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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.
Structural insights into VLDLR recognition by western equine encephalitis virus. Western equine encephalitis virus (WEEV), a group of encephalitic alphaviruses that cause severe diseases in humans and equids, historically used the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection. However, current epidemic strains no longer use VLDLR as a receptor. In this study, we identify that LA1, LA2, LA3, and LA5 of VLDLR can directly interact with WEEV. Using cryo-electron microscopy, we investigate the structures of complexes formed between WEEV and VLDLR-LBD or other VLDLR fragments. Our findings show that LA1 and LA2 insert into a cleft formed by two ad...
Quantitative holographic analysis in stallion spermatozoa following cryopreservation. This study employs Holographic tomography (HT) to examine structural and biophysical changes occurring during the cryopreservation of stallion sperm. HT is an advanced imaging technique that integrates digital holography with tomography to achieve three-dimensional, quantitative reconstructions of objects without the need for treatment or reporter dyes. By using refractive index (RI) intervals to represent specific structural regions of sperm cells, variations in optical density, surface area, volume, and dry mass across different cryopreservation extenders and donors have been quantified. Thr...
Naloxone supplementation during vitrification of equine in vitro matured oocytes after overnight holding: insights from a comparative study with the bovine model. Cryopreservation of equine mature oocytes remains a major challenge in assisted reproduction, mainly due to the limited availability of material and logistical constraints requiring a holding phase. To address these issues, this study evaluated whether naloxone (NX), an opioid receptor antagonist with reported antioxidant properties, could improve vitrification outcomes and whether bovine oocytes could serve as a suitable preliminary and supportive model. Two experiments were conducted. In experiment 1, immature (bGV) and mature (bMII) bovine oocytes were vitrified without (VIT) or with NX (VI...
Incubation of Frozen-Thawed Semen Under Capacitating Conditions Supports Successful In Vitro Fertilization and Improves Intracytoplasmic Sperm Injection-Results in Horses. In 2022, a repeatable protocol for in vitro fertilization (IVF) using fresh semen was established in horses. This facilitated successful capacitation of equine semen allowing to explore novel applications. Objective: We aimed to extend this technique to IVF with frozen-thawed semen and intracytoplasmic sperm injection (ICSI), and determine the outcome parameters such as blastocyst production and euploidy rates. Methods: A total of 221 oocytes were subjected to either IVF with frozen-thawed semen, ICSI with frozen-thawed semen incubated under capacitating conditions (ICSI cap) or control ICSI w...
Optimising Stallion Semen Cryopreservation: Preliminary Insights Into Pre-Centrifugation Extender Effects. This study evaluated the effects of cholesterol, pentoxifylline and casein, with or without skim milk, added to extenders during pre-centrifugation on equine sperm cryosurvival. Seven ejaculates from four stallions (n = 28) were divided into four groups: SM (skim milk), SMP (SM + pentoxifylline), SMCho (SM + cholesterol) and ChoCa (cholesterol + casein). After centrifugation and freezing, sperm kinematics and plasma membrane integrity were assessed immediately and 30 min post-thaw. SMCho and ChoCa showed superior results compared with SM and SMP. These findings indicate that chol...
Ultra-rapid freezing in spheres yields a higher cryoresistance than in straws but remains inferior to conventional slow freezing of stallion sperm. This study evaluated the cryoresistance of stallion sperm frozen by ultra-rapid (UR) methods using microspheres and straws or by the conventionally-slow (CS) method. Sixteen ejaculates from four stallions were each divided into three aliquots according to the freezing method: UR freezing in 30-μL spheres (UR-Spheres) by direct immersion in liquid nitrogen (LN); UR freezing in 0.25-mL straws (UR-Straws) by direct horizontal submersion in LN; and CS freezing in LN vapors. Ultra-rapid freezing medium included 100 mM trehalose +1 % BSA, and the CS freezing medium contained 5 % dimethylformamid...
Butylated hydroxytoluene (BHT) improved semen quality and sperm DNA of frozen-thawed Arabian stallions preserved in modified INRA-82 extender. Alpha-tocopherol is one of the non-enzymatic lipophilic antioxidants. Butylated hydroxytoluene (BHT) is a synthetic analog with similar modes of action in protecting the cryopreserved sperms. This study hypothesized that a certain concentration of any antioxidant is suitable for improving the post-thaw semen quality of stallions. To determine the optimum BHT concentration, a synthetic antioxidant similar to vitamin E in potency and scavenging oxidative stress power in concentrations of 0.0, 0.25, 0.50, 1.0, 2.0, and 4.0 mM/ml were added to semen extender. The post-thaw sperm progressive moti...
Direct Warming of Vitrified In Vivo Equine Embryos. Vitrified in vitro-produced embryos can be successfully warmed in isotonic media at room temperature (RT; 22°C). However, this protocol has not been reported for in vivo embryos, which are more challenging to vitrify and warm. Study objectives were to see if vitrified in vivo embryos warmed in RT isotonic medium gave equivalent pregnancy rates to stepwise serial dilution warming, and if embryo size influenced the results. One hundred and seventeen embryos were divided into groups by size (G1:≤ 300 μm, n = 59; G2:> 300-400 μm, n = 33; G3:> 400-500 μm, n = ...
Analysis of Motion Characteristics and Plasma Membrane Intactness (Viability) in Sperm from Domestic Animals. Sperm quality analysis using computer-assisted sperm analysis (CASA) systems and fluorescence-based techniques has become common in the animal reproduction industry, particularly for large animals (i.e., bovine, porcine, equine). In this chapter, the methods commonly utilized in the author's laboratory to examine sperm motion characteristics via CASA and plasma membrane intactness by flow cytometry will be described. These include methods to properly dilute fresh (stallions, bulls, boars), cool-stored (stallions, boars), or frozen/thawed (stallions, bulls, boars) sperm for assessment of sperm ...
Influence of Cryopreservation on the Acrosome Reaction in Hucul Stallion Spermatozoa. The Hucul horse is a Polish primitive breed with a small population size, which highlights the importance of preserving the genetic resources. The cryopreservation of semen is essential for creating gene banks, but its effect on the acrosome reaction in Hucul stallions has not yet been investigated. The acrosome reaction is one of the most important physiological events associated with the fertilization process. Therefore, our goal was to determine the level of acrosome reaction in chilled and frozen/thawed Hucul stallion semen using the FluoAcro test and the SCA semen analysis system. We foun...
A comparison of the efficacy of three commercial human embryo vitrification kits for cryopreservation of in vivo produced equine embryos. Different cryoprotectants can influence the ability of embryos to successfully survive vitrification and subsequent warming before transfer. Objective: To compare pregnancy rates for embryos ≤500 μm vitrified, without puncture or aspiration of the blastocoele cavity, with one of three commercial human embryo vitrification kits containing the same penetrating cryoprotectants (DMSO and EG) but varying in their non-penetrating cryoprotectants (NPCPAs; sucrose, trehalose, dextran serum supplement [DSS], and hydroxypropyl cellulose [HPC]). Methods: In vivo experiments. Methods: Embryos (n =...
Effect of Centrifugation of Stallion Semen Through a Low Density Colloid Prior to Freezing on Sperm Cryosurvival. Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation)...
Isolation and Characterization of Equine Mesenchymal Stromal/Stem Cells. Regenerative medicine is a relatively new branch of therapeutics in equine medicine, which aims to restore and reconstitute tissue function and structure via cellular and/or noncellular approaches. Biological constituents such as mesenchymal stromal/stem cells (MSCs) are potent therapeutics, which can aid in damaged tissue regeneration due to their differentiation capacity into many different cell types such as adipose tissue, bone, and cartilage. MSCs can be successfully and conveniently isolated from equine subcutaneous adipose tissue (adipose-derived stromal cells, ASCs). In horses, there a...
Tenogenic potential of tendon-derived mesenchymal stem cells isolated post-mortem: Impact of cryopreservation. In situ injection of mesenchymal stem cells appears as a promising treatment of tendinopathies. Tendon-derived mesenchymal stem cells (TDSCs) are widely studied and show a lot of interesting characteristics for clinical use. The aim of this study is to confirm the tenogenic potential of cryopreserved TDSCs and to confirm their ability to produce type I and/or type III collagens fibers in culture. Tendon-derived mesenchymal stem cells are harvested from the tendon no later than 72 h post-mortem. Their tenogenic potential has been assessed by quantitative reverse transcriptase polymerase chain ...
In vitro embryo production via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) in pure and crossbred Japanese Hokkaido native ponies. This study evaluated the viability of in vitro embryo production using ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) as breeding techniques for pure and crossbred Hokkaido native ponies (n = 9). Oocytes were collected using transvaginal ultrasound-guided follicle aspiration. ICSI was performed on in vitro matured oocytes using frozen semen. Embryonic cultures were monitored using time-lapse cinematography. Blastocysts were cryopreserved and, after thawing, were transferred non-surgically into recipient mares. Over nine OPU sessions, the mean number of aspirated follicles was 2...
Metformin and rosiglitazone affect motility, lipid peroxidation and mitochondrial activity of thawed equine spermatozoa. Maintaining sperm energy homeostasis in vitro is very important to improve the efficacy of stallion sperm preservation. Equine spermatozoa preferentially utilize oxidative phosphorylation over glycolysis to generate ATP. Metformin and rosiglitazone are antidiabetic compounds that enhances metabolic flexibility and glucose utilization. The aim of this study was to evaluate metformin and rosiglitazone supplementation of the freezing medium on quality and oxidative status of thawed stallion semen. A total of 15 ejaculates from five horses were collected and supplemented before freezing with metfo...
Sperm Vitrification in Horse and Donkey. Sperm vitrification is a novel-assisted reproductive technique that is increasingly gaining relevance in the last years. This technique allows to cryopreserve sperm from valuable stallions and donkeys without the exposure to permeable cryoprotectants, particularly toxic for the gametes of these species.This chapter aims to describe the current range of methodologies available that are key to ensure sperm quality after vitrification and warming of stallion and donkey sperm.
Cryopreservation of Horse Sperm. Cryopreservation is currently the only strategy for long-term conservation of equine sperm. To get optimal post-thaw sperm survival, carefully following each step of the freezing protocol is crucial. First, one needs to obtain and exhaustively analyze an ejaculate of good sperm quality. Then, the seminal plasma is removed by centrifugation, and the resulting pellet is resuspended in a certain volume of the freezing medium to reach the right sperm concentration. Finally, sperm samples are packaged into 0.5-mL straws, cooled, and frozen using an automatic, controlled-rate freezer. Once the tempe...
Effect of pre-freeze sperm concentration, freezing extender, and epididymal flushing technique on post-thaw quality of cryopreserved epididymal stallion sperm. Cryopreservation of stallion epididymal sperm has become a common clinical procedure after routine castration, euthanasia, or acute death. Unique features of epididymal sperm compared to ejaculated sperm include the requirement to remove sperm from the cauda epididymis, lack of exposure to seminal plasma, and potential recovery of large sperm numbers. In this study, the effect of the flushing technique (Extender (INRA) vs. AIR), freezing extender (LE, CMLE, MFR5, CFR5, or BOTU), and the concentration at which sperm are cryopreserved (200, 400, or 800 × 10 sperm/mL) on post-thaw epididymal s...
Sperm Motility Evaluation in Stallion Fresh, Cooled and Frozen Semen Using a Portable Computer-Assisted Sperm Analysis System. Semen analysis is an important laboratory diagnostic test for stallions. Evaluation of sperm motility is integral to basic semen analysis and results are important for breeding management and clinical practice. Computer-assisted sperm analysis (CASA) allows objective sperm motility evaluation and increases analytical precision. The objective of the present study was to validate a portable CASA system (AndroScope). Fresh/cooled semen samples (33 ejaculates, 18 stallions) and frozen semen (40 ejaculates and one epididymal flush, 27 stallions) were evaluated using the AndroScope and the IVOS II C...
Cryoprotective effect of zinc and gold nanoparticles during cooling and freeze-thawing on Marwari stallion sperm parameters and reactive oxygen species production. Sperm cryopreservation is one of the most important procedures in the development of biotechnologies for assisted reproduction. Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Different substances and compounds can be added to semen extenders to improve sperm quality. Objective: To investigate the effect of supplementing semen extender with zinc nanoparticles (ZnONPs) and gold nanoparticles (AuNPs) on cooled and frozen-thawed spermatozoa of Marwari stallion. Methods: A total of 20 ejaculates from four Marwari ...
Equine in vitro fertilization with frozen-thawed semen is associated with shortened pre-incubation time and modified capacitation-related changes. We recently reported successful equine IVF using fresh semen pre-incubated for a prolonged period (22 h) before co-culture with oocytes. In this study, we evaluated the feasibility of equine IVF with frozen-thawed sperm and evaluated capacitation-related changes in these sperm over the pre-incubation period. Sperm selected via a commercial sperm separation device (SSD) yielded significantly higher fertilization than did sperm selected by swim-up or by colloid centrifugation. Using the SSD method, fertilization rates with sperm pre-incubated for 15 min, 3 h, 6 h, and 9 h were 7.1, 22.2, 38...
Results of the “test-freeze” approach in a commercial program of stallion sperm cryopreservation and the relationship between pre-freeze sperm quality and “freezability.”. In the current study, we report the effect of different commercially available semen freezing extenders utilized for the "test-freeze" procedure for 13 years (2010-2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility - TM [%]; progressive motility - PM [%]; and curvilinear velocity - VCL [μm...
Effects of holding and the addition of naloxone on vitrification of equine immature oocytes. This study investigates the effects of overnight holding and naloxone (Nx) supplementation on the vitrification outcomes of equine immature oocytes. Oocytes were divided into six experimental groups based on treatment combinations: fresh (F) and held (H) control oocytes, oocytes vitrified with or without Nx (10 M) (VIT and VIT-Nx), oocytes vitrified after overnight holding with or without Nx (10 M) (H-VIT and H-VIT-Nx). They were assessed for survival, meiotic competence, intracellular oxidative stress, mitochondrial activity and distribution, apoptosis, and apoptotic gene expression. At sur...
Changes in bacterial viability after preparation and storage of fecal microbiota transplantation solution using equine feces. Fecal microbiota transplantation (FMT) has been used as a treatment option for horses (Equus caballus) with gastrointestinal diseases. Several preparation and conservation protocols to improve bacterial survival have been studied in other species. Unassigned: This study aimed to evaluate the impact of oxygen exposure and different protectant solutions on bacterial viability before and after freezing using horse feces. Fecal samples from 10 healthy horses were aliquoted and diluted in cryoprotectant solutions containing antioxidants (n = 40) or 10% glycerol (n = 40). Half of the aliquots from e...
Endangered Przewalski’s Horse, Equus przewalskii, Cloned from Historically Cryopreserved Cells. Cloning from historically cryopreserved cells offers a potential means to restore lost genetic variation or increase the representation of particular lineages within bottlenecked species, provided such biobanked materials are archived for such genetic rescue applications. One species for which cloning can provide genetic management benefits is Przewalski's horse, . All ~1800 living Przewalski's horses, distributed across ex situ breeding facilities and in situ reintroduction sites, are descended from one or more of the five founder lineages established by the 12 horses captured from the wild b...
Sperm vitrification in horses and donkeys. Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
The effect of resveratrol on the cryopreservation of Mongolian horse semen. Cryopreservation of semen has advanced significantly with the development of artificial insemination techniques, but post-thawed sperm often exhibit reduced viability, membrane integrity, and acrosome integrity compared to fresh sperm, leading to decreased fertilization capacity. Oxidative stress is a major concern during cryopreservation. This study investigated the use of resveratrol (RSV), a potent antioxidant, in the cryopreservation of Mongolian horse semen. Different concentrations of RSV were incorporated into semen cryopreservation extenders, and the morphological and antioxidant indic...
Molecular basis for shifted receptor recognition by an encephalitic arbovirus. After decades of inactivity throughout the Americas, western equine encephalitis virus (WEEV) recently re-emerged in South America, causing a large-scale outbreak in humans and horses. WEEV binds protocadherin 10 (PCDH10) as a receptor; however, nonpathogenic strains no longer bind human or equine PCDH10 but retain the ability to bind avian receptors. Highly virulent WEEV strains can also bind the very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as alternative receptors. Here, by determining cryo-electron microscopy structures of WEEV strains isolated from...
Marked blastomere herniation is the only post-thaw morphological characteristic associated with a reduced pregnancy rate for in vitro produced equine blastocysts. It is not known whether the likelihood of pregnancy after transfer of cryopreserved equine in vitro produced (IVP) blastocysts is related to post-thaw morphological characteristics, or could be impacted by other factors such as embryo biopsy prior to cryopreservation. This study examined associations between post-thaw morphological appearance of equine IVP embryos, and biopsy prior to slow-freezing, on the likelihood of pregnancy. Biopsy of IVP blastocysts was performed by harvesting cells herniated through the intra-cytoplasmic sperm injection (ICSI)-derived hole in the zona pellucida (ZP). M...
