental models and reduces the incidence of laminitis in clinical colitis cases. This article reviews the recent literature support...
Therapeutic effects of equine amniotic membrane suspension on corneal re-epithelialization and haze in a modified lagomorph ex vivo wound healing model. To investigate the therapeutic effects of topical equine amniotic membrane (eAM) suspension following corneal wounding in a controlled experimental setting. Methods: Equine amniotic membrane was collected, gamma irradiated, homogenized for topical suspension preparation, and cryopreserved. Corneoscleral rims harvested from fresh rabbit globes were wounded via keratectomy and were maintained in an air-liquid interface ex vivo corneal culture model. Treatment groups included topical gamma irradiated eAM suspension (n = 20) and a control group (n = 20). Re-epithelialization of the wound was a...
Evaluation of growth, viability, and structural integrity of equine endometrial organoids following cryopreservation. Reproductive diseases in mares are a significant cause of subfertility and profound economic loss in the equine industry. Utilizing a 3D in vitro cell culture system that recapitulates the in vivo physiology will reduce time, cost, and welfare concerns associated with in vivo reproductive research in mares. If this 3D model is combined with effective cryopreservation, reproductive research on mares can occur year-round, which is not currently possible in this seasonal species. Endometrial organoids, 3D in vitro cell clusters that exhibit in vivo uterine physiology, have been established in mic...
Edible bird’s nest supplementation in chilled and cryopreserved Arabian stallion semen. Diluents and various biological products have been used in different animal species, with promising outcomes in post-thaw sperm quality. Nevertheless, only a few reports are available for the semen of Arabian horses. Edible bird's nest (EBN) - a product of the salivary secretions of swiftlet species is widely known to have both antioxidant and anti-inflammatory properties. Presently, there is no data available on the role of EBN supplemented in different extenders and its effect on semen quality in stallion semen. Two in vitro experiments were conducted to examine the effects of edible bird's ...
Towards increasing stallion sperm longevity by storage at subzero temperatures in the absence of ice. The aim of cell preservation technologies is to slow down damaging reactions by lowering the storage temperature. Upon dilution in a stabilizing extender, stallion sperm can be stored at refrigerator temperatures for several days. Cryopreservation allows storage for decades, but freezing and thawing cause damage and viability losses. It is assumed that by storing cells at subzero temperatures in a non-frozen supercooled state, the damaging effects of ice formation can be avoided. In this study, we have investigated if stallion sperm can be stored at -10°C in the absence of ice, and compared v...
INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling. Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes pr...
Use Low Ozone Dosages has Positive Effects on the Cooling and Cryopreservation of Equine Semen. The aim of this study was to determine an ozone dosage capable of inducing pro-oxidation, and to verify its action on sperm cells during the process of cooling and cryopreservation of equine semen. In this study, we evaluated the ozone concentrations of 2µg/mL,15µg/mL, 30µg/mL e 60 µg/mL added in equine semen cooling and freezing extenders. Samples were evaluated for sperm kinetics patterns, function of sperm structures and lipid peroxidation. In the experiment, the concentration of 15 µg/mL showed higher total and progressive motility when comparing to control (60.3±3 and 40.7±3.4 vs. ...
New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification. Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using th...
The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions. Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism. In view of these facts, we hypothesized that differences in protein composition of the seminal plasma among stallions may help to explain differences in freeze-ability seen among them. Three independent ejaculates from 10 different stallions of varying breeds were frozen using standard protocols in our lab...
Loading equine oocytes with cryoprotective agents captured with a finite element method model. Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane...
A Novel QTL and a Candidate Gene Are Associated with the Progressive Motility of Franches-Montagnes Stallion Spermatozoa after Thaw. The use of frozen-thawed semen is an important reproduction tool to preserve the biodiversity of small, native horse breeds such as the Franches-Montagnes (FM). However, not all stallions produce cryotolerant semen with a progressive motility after thaw ≥ 35%. To improve our understanding of the genetic background of male fertility traits in both fresh and frozen-thawed semen, we performed genome-wide association studies (GWAS) on gel-free volume, sperm cell concentration, total sperm count, and progressive motility in fresh and frozen-thawed semen from 109 FM stallions using 335,494 genome-...
Equine ovarian tissue xenografting: impacts of cooling, vitrification, and VEGF. Ovarian tissue transplantation methods using cooled and cryopreserved samples have been attractive options for fertility preservation in animal models and humans. The aim of this study was to evaluate the impact of previous exposure to cooling, cryopreservation, and VEGF on the overall efficiency of equine ovarian tissue after heterotopic xenotransplantation in mice. The end points evaluated were follicular morphology and development, follicular and stromal cell densities, angiogenesis (i.e. the density of new and mature blood vessels), collagen types I and III fiber densities, and total fibro...
Cryotherapy provides transient analgesia in an induced lameness model in horses. The analgesic effect of cryotherapy in an induced lameness model was evaluated. Lameness was induced with solar pressure from a custom-made shoe in a 10-horse, cross-over study. The degree of lameness was recorded with a commercial non-invasive inertial sensor. The distal limbs were maintained in an ice and water slurry (cryotherapy) or at ambient temperature (control) for 1 hour. Lameness was assessed serially over the following hour. Lameness at each time point was compared to the baseline induced lameness, within and between groups. Lameness had improved significantly in all horses 5 minute...
First results about ProAKAP4 concentration in stallion semen after cryopreservation in two different freezing media. The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant di...
Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos. The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B 300 µm). However, more research is required to find the best method for embryos ≥700 µm.
Sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa. Different stallions exhibit a high level of variation in the ability of their sperm to survive cryopreservation. A large fraction of stallions show poor post-thaw sperm motility, and their semen is not suitable for commercial freezing. In this study, we hypothesized that the presence of sperm-bound antisperm antibodies (ASAs) was associated with poor cryosurvival of stallion sperm. Our objective was to assess the level of ASA binding to stallion sperm, and determine if it was associated with good or poor sperm cryosurvival. In Experiment 1, cooled shipped semen from 27 stallions was frozen usi...
high-throughput droplet vitrification of stallion sperm using permeating cryoprotective agents. Stallion sperm is typically cryopreserved using low cooling rates and low concentrations of cryoprotective agents (CPAs). The inevitable water-to-ice phase transition during cryopreservation is damaging and can be prevented using vitrification. Vitrification requires high cooling rates and high CPA concentrations. In this study, the feasibility of stallion sperm vitrification was investigated. A dual-syringe pump system was used to mix sperm equilibrated in a solution with a low concentration of CPAs, with a solution containing a high CPA concentration, and to generate droplets of a defined si...
Specific Seminal Plasma Fractions Are Responsible for the Modulation of Sperm-PMN Binding in the Donkey. While artificial insemination (AI) with frozen-thawed sperm results in low fertility rates in donkeys, the addition of seminal plasma, removed during cryopreservation, partially counteracts that reduction. Related to this, an apparent inflammatory reaction in jennies is induced following AI with frozen-thawed sperm, as a high amount of polymorphonuclear neutrophils (PMN) are observed within the donkey uterus six hours after AI. While PMN appear to select the sperm that ultimately reach the oviduct, two mechanisms, phagocytosis and NETosis, have been purported to be involved in that clearance. ...
Use of nutraceuticals in the stallion: Effects on semen quality and preservation. Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from differ...
Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm. Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batc...
