Topic:Freezing Technique
Freezing techniques in horses involve the controlled application of low temperatures to preserve equine biological samples, tissues, or cells for research and clinical purposes. These techniques are employed in various contexts, including the preservation of semen for artificial insemination, the storage of embryos for breeding programs, and the conservation of genetic material. The process typically involves the use of cryoprotectants to prevent ice crystal formation, which can damage cellular structures. Research in this area focuses on optimizing freezing protocols to enhance viability and functionality post-thaw. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and outcomes of freezing techniques in equine science.
Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa. The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sp...
[Successful direct transfer of a deep frozen-thawed equine embryo]. Embryos were flushed on day 7 after ovulation from two mares, and frozen using a conventional slow freezing procedure in phosphate buffered (PBS) saline supplemented with 10% FCS, 1.5 mol/L ethylene glycol and 0.25 mol/L sucrose. One of the two embryos was thawed after 10 months of storage in liquid nitrogen and transferred directly (without dilution of the cryoprotectant and quality examination) to a synchronized recipient. This transfer resulted in the birth of a live female foal. To our knowledge, this is the first live foal born after direct transfer of a frozen-thawed equine embryo.
Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis. Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were fro...
Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm a...
Advances in cooled semen technology. In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in ord...
The equine frozen semen industry. Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major imped...
Management and fertility of mares bred with frozen semen. Semen quality, mare status and mare management during estrus will have the greatest impact on pregnancy rates when breeding mares with frozen semen. If semen quality is not optimal, mare selection and reproductive management are crucial in determining the outcome. In addition to mare selection, client communication is a key factor in a frozen semen program. Old maiden mares and problem mares should be monitored for normal cyclicity and all, except young maidens, should have at least a uterine culture and cytology performed. Mares with positive bacterial cultures and cytologies should be treate...
Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glyce...
Effect of insemination time of frozen semen on incidence of uterine fluid in mares. Ninety five mares were inseminated with frozen semen either within 12 h before ovulation or within 8 h after ovulation. The effect of preovulatory versus postovulatory insemination (AI) on the subsequent detection of uterine fluid was studied. The overall pregnancy rate was 43% and this was not significantly influenced by preovulatory or postovulatory insemination. When mares were first examined 12 h after AI, 18 of 52 mares (35%) had accumulated uterine fluid. However, when mares were first examined 18 to 24 h after AI, only 6 of 43 mares (14%) had uterine fluid. Presence of intrauterine flui...
Generation of reactive oxygen species by equine spermatozoa. To characterize generation of reactive oxygen species (ROS) by equine spermatozoa. Methods: Multiple semen samples collected from 9 stallions. Methods: Equine spermatozoa were separated from seminal plasma on a discontinuous polyvinylpyrrolidone (PVP)-coated silica gradient and resuspended in a modified Tyrode albumin-lactate-pyruvate medium. Amount of hydrogen peroxide (H2O2) generated was assayed by use of a 1-step fluorometric assay, using 10-acetyl-3,7-dihydroxyphenoxazine as a probe for detection of H2O2 in a microplate assay format. Concentration of H2O2 was determined by use of a fluore...
Influence of Eqvalan (ivermectin) on quality and freezability of stallion semen. The objective of this study was to evaluate the effect of Eqvalan (ivermectin) on stallion semen quality and freezability. Experiments were performed using 22 Freiberger stallions, randomly divided into a control and test group. Semen was collected once a week for 17 weeks from October 1997 to February 1998. Eqvalan was given orally to all stallions of the test group at a therapeutic dose of 0.2 mg ivermectin/kg. Besides measuring the scrotal width, ejaculates were collected to determine the volume, concentration, and the motility and morphology (normal sperm, major defects, vacuoles and acros...
Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results. The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first...
Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis. Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of ea...
Morphological and functional changes of stallion spermatozoa after cryopreservation during breeding and non-breeding season. The study compared quality and freezability of stallion semen during breeding and non-breeding seasons. Ejaculates were collected twice per week from four stallions during May (n = 24) and December (n = 24). The semen was mixed with skim milk extender, centrifuged and resuspended in fresh extender. Aliquots of this sperm suspension were separated from extender and diluted in TALP medium for sperm evaluation or with cryoextender (type "Gent" or a combination of Triladyl and skim milk). Samples of 0.5ml were cryopreserved in straws using a programmed freezer. Parameters of sperm quality were eva...
Absence of selective brain cooling in free-ranging zebras in their natural habitat. We used implanted miniature data loggers to measure brain and arterial blood temperatures in three free-ranging zebras (Equus burchelli) in their natural habitat, every 5 min for 9 days. The animals experienced globe temperatures exceeding 40 C, and radiant heat load of about 1000 W m-2. Arterial blood exhibited a moderate amplitude (1.7 C) nychthemeral rhythm, with an acrophase at 19.00 h and a nadir late in the morning, at 10.00 h. Brain temperature consistently exceeded blood temperature, on average by 0.2-0.4 C, and changes in brain temperature closely tracked changes in blood temperature....
Significance of plasmalemma disruption in bovine and equine spermatozoa. We have investigated fresh and cryopreserved bovine and equine spermatozoa using light and transmission soft X-ray microscopy. Spermatozoa were examined, in the presence or absence of semen, after using Percoll gradient centrifugation and re-suspending in medium. X-ray microscopy provided high resolution (30 nm) transmission images of whole cells in solution with high contrast, while retaining the simple preparation techniques used in light microscopy. We demonstrated translucent, membrane-bound vesicles in the acrosomal and midpiece regions that were similar in size and we noted their inciden...
Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa. The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addit...
Acrosomal ultrastructure of stallion spermatozoa cryopreserved with ethylene glycol using two packaging systems. The present experiments aimed to examine the substitution of glycerol (G) by ethylene glycol (E) as a cryoprotective agent for stallion spermatozoa. Two different ethylene glycol concentrations (5% and 10%) and also the association of glycerol (2%) and ethylene glycol (3%) (E/G) were studied (Experiment 1). In Experiment 2, two packing systems (0.5 x 4.0 ml) were evaluated using both cryoprotectors. In both experiments, the sperm membrane integrity after freezing was evaluated using transmission electron microscopy. The mean post-thaw motility was 34.25, 36.5, 29.25 and 34.75% for G5%, E5%, E1...
Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene...
Assessment of equine sperm mitochondrial function using JC-1. The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa. This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loa...
Effects of glutamine, proline, histidine and betaine on post-thaw motility of stallion spermatozoa. The supplementation of the freezing diluent with 3 amino acids (glutamine, proline and histidine) and 1 amino acid-related compound (betaine) in preserving stallion spermatozoa diluted in INRA82 extender containing 2.5% (v/v) glycerol and 2% (v/v) egg yolk (control extender) during freezing and thawing was studied at 0, 40, 80, 120 and 160 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 1). Glutamine and proline were studied at 0, 10, 20, 30, 40, 50, 60, 70 and 80 mM in 20 split ejaculates (10 stallions x 2 ejaculates; Experiment 2). In each experiment, spermatozoa were eval...
Sperm transport and survival in the mare: a review. After the deposition of semen in the mare's uterus, spermatozoa must be transported to the site of fertilization, be maintained in the female tract until ovulation occurs, and be prepared to fertilize the released ovum. Sperm motility, myometrial contractions, and a spontaneous post-mating uterine inflammation are important factors for the transport and survival of spermatozoa in the mare's reproductive tract. Fertilizable sperm are present in the oviduct within 4 h after insemination. At this time, the uterus is the site of a hostile inflammatory environment. Our data suggest that spermatozoa...
Effect of spermatozoal concentration and number on fertility of frozen equine semen. Information on the number of motile spermatozoa needed to maximize pregnancy rates for frozen-thawed stallion semen is limited. Furthermore, concentration of spermatozoa per 0.5-mL straw has been shown to affect post-thaw motility (7). The objectives of this study were 1) to compare the effect of increasing the concentration of spermatozoa in 0.5-mL straws from 400 to 1,600 x 10(6) spermatozoa/mL on pregnancy rate of mares, and 2) to determine whether increasing the insemination dose from approximately 320 to 800 million progressively motile spermatozoa after thawing would increase pregnancy r...
Hypoosmotic test in equine spermatozoa. The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and ...
The current status of equine embryo transfer. The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulati...
In vitro and xenogenous capacitation-like changes of fresh, cooled, and cryopreserved stallion sperm as assessed by a chlortetracycline stain. Like the human female, the mare experiences reproductive tract pathology that may sometimes be circumvented by the use of assisted reproductive technologies (ARTs). One such technology, gamete intrafallopian transfer (GIFT), may be used in mares that exhibit ovulatory, oviductal, or uterine abnormalities that limit the use of common ARTs, such as embryo transfer. Homologous GIFT has been successfully performed in the horse; however, the logistics, costs, and associated risks of surgically transferring gametes to the oviducts of a recipient mare are considerably high. Use of a less costly speci...
Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing. The aim of the present study was to investigate the cryoprotectant properties of cholesterol after incorporation into the plasma membranes of equine spermatozoa. A cholesterol-methyl-beta-cyclodextrin complex was used to alter sperm plasma membrane cholesterol content. Ejaculates from six stallions were centrifuged in a non-fat skimmed milk glucose-sucrose extender (MK) or a modified Tyrode's medium (TALP). The sperm pellets were resuspended in the appropriate extender with or without added cholesterol (0.125 mmol cholesterol-methyl-beta-cyclodextrin complex l(-1)) and incubated at 24 degrees ...
Pregnancies produced from fertile and infertile stallions by intracytoplasmic sperm injection (ICSI) of single frozen-thawed spermatozoa into in vivo matured mare oocytes. The use of intracytoplasmic sperm injection (ICSI) for in vitro fertilization of equine oocytes and the developmental potential of these oocytes after transfer to the Fallopian tubes of synchronized mares were examined. Oocytes were aspirated from mature follicles 39 h after injection of a GnRH analogue and transported 190 km at 39 degrees C. Semen from a fertile and an infertile stallion was frozen and prepared for injection. Successfully injected oocytes were transferred surgically into the ampulla of the Fallopian tube either: (i) 4-8 h after semen injection; or (ii) after 24-48 h culture b...
Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions. In the present study, the interactions among stallions, semen extenders and cooling treatments before stallion semen samples were frozen were studied. In Expt 1, the effects of four cooling treatments and three semen extenders were investigated (11 stallions x four split ejaculates), whereas in Expt 2, the effects of two semen extenders, two egg yolk concentrations and two glycerol concentrations were investigated (six stallions x five split ejaculates). Sperm motility after thawing was evaluated. In Expt 1, the extender x cooling treatment interaction was significant. Centrifugation and addit...