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.
Effect of freeze-drying on measurements of pH in biopsy samples of the middle gluteal muscle of the horse: comparison of muscle pH to the pyruvate and lactate content. Muscle biopsies taken after exercise, in comparison to those at rest, contain increased amounts of blood and this is a particular problem in studies of the horse. The inclusion of blood in muscle will introduce an upward bias in values of pH measured in muscle homogenates. In an attempt to control this, muscle biopsy samples of the middle gluteal from Thoroughbred horses were freeze-dried and dissected free of blood before determination of pH. Following exercise, muscle pH measured after freeze-drying was similar to that measured in homogenates prepared from frozen samples. In contrast, freeze...
Radioimmunoassay for etorphine in horses with a 125I analog of etorphine. To improve the sensitivity and specificity of screening for etorphine in horses, an 125I-labeled etorphine analog was synthesized and an antibody to etorphine was raised in rabbits. A radioimmunoassay (RIA) for etorphine was developed, using these reagents. Bound and free 125I-labeled etorphine was separated by a double-antibody method that reduced interference from materials associated with equine urine. The 125I-labeled etorphine binding was rarely greater than 250 pg of background etorphine equivalents/ml in raw urine and was 100 pg/ml in hydrolyzed urine. The 125I-RIA was capable of detect...
Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa. A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. ...
Effect of sample freezing on the isolation of Mycoplasma spp. from the clitoral fossa of the mare. The growth of Mycoplasma equigenitalium and Mycoplasma subdolum from specimens collected from the clitoral fossa of each of four Standardbred mares was not diminished by freezing of the specimens in liquid nitrogen (-196 degrees C) for up to 30 days when compared to samples cultured immediately.
Effects of cooling rate and storage temperature on equine spermatozoal motility parameters. Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storag...
Structural, histochemical and biochemical observations on horse milk-fat-globule membranes and casein micelles. Horse milk fat globules (MFGs) and casein micelles were studied using freeze fracturing, freeze etching and thin-section electron microscopy, as well as lectin histochemistry, gel electrophoresis, and Western blotting. Horse MFGs were found to be relatively small, their average volume-surface diameter being about 2.75 microns. The MFG membrane is composed of three layers: an inner proteinaceous coat occasionally having a paracrystalline substructure, a unit membrane, and a prominent filamentous glycocalyx. The last is rich in glycoconjugates, as revealed by its binding of various lectins. In a...
Analysis of the physiological processes connected with sexual maturation of stallions. Physiological processes connected with sexual maturation of stallions were observed on 10 half-breed Anglo-Arab stallions beginning from 8 months of age, until 4.5 years of age. It was found that there is full somatic and sexual development in the stallion reached around the age of 3.5 years, and the sperm morphology stabilized in the range of the physiological norm around 3.0 years of age. On the other hand biochemical components of the semen plasma such as glycerylphosphorylcholine (GPC), ergothioneine (EGT), total protein (PRT), up to age 4.5 years, reach significantly lower value than in m...
Practicalities of insemination of mares with deep-frozen semen. From 341 stallions examined for sperm quality, 61% of warm-blooded stallions and 47% of cold-blooded stallions fulfilled the pre-existing criteria for their occasional use in insemination. From these stallions 51-71% of acceptable ejaculates were obtained. Altogether 959 mares were inseminated in an average of 1.36 oestrous cycles. For the insemination of one mare in one oestrous cycle on the average 2.2 insemination doses were used. These inseminations were carried out by 41 cattle insemination technicians trained in mare insemination. A pregnancy rate of 56% and a foaling rate of 48% were ac...
Ultrastructure of cryopreserved horse embryos. Embryos were recovered non-surgically at about Day 6 after ovulation from 15 Quarter horse-type mares and were evaluated for morphological changes which may occur because of exposure to the cryoprotectant and/or cryopreservation. Electron microscopy was used to elucidate the fine structure of intracellular organelles which, if damaged, could cause cellular death. The horse embryo does not totally re-expand in the 10% glycerol freezing medium, nor will it completely re-expand in the isotonic holding medium following glycerol removal whether or not the embryo has been frozen. Embryos in this stu...
Influence of season and frequency of ejaculation on production of stallion semen for freezing. In an attempt to define optimal season and ejaculation frequency for frozen semen, semen was collected from 6 stallions (3 horses and 3 ponies) 3 times per week or every day, alternating every week, for 1 year. The semen was evaluated and frozen. All the samples were thawed at the end of the experiment. At collection, fresh semen evaluations showed that winter (as opposed to spring and summer) was associated with low sexual behaviour, small volumes of spermatozoa and gel, high sperm concentration and lower motility. The high ejaculation frequency yielded a decreased volume, concentration of sp...
Fertility of stallion semen frozen in 0.5-ml straws. Semen of 2 pony stallions was frozen by 2 methods in 0.5 ml PVC straws. The fertility of the frozen-thawed semen was evaluated by inseminating 60 mares during 69 oestrous cycles. An overall single cycle pregnancy rate of 55% was achieved. Freezing method, stallion, insemination during steroid-synchronized oestrus or insemination only every 2nd day during oestrus did not significantly influence pregnancy rates. Pregnancy rates were significantly improved from a mean 44% to a mean 73% when the mean number of progressively motile spermatozoa per insemination was increased from 175 x 10(6) to 249 ...
Use of different nonglycolysable sugars to maintain stallion sperm viability when frozen or stored at 37 degrees C and 5 degrees C in a bovine serum albumin medium. Bovine serum albumin (BSA) diluents containing lactose, raffinose or sucrose were not different (P greater than 0.05) in their ability to maintain stallion sperm viability, as determined by percentage motile spermatozoa (PMS) and their rate of forward movement (RFM), when stored at 37 or 5 degrees C for 24 h. These diluents did promote a higher (P greater than 0.05) PMS and RFM, when compared with BSA diluents containing arabinose or galactose. The BSA-arabinose and BSA-galactose diluents did not differ (P less than 0.05) in their ability to support sperm viability and were detrimental to sper...
Deep freezing of horse embryos. Fourteen horse embryos recovered non-surgically on Days 6-8 after ovulation (Day 0) were cooled slowly to - 35 degrees C (7 embryos) or - 40 degrees C (7 embryos) and stored in liquid nitrogen (- 196 degrees C) for 4-98 days. Surgical transfer of the thawed embryos to unmated recipient mares that had ovulated - 2 to + 1 days with respect to the embryo donors resulted initially in the establishment of 4 conceptuses. However, only one mare maintained her pregnancy to term.
An investigation on the use of cryosurgery for treatment of bone spavin, splint, and fractured splint bone injuries in standardbred horses. Bone spavin, splint, and fractured splint bone injuries have been treated with varying methodologies at Wheatley Hall Farm Equine Clinic. Cryosurgery is the most successful. With cryosurgery the small, pain-producing afferent C fibers are destroyed, and painful neuromas do not return. Injured sites were cryosurgically treated with liquid nitrogen for a double freeze-thaw period of 45 sec. 5 sec, 45 sec. Before and after treatment comparisons were conducted on study standardbreds. In all three injury groups, results showed that the standardbreds tended to race as well or with improved times and...
Effects of cryotherapy on the palmar and plantar digital nerves in the horse. The duration of anesthetic effect and the histopathologic changes resulting from a controlled freeze of the palmar and plantar digital nerves in the horse were evaluated. Two techniques were compared: (i) nerves were frozen by direct application of the cryoprobe after surgical exposure and (ii) nerves were frozen by percutaneous application of the cryoprobe to the overlying skin. Return of skin sensation and ability to detect a stimulus were used to determine return of nerve function. The duration of anesthetic effect was significantly (P less than 0.005) longer for nerves frozen after surgica...
Equine plasma banking: collection by exsanguination. A procedure was developed for the collection, preparation, storage, and administration of equine plasma. The technique involved exsanguination of anesthetized donor horses via carotid artery catheterization with a large-bore cannula. Blood was collected into plastic bags, allowed to settle by gravity, then transferred into storage bags and frozen. These were quickly thawed when needed.
Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm mo...
Horse red blood cells frozen with 20% (w/v) glycerol and stored at -150 C for five years. When equine RBC were frozen with 20% (w/v) glycerol and stored at -150 C for as long as 5 years, there were no adverse effects on freeze-thaw or freeze-thaw-wash recovery or oxygen transport function. The manner in which the glycerol was added to, and removed from, the equine RBC was shown to be an important consideration in ensuring optimal freeze-thaw-wash recovery values.
Fertility of frozen equine semen. Semen of 16 stallions collected by the fractionated method and frozen in liquid nitrogen was used to inseminate 175 mares of different ages and in various reproductive conditions. Pregnancy was recorded in 91 mares of which 72 delivered a foal. Pregnancy followed by resorption occurred in another 10 mares and 9 aborted. The best results were obtained in the young primiparous and in older mares inseminated in the oestrous cycle that followed the post-partum oestrus. Overall, 64% of mares became pregnant and 56% gave birth to a living foal. The highest occurrence of fetal death and resorption we...
An investigation of sperm migration into the oviducts of the mare. A total of 23 mares were inseminated once within 0-6 h after clinical detection of ovulation, 14 with fresh and 9 with deep-frozen semen containing 0.1 x 10(9) to 4.7 x 10(9) motile spermatozoa. Within these two groups, the mares were slaughtered 2, 4 or 6 h after insemination and their genital tracts removed. The utero-tubal junction, isthmus and ampulla ipsilateral to the ovary in which ovulation occurred were flushed separately for sperm recovery. In 1 or 2 mares of each group, the uterine horn and corpus uteri, the cervix and vagina were also flushed. Tissue samples were collected from the...
Effect of glycerol on motility, viability, extracellular aspartate aminotransferase release and fertility of stallion semen before and after freezing. The effect of different glycerol concentrations (0 to 5.3 per cent) on motility, viability and aspartate aminotransferase (AST) release of stallion spermatozoa was studied before and after deep-freezing. Addition of glycerol to a TRIS-fructose-egg yolk diluent used to extend stallion semen had no effect on motility and viability of spermatozoa and it did not increase AST release. Inclusion of glycerol in the extender only partially preserved the motility and viability of stallion semen during deep-freezing. A fertility trial revealed that concentrating stallion semen by centrifugation, followe...
A freeze-thaw method for concentrating plasma and serum for treatment of hypogammaglobulinaemia. Components of plasma or serum, including immunoglobulins, were concentrated two-fold by freezing then collecting 40-50% of the initial volume during thawing. This concentrated plasma (or serum) was administered intravenously to treat hypogammaglobulinaemic foals and calves. An adaptation of this method suitable for field use is described.