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.
Continuous in vitro cultivation of erythrocytic stages of Babesia equi. The protozoan parasite Babesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37 degrees C. Parasites were detected after 2 days in culture. When the percentage of parasitized erythrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosp...
Large equine blastocysts are damaged by vitrification procedures. Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters ( 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts w...
[Embryo transfer in horses–current status and future perspectives]. Although foals born after embryo transfer are eligible for registration in the majority of horse breeds, application of embryo transfer is still rare. This is mainly due to the lack of a possibility for superovulation. Uterine stage embryos can be recovered by a non-surgical flushing technique. Transfer can be accomplished by non-surgical as well as surgical methods. In contrast to the situation in cattle, most related technologies are scarcely available. Methods of cryopreservation as well as bisection of embryos are hampered by the fact that suitable embryos (morula) can be collected from th...
Recent developments in cryopreservation of stallion semen with special emphasis on thawing procedure using thermal hysteresis proteins. This research study explores the process of cryopreservation of stallion semen, focusing on improving the thawing procedures using thermal hysteresis proteins (THPs) from Antarctic and Arctic fish in order to […]
In vitro viability of cryopreserved equine embryos following different freezing protocols. The main objective of this study was to evaluate two freezing protocols and the effect of agar embedding on survival of day 6.5 equine embryos. A total of 133 embryos were used, in one group (n = 51), embryos were first embedded in agar before the freezing protocol was started. A freezing protocol to -30 degrees C or -33 degrees C was used before plunging embryos into liquid nitrogen (LN2). The embryos were thawed in water at 37 degrees C, evaluated and placed in culture. After 24 h culture, the embryos were evaluated for their morphology and development. No differences were observed between e...
Effect of seminal plasma on motion characteristics of epididymal and ejaculated stallion spermatozoa during storage at 5 degrees C. The objective of this experiment was to examine the effect of seminal plasma on motion characteristics of epididymal and ejaculated equine spermatozoa during storage at 5 degrees C. Epididymal spermatozoa were flushed with either seminal plasma or a skim milk-glucose extender. Ejaculated spermatozoa were collected with extender added 10 minutes after semen collection and addition of extender during ejaculation by placing 50 ml extender in the collection bottle. Semen samples were centrifuged and resuspended with a skim milk-glucose extender containing seminal plasma (0, 5 and 25%; v/v), prepar...
Babesia equi erythrocytic stage continuously cultured in an enriched medium. Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 1...
Pregnancies following transfer of equine embryos cryopreserved by vitrification. The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml str...
Post-thaw motility and longevity of motility of imipramine-induced ejaculates of pony stallions. Imipramine-induced ex copula ejaculates (11) and fractionated in copula ejaculates were collected from each of 5 pony stallions for freezing in 5-ml straws (6), using a modified Kenney glucose skim-milk extender (2). Initial post-thaw total and progressive motilities and daily post-thaw total and progressive motilities, as well as the number of days to reach 0 progressively motile spermatozoa, were also similar for the 2 methods of collection. The percentage of morphologically normal spermatozoa both before freezing and after thawing were also similar for in copula and ex copula ejaculates. Co...
Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing. The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoa...
Cryopreservation of equine oocytes by 2-step freezing. Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for...
[Cryopreservation trial with semen of purebred Arabian and Haflinger stallions in the Turkish national stud in Karacabey]. Within a German-Turkish university partnership deep freezing preservation of stallion semen was performed as a part project of the cooperation contract. In this study a modification of the introduced Makrotüb method was used for semen freezing. The investigated characteristics of fresh semen of the Arab stallions were in the normal range cited in the international literature. However, the semen data obtained from the Haflinger stallions were markedly and partially significantly in lower range than measured for the Arab stallions. This may reflect an incomplete adaptation process of the import...
Quantitative analysis of morphological modifications of day 6.5 horse embryos after cryopreservation: differential effects on inner cell mass and trophoblast cells. Sixteen embryos were recovered nonsurgically at day 6.5 after induced ovulation from Welsh pony mares and were evaluated for cellular changes that occur because of exposure to the cryoprotectant with or without the freeze and thaw process. Day 6.5 horse embryos were either (i) frozen and thawed using glycerol as cryoprotectant (n = 6), (ii) given only the glycerol treatment (n = 5), or (iii) washed in phosphate-buffered saline (PBS) the same number of times as in the glycerol treatment (n = 5). After treatments, embryos were incubated in Minimum Essential Medium (MEM), supplemented with BSA, g...
Culture confirmation of the carrier status of Babesia caballi-infected horses. Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.
Isolation, propagation, and cryopreservation of equine articular chondrocytes. Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffere...
Artificial insemination and preservation of semen. Artificial insemination is an effective technique for improving utilization of stallions in breeding programs. When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable. When AI techniques are employed for mares and stallions with marginal fertility, pregnancy rates may be improved in comparison with natural mating. Preservation of stallion semen in the liquid or frozen state reduces the costs and potential health hazards incurred by transporting mares and provides easier access to genetic material that may otherwise be unavailable. Acceptable preg...
The role of selected biochemical components of equine seminal plasma in determining suitability for deep-freezing. Experiments conducted on the freezability of 400 ejaculates collected from 64 stallions demonstrate the possibility of predicting the semen's ability to withstand the freezing/thawing process. If the sperm concentration, AspAT activity and total protein content in the seminal plasma of raw ejaculates are determined before freezing, the effects of freezing may be forecast in about 80% of the ejaculates.
Incorporation of fresh and cryopreserved bone in osteochondral autografts in the horse. The structural integrity of subchondral bone in fresh and frozen osteochondral autografts was investigated at month 3 in 10 horses. Two osteochondral autografts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. Grafts were frozen in 7.5% DMSO. After 14 days, the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifles. A fresh graft from the right hock was implanted in each left stifle. To control for the effects of surgery, a fresh graft was transferred from the right stifle to the le...
Heterotopic transfer of fresh and cryopreserved autogenous articular cartilage in the horse. Two 10 mm thick osteochondral grafts were harvested from the lateral aspect of the lateral trochlear ridge of the left talus in each of 10 anesthetized horses. The grafts were frozen in a 7.5% DMSO solution and stored in liquid nitrogen. The horses were anesthetized again on day 14 and the thawed grafts were press-fitted into drill holes in the trochlear ridges of the right stifle. A fresh graft was transferred from the right hock to the left stifle. To control for the effects of surgery, another fresh graft was transferred from the right stifle to the left stifle. The result was two grafts in...
Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa. Slow-cooled stallion spermatozoa, with and without seminal plasma removed by centrifugation, were diluted in Kenney's extender (KE) containing nonfat dry skim milk with glucose and antibiotics or in KE supplemented by adding a modified high-potassium Tyrode's medium (KMT). Four ejaculates from each of four stallions were collected and divided factorially across these four treatments. Percentage of motile sperm, path velocity, and linearity immediately after treatment (0 h) and after storage at 4 degrees C for 24, 48, and 72 h were evaluated objectively by use of a HTM-2030 sperm motility analy...
Cryoglobulinemia in a horse. Cryoglobulin was isolated from a horse which had glomerulo-nephritis and a history of swelling and skin ulcers of the limbs in the winter. The isolated cryoglobulin showed a single peak on a gel permeation chromatography column with an apparent molecular mass (Mr) of 180,000 which could be divided into two gamma bands by cellulose acetate electrophoresis. Immunoelectrophoretic analysis revealed that the cryoglobulin formed two precipitation lines with anti-horse IgG. Spur formation was observed when the cryoglobulin and the IgG purified from a normal healthy horse were cross-reacted with anti-...
Penetration of frozen-thawed, zona-free hamster oocytes by fresh and slow-cooled stallion spermatozoa. A method for preparing stored unfrozen stallion spermatozoa for the zona-free hamster oocyte penetration test (HOPT) and a subsequent comparison of fresh and stored sperm by the HOPT were evaluated. In Experiment 1, sperm from 4 stallion ejaculates, cooled to 4 degrees C and stored for 24 h, were treated with 60, 90 and 120 microM of dilauroylphosphatidyl-choline (PC12) liposomes to initiate the acrosome reaction. The percentage of motile and acrosome-reacted (AR) sperm were recorded after 8, 15 and 30 min of incubation at 39 degrees C, by automated image analysis. Liposome concentration did n...
The effect of cryopreservation on the metabolic activity of day-6.5 horse embryos. The decrease in embryo viability caused by cryopreservation may be due, in part, to metabolic disturbances. To determine the effect of cryopreservation on metabolism, Day -6.5 horse embryos were either frozen and thawed using glycerol as the cryoprotectant, given only the glycerol treatment or washed an equal number of times in phosphate buffered saline (PBS). Before and after treatment, individual embryos were incubated with L-[14C(U)]-glutamine, to measure Krebs cycle activity, and D-[5-3H]-glucose, to measure Embden-Meyerhof pathway activity. Before treatment, glucose metabolism ranged from...
Use of concanavalin A for coating the membranes of stallion spermatozoa. Semen from three ejaculates from each of 4 stallions was frozen in liquid nitrogen. Morphology was evaluated by coating the spermatozoa with fluorescein-labelled Concanavalin A (FITC-ConA2) and motility was measured by computer-assisted image analysis. Coating was performed at each step of the freezing procedure (dilution, cooling, addition of glycerol and freeze-thawing) and observations were made after each step, to evaluate changes, or after subsequent steps, to determine protection provided by the coating method. All the parameters showed progressive changes during the freezing procedure. ...
[Successful use of deep-frozen stallion sperm after 23 years of storage at -196 degrees C]. A reduction in the motility of the spermatozoa in stallion semen stored in pellet form for 23 years in liquid nitrogen at -196 degrees C could not be seen after thawing. The insemination of a mare with this semen resulted in a normal pregnancy. A normally developed, healthy male foal was born after a gestational period of 321 days.