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.
Dynamics of sperm DNA fragmentation in domestic animals II. The stallion. The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. ...
Electrolyte distribution and yolk sac morphology in frozen hydrated equine conceptuses during the second week of pregnancy. To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concep...
Membrane transport properties of equine and macaque ovarian tissues frozen in mixtures of dimethylsulfoxide and ethylene glycol. The rate at which equine and macaque ovarian tissue sections are first cooled from +25 degrees C to +4 degrees C has a significant effect on the measured water transport when the tissues are subsequently frozen in 0.85 M solutions of glycerol, dimethylsulfoxide (DMSO), or ethylene glycol (EG). To determine whether the response of ovarian tissues is altered if they are suspended in mixtures of cryoprotective agents (CPAs), rather than in solutions of a single CPA, we have now measured the subzero water transport from ovarian tissues that were suspended in mixtures of DMSO and EG. Sections of fr...
A comparison between freezing methods for the cryopreservation of stallion spermatozoa. The effects of sperm freezing concentration (40 x 10(6)mL(-1) vs. 400 x 10(6)mL(-1)), straw size (0.25 mL vs. 0.5 mL) and freezing method (liquid nitrogen vapour in a Styrofoam box vs. programmable freezing machine) were evaluated in a 2 x 2 x 2 factorial experimental design using 3 split ejaculates from each of 4 stallions. Immediately after thawing, the total motility and forward progressive motility of spermatozoa frozen at a concentration of 40 x 10(6)mL(-1) was higher than for spermatozoa frozen at 400 x 10(6)mL(-1). No significant differences were observed in the semen parameters assesse...
Characterization and differentiation of equine umbilical cord-derived matrix cells. Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton's jelly). Equine umbilical cord matrix (EUCM) cells can be ...
Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. An alternative mechanism has been proposed by Morris (2006) that cell damage is a result of an osmotic imbalance encountered during thawing. This paper examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. Horse spermatozoa were cooled at a range of cooling rates from 0.3 to 3,000 degrees C/min in the presence of a cryoprotectant. The ultra...
Comparison of different extenders with defined protein composition for storage of stallion spermatozoa at 5 degrees C. To maintain the fertility of stallion spermatozoa during cooled storage, extender media are added to semen. In this study, three semen extenders were compared: EquiPro which contains defined caseinates and whey proteins instead of dried skim milk. The extender is provided in dry form and dissolved in distilled water prior to use. EquiPro TM has the same composition as EquiPro but is provided in a sterilized ready-to-use liquid form. AndroMed-E contains soybean lecithin as protein source. Semen was collected from seven stallions. Ejaculates were divided into three aliquots, diluted with the dif...
Influence of cryopreservation on mitochondrial functions in equine spermatozoa. Cryopreservation of spermatozoa is of essential importance for artificial insemination and breeding programs in horses. Besides other factors, spermatozoal motility depends on mitochondrial energy metabolism. Based on changes of single mitochondrial functions it has been suggested that mitochondrial damage during cryopreservation could be a major reason for diminished post thaw semen quality. However, it is still unclear to which extent this influences the whole bioenergetic performance of mitochondria and whether this plays a role during routine cryopreservation procedures. Therefore, it was ...
The costs of breed reconstruction from cryopreserved material in mammalian livestock species. The aim of this work was to compare costs, in the horse, cattle, sheep, swine, and rabbit species, for the creation of gene banks for reconstruction of an extinct breed, using different strategies: embryos-only, embryos in combination with semen, and semen-only. Three cost measures were used: time required for population reconstruction, cost for creation of the gene bank, number of years-keeping-female to reach reconstruction. Semen costs were estimated across four scenarios: the presence or absence of a commercial market for semen, purchase of semen donors, and semen extracted from the epidid...
A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm. Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. Methods: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after tha...
In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection. In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates...
Hypothermic storage of equine isolated hepatocytes. The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte...
Vitrification of equine embryos. Vitrification can be used successfully to cryopreserve equine embryos. Embryos for vitrification should be collected from donor mares' uteri when they are 300 mm or less in diameter, however,and at the morula or early blastocyst stage of development. No special equipment is required for vitrification; the straw containing the embryo is exposed to vapor for 1 minute before plunging it into liquid nitrogen. Warming of the straw requires no special equipment,and the embryo can be transferred directly from the straw into a recipient's uterus. Vitrification has been repeatedly successful when the p...
Collection and freezing of epididymal stallion sperm. The ability to harvest and preserve epididymal sperm from a stallion after simple elective castration, a catastrophic injury, or severe acute illness and subsequent death has been realized, allowing for the preservation of genetics that would have been lost otherwise.Currently, the care taken to collect the testes and epididymides properly, coupled with proper packaging and shipping, could make the greatest contribution to salvaging viable sperm. As advances in assisted reproductive techniques continue, more offspring may be obtained from stored epididymal sperm from valuable stallions.
Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep. Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and sign...
Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes. Oocyte cryopreservation is a potentially valuable way of preserving the female germ line. However, the developmental competence of cryopreserved oocytes is presently poor. This study investigated whether the morphology of the cumulus complex surrounding an immature equine oocyte and/or the oocyte's stage of maturation affect its cryopreservability. Compact (Cp) and expanded (Ex) cumulus oocyte complexes (COCs) were vitrified either shortly after recovery (germinal vesicle stage, GV) or after maturation in vitro (IVM); cryoprotectant-treated and -untreated non-frozen oocytes served as controls....
Effect of seminal plasma fractions on stallion sperm survival after cooled storage. This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP...
Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo devel...
Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods. The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured wit...
Cryopreservation of horse semen under laboratory and field conditions using a Stirling Cycle freezer. A Stirling Cycle freezer has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Horse semen samples were cooled in 0.25 ml straws and 15 ml bags in the Stirling Cycle freezer under laboratory conditions and as a portable device, powered from a car battery. For comparison, straws were frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, motility and viability of samples frozen in the Stirling Cycle freezer were not significantly different when compared to samples frozen in the liquid nitrogen freezer. Unlike liquid nitrogen syst...
Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Oocyte cryopreservation is a potentially valuable technique for salvaging the germ-line when a valuable mare dies, but facilities for in vitro embryo production or oocyte transfer are not immediately available. This study examined the influence of maturation stage and freezing technique on the cryopreservability of equine oocytes. Cumulus oocyte complexes were frozen at the immature stage (GV) or after maturation in vitro for 30 hr (MII), using either conventional slow freezing (CF) or open pulled straw vitrification (OPS); cryoprotectant-exposed and untreated nonfrozen oocytes served as contr...
Motility and plasma membrane integrity of spermatozoa in fractionated stallion ejaculates after storage. With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with exten...