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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.
Factors affecting motion characteristics of frozen-thawed stallion spermatozoa. Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, T...
[Reproductive medicine in transition: new developments in embryo transfer]. Successful application of embryo transfer (ET) has become common practice in cattle, horses, sheep, goats and a variety of other species held in captivity. Yet in cattle only has the technique been established commercially. In 1994 more than 100,000 bovine embryos have been transferred in European countries. Important progress in transvaginal ovum pick up (OPU), in vitro production (IVP) and cryopreservation have further improved the applicability of ET. Direct transfer simplifies the procedure considerably allowing individual transfers and eliminating the need of synchronizing recipients. In ...
Effect of various extenders and taurine on survival of stallion sperm cooled to 5 degrees C. Stallion semen was diluted in five different extenders (dimitro-poulus onze (Dimitro's), Kenney's modified tryode (Kenney's), modified INRA82 (INRA82), egg yolk-citrate-taurine (Citrate) and EZ-Mixin) and evaluated for motility after cooling and storage at 5 degrees C for 0, 24, 48, 72 and 96 h. EZ-Mixin extender was used as control while 70 and 100 mM of taurine were added to Dimitro's, Kenney's and INRA82 to study its effect under conditions of storage at 5 degrees C and varying processing modifications. Motility in INRA82 was 57.0, 58.4, 61.1, and 56.1% after 24, 48, 72 and 96 h, respective...
Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. Two bioassays were used to evaluate the interaction of fresh and cryopreserved equine semen with oviductal epithelial cells (OEC) and with the zona pellucida (ZP). Split ejaculates were either stored at room temperature or frozen and thawed. In experiment 1, progressive motility and membrane integrity were evaluated for each treatment. Fluorescent labeled spermatozoa were cocultured with monolayers of OEC for 30 minutes, and the number of sperm attached to OEC was counted by fluorescence microscopy and analysis of digitized images. Motility of spermatozoa attached to OEC was observed at 0.5, 3...
Effect of different protein supplements on motility and plasma membrane integrity of frozen-thawed stallion spermatozoa. Three experiments were conducted to evaluate the effect of different macromolecule components (egg yolk, skim milk, and BSA) in a widely employed extender for cryopreservation of horse semen. Spermatozoal motility (MOT) and the percentage of spermatozoa with an intact plasma membrane (IPM) were evaluated in frozen-thawed samples. In the first experiment (four Draft Horse stallions, four ejaculates each) a standard freezing extender containing 20% whole egg yolk was modified by replacing extender components (glucose-EDTA solution, 11% lactose solution) with an increasing volume of a skim milk d...
Relationship among seminal characteristics, fertility and suitability for semen preservation in draft stallions. Seminal characteristics, fertility and the response to semen preservation (liquid storage and cryopreservation) were evaluated in 4 Draft stallions (Percheron 2, Breton 2). Seminal characteristics (gel-free volume, sperm concentration, sperm morphology, percentage of motile spermatozoa) were assessed in 5 ejaculates from each of the 4 stallions. The fertility of the stallions was calculated retrospectively as the accumulated pregnancy rate over 3 breeding seasons. Five ejaculates from each of the stallions were subjected to liquid storage at 5 degrees C. The percentage of motile spermatozoa (P...
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...
