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.
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...
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...
Curcumin-loaded niosomal nanocarriers offer a promising approach to improve quality characteristics, apoptotic gene expression, and flow cytometry assessments of stallion spermatozoa after thawing. The optimization of cryopreservation media to reduce oxidative damage on post-thaw spermatozoa is crucial. This research aimed to assess the antioxidant properties of curcumin-loaded niosomal nanocarriers (CurLNN) on the functional characteristics, the relative expression of apoptotic genes, and flow cytometry assessments of apoptotic-like changes, reactive oxygen species production (ROS), mitochondrial membrane potential, and chromatin integrity in stallion spermatozoa following thawing. Twenty-five ejaculates were diluted in INRA96 freezing media supplemented with 20 μM of either curcumin ...
Ice application without water drainage supports optimal hoof cooling in adult horses. Cryotherapy is often used to reduce inflammation in acute equine laminitis cases. Certain hoof temperatures have been suggested as effective in minimizing the inflammatory process; however, there is limited evidence on which methods are best at achieving these temperatures. Our objective was to determine how different methods of cryotherapy influence the rate and extent of cooling for the equine hoof wall. Four horses received three hoof cooling treatments and a control (CON; no treatment application) in a 4 × 4 Latin square design. Treatments included (1) ice surrounding the hoof in a 5 L fl...
The use of sex-sorted semen in horses. The application of sex-sorted semen in horses has historically lagged the bovine industry due to differences in the reproductive physiology and grater variability in equine semen quality. Recent advancements, including SuperGen™ sorters and Ultraplus™ semen processing methods developed by STgenetics® (Navasota, Texas), have significantly improved the efficiency of the sex-sorting process and reduced the sperm damage previously reported. To facilitate the broader use sex-sorted semen in the equine industry, it is essential to address challenges such as shipping cooled semen to a central so...
Niosomal nanocarriers loaded with artemisinin has potential to enhance the functional characteristics of cryo-preserved equine spermatozoa. Cryopreservation is a crucial method for the preservation of genetic materials. Nevertheless, this technique can have adverse effects due to cryo-oxidative damage. The primary objective of this study was to investigate the cryo-protective effects of niosomal nanocarriers loaded with artemisinin (NN-Art) on the functional attributes of equine spermatozoa. The parameters examined included sperm motility and kinematics through computer-assisted sperm analysis (CASA), plasma membrane integrity via the hypoosmotic swelling test, viability using the eosin-nigrosin staining technique, morphology with...
Optimization of vitrification methods for equine oocytes. An important method for preserving equine germplasm is the cryopreservation of equine oocytes. Due to its ease, rapidity and affordability, vitrification freezing has taken over as the primary method of horse oocyte cryopreservation. The vitrification cryoprotectants utilized in this investigation were Ethylene glycol (E), Dimethyl sulfoxide (D), Sucrose (S), and Ficoll (F). According to the oocyte volume alteration, the treatment time was 39 s in equilibrium solution ED10 (10 % EG + 10 % DMSO), 32 s in equilibrium solution ED15 (15 % EG + 15 % DMSO), while 20 s in equilibrium so...
IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study. Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they...
Effect of refreezing extender on stallion sperm quality and embryo production after intracytoplasmic sperm injection. Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
Reimagining stallion sperm conservation: Combating carbotoxicity through pyruvate-induced Warburg effect to enhance sperm longevity and function. Although stallion spermatozoa are now recognized as highly dependent on oxidative phosphorylation for ATP production in the mitochondria, most extenders in use contain supraphysiological concentrations of glucose as the main energy source. While the toxicity of cryoprotectants has been well documented in the literature, the potential toxicity of excessive glucose in extenders is largely ignored. However, the toxicity of excess glucose, known as "carbotoxicity", is well-established in many areas of medicine. In this paper, we review the basic aspects of stallion spermatozoa metabolism, focusing...
Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters. The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylf...
Exploring the Dissolution, Solid-state Properties, and Long-term Storage Stability of Cryoprotectant-free Fenbendazole Nanoparticles. Fenbendazole is an antiparasitic drug widely used in veterinary medicine to treat parasitic infections caused in animals like cattle, horses, sheep, and dogs. Recently, it has been repositioned as a potential alternative for cancer treatment. However, it is a highly hydrophobic molecule (0.9 ug/mL), which can compromise its dissolution rate and absorption. Thus, this work aimed to apply a nanotechnological approach to improve drug solubility and dissolution performance. Fenbendazole nanoparticles stabilized by different poloxamers were obtained by lyophilization without cryoprotectants. The be...
Factors affecting pregnancy rates in mares bred with cryopreserved semen. Historically, 8 × 0.5 ml straws, containing approximately 800 million sperm and 250 million progressively motile sperm were provided as a single 'breeding dose' of cryopreserved stallion semen. With the use of deep horn artificial insemination, there is a trend to reduce the number of 0.5 ml straws sold as a breeding dose, sometimes down to as little as one straw. Our aims were to determine if the number of straws provided as a breeding dose, as well as other mare, stallion and management factors, have an impact on pregnancy outcome in mares inseminated with cryopreserved semen. Unexpectedly,...
Effect of Mitoquinone on sperm quality of cryopreserved stallion semen. This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated ...
Impact of low-dose ozone supplementation on motility parameters and bacterial growth in horse cryopreserved semen. Two studies were conducted to evaluate the use of medical ozone (O) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent...
Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen. Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifu...
Effect of novel lyophilized extenders on stallion post-thaw sperm motility. The aim of this study was to assess the effect of lyophilized freezing extenders, which can be stored at room temperature, on stallion post-thaw sperm total motility (TM). Ejaculates of 28 stallions were frozen with four different extenders: two commercial freezing extenders offered worldwide and two novel lyophilized extenders (STAR and MX3), and two different cryopreservation protocols (CP1 with an equilibration period of 20 min. and CP2 with an equilibration period of 60 min.). The TM was assessed after thaw. Mean TM did not show significant differences between cryopreservation protocols wi...
The Effects of Supplementation of the Freezing Extender with Silymarin on the Quality Parameters of Frozen-Thawed Arabian Stallion Sperm: A Preliminary Evaluation. This study evaluated the effects of supplementation of the freezing extender with different concentrations of silymarin on the quality of frozen-thawed Arabian stallion spermatozoa. Semen samples from three stallions (1, 2, and 3) were suspended in the freezing extender without or with silymarin (0, 25 μg/mL, 50 μg/mL, 75 μg/mL, and 100 μg/mL) and cryopreserved in 0.5 mL straws. After 1 month of storage, the frozen semen samples in straws were thawed and evaluated in terms of viability, mitochondrial membrane potential, kinematic parameters, total and progressive motility, plasma membrane ...
Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa. Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Methods: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumi...
Impact of Seminal Plasma Antioxidants on DNA Fragmentation and Lipid Peroxidation of Frozen-Thawed Horse Sperm. Cryopreservation is a stressful process for sperm, as it is associated with an increased production of reactive oxygen species (ROS). Elevated ROS levels, which create an imbalance with antioxidant capacity, may result in membrane lipid peroxidation (LPO), protein damage and DNA fragmentation. This study aimed to determine whether the membrane LPO and DNA fragmentation of frozen-thawed horse sperm relies upon antioxidant activity, including enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and paraoxonase type 1 (PON1)); non-enzymatic antioxidant capacity (Trolo...
Freezing Stallion Semen-What Do We Need to Focus on for the Future? Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics ...
Freezing of equine semen is influenced by exposure time and concentration of the cryoprotectant glycerol. Glycerol is a cryoprotectant widely used in the freezing of mammalian semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. Objective: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. Methods: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Anal...
Effect of different manual puncture methods on donkey embryo before vitrification. The application of embryo recovery and transfer technology in the donkey industry is far lower than that of horses and cattle. Sometimes the recovered embryos could not be transferred in time, which required embryo cryopreservation. The embryo cryopreservation technology is more conducive to the preservation and transportation of recovered embryos with excellent genetic traits. However, this technique for donkey embryos is not efficient and needs further optimization. The objective of this study was to evaluate the effect of different manual puncture methods on the viability and pregnancy rate...
Continuous digital hypothermia for prevention and treatment of equine acute laminitis: A practical review. Laminitis is a severely debilitating and life-threatening condition that occurs as a consequence of different primary triggering factors. Continuous digital hypothermia (CDH) is recommended in horses at risk of, or diagnosed with, acute laminitis due to its several physiological and biochemical alterations that may be positive for the prevention and early treatment of the condition, representing a low risk of adverse effects. Modulation of the inflammatory response, profound vasoconstriction, and prevention of tissue damage are the most notable protective effects of cryotherapy on the lamellae...
Shipping duration and temperature influence the characteristics of cryopreserved horse semen stored in different shipping devices for up to 14 days. This study aimed to investigate the effects of storing horse semen either in a dry shipper (≤ -150 °C) or on dry ice (≤ -78 °C) for up to 14 days. A total of 264 frozen semen straws from male horses (n = 8) stored in liquid nitrogen were transferred on day 0 (d0) to a dry shipper or a dry ice styrofoam box. On d1, d3, d7, d10, and d14, straws from the dry shipper and dry ice were returned to the liquid nitrogen container. Semen was evaluated by CASA for total (TMot), progressive motility (PMot) and sperm velocity parameters, by fluorescence microscopy for percentage of membrane-intac...
Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa. Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from...
Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests. Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, suff...
Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation. In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), a...
Carboxymethylchitosan with medium-molecular-weight affects kinectics and acrosome of stallion sperm after freezing/thawing. Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were ...