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.
Hochi S, Fujimoto T, Choi YH, Braun J, Oguri N.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...
Tekin N, Yurdaydin N, Klug E, Daskin A, Keskin O, Kücük H.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...
O'Rielly JL.The IgG concentration of plasma from 13 mares was measured by radial immunodiffusion when fresh and after storage at -4 degrees C and thawing by 3 methods. There was no significant (P > 0.05) reduction in the IgG concentration when plasma was thawed over 6 hours at 22 degrees C (1352.9 +/- 101.6 mg/dL) (mean +/- SEM) compared with the fresh sample (1369.5 +/- 88.1 mg/dL). In contrast, there was a significant (P < 0.05) reduction in the IgG concentration of plasma that was rapidly thawed at 57 degrees C over 50 minutes (1142.9 +/- 66.2 mg/dL), or placed in a microwave oven for 20 to 25 minutes ...
Horney BS, Honor DJ, MacKenzie A, Burton S.Serum sorbitol dehydrogenase (SDH) activities in 10 cows and nine horses were measured using an automated clinical analyzer. The serum samples were divided into aliquots that were stored at room temperature (21 degrees C), refrigerated (0-5 degrees C), or frozen (-30 degrees C). The stability of the SDH activity was monitored at various intervals. SDH activity in bovine sera remained stable for at least 5 hours at room temperature, 24 hours refrigerated, and 72 hours frozen without any significant (p < 0.05) differences from the initial serum values. In equine sera, SDH activity remained st...
Rojas G, Vargas M, Robles A, Gutiérrez JM.Twenty batches of polyvalent antivenom produced at the Instituto Clodomiro Picado were analyzed for turbidity, both before and after freezing-thawing and lyophilization. Eight batches became turbid upon freezing-thawing, and this change correlated with high levels of cholesterol, triglycerides and lipoproteins, especially beta-lipoprotein. Since normal horse serum does not become turbid after freezing-thawing, despite the fact that it has high lipoprotein levels, the possibility was raised that phenol, used as a preservative during serum fractionation, might affect lipoproteins, inducing the a...
Craig AM, Blythe LL, Rowe KE, Lassen ED, Barrington R, Walker KC.Recent evidence concerning the pathogenesis of equine degenerative myeloencephalopathy indicated that low blood alpha-tocopherol values are a factor in the disease process. Variables that could be introduced by a veterinarian procuring, transporting, or storing samples were evaluated for effects on alpha-tocopherol concentration in equine blood. These variables included temperature; light; exposure to the rubber stopper of the evacuated blood collection tube; hemolysis; duration of freezing time, with and without nitrogen blanketing; and repeated freeze/thaw cycles. It was found that hemolysis...
Parks JE, Lynch DV.Composition and thermotropic phase behavior of sperm membrane lipids from species ranging in sensitivity to cold shock were determined. Lipids from whole sperm and sperm plasma membrane were fractionated into neutral lipid, glycolipid, and phospholipid fractions. Compositional analyses were completed for free sterols, phospholipids and phospholipid-bound fatty acids. Phase transition temperatures were determined for phospholipid and glycolipid fractions using differential scanning calorimetry. Cholesterol was the major sterol in sperm lipids of all species. Cholesterol to phospholipid molar ra...
Brinsko SP, Varner DD.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...
Bittmar A, Kosiniak K.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.
Borovanský J, Vedralová E, Hach P.Concentration of melanosomes in various tissues has been unknown because of the impracticability of their direct quantification. Using an indirect approach comprising the estimation of melanin both in freeze-dried tissue samples and in isolated melanosomes, we obtained data on the amount of melanosomes in various pigment tissues. The concentrations of melanosomes found in the tissues were relatively high, not only reflecting the dark color of pigment tissues but also explaining their capacity to perform various functions ascribed to the presence of melanin.
Desjardins MR, Hurtig MB, Palmer NC.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...
Desjardins MR, Hurtig MB, Palmer NC.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...
López ML, de Souza W.The presence and distribution of cholesterol in mature and immature epididymal spermatozoa was analyzed using filipin as a cytochemical tool in freeze-fracture replicas and thin section preparations. The polyenic-antibiotic filipin formed complexes with 3, beta -OH sterols, producing characteristic protrusions, or pits, that were heterogeneously distributed in the plasma membrane of stallion spermatozoa, revealing a specific organization in a functionally specialized area of the gamete. The acrosomal region of the sperm head presented a significantly higher density of filipin sterol complexes ...
Samper JC, Hellander JC, Crabo BG.Studies were designed to investigate whether sperm motility determined with a Hamilton-Thorn HTM-2000 motility analyzer (HTM), or the percentage of spermatozoa that passed through glass wool (GW), Sephadex (S), or glass wool/Sephadex (GWS) filters could be used to predict the fertilizing potential of fresh or frozen semen. In the fresh semen study, 10 randomly selected ejaculates from 4 stallions exclusively used for A.I. breeding were assayed during the season. The 521 mares used were inseminated with 500 x 10(6) motile spermatozoa after gynaecological examination every 2 days. In the frozen ...
Padilla AW, Tobback C, Foote RH.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...
Rieger D, Bruyas JF, Lagneaux D, Bézard J, Palmer E.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...
Blanc G, Magistrini M, Palmer E.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. ...
Krause HD, Sieme H, Merkt H, Bader H, Wöckener A.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.
Braun J, Schefels W, Stolla R.In 1970 semen from a Haflinger-stallion was frozen by the pellet method. 18 years later semen samples were used to inseminate 4 mares. Inseminations were performed shortly after ovulation with a total number of motile spermatozoa between 150 and 636 x 10(6), the percentage of motile spermatozoa being 20% to 40%. Three mares conceived after a single insemination, one mare got pregnant after 4 inseminations during 3 oestrous periods. Meanwhile, 3 foals were born and one of the mares is still pregnant. The results demonstrate that long-term storage of frozen semen in liquid nitrogen does not impa...
Truax RE, Powell MD, Montelaro RC, Issel CJ, Newman MJ.A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples b...
Blach EL, Amann RP, Bowen RA, Frantz D.Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender w...
Harris RC, Snow DH, Katz A, Sahlin K.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...
Tai CL, Wang C, Weckman TJ, Popot MA, Woods WE, Yang JM, Blake J, Tai HH, Tobin T.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...
Kloppe LH, Varner DD, Elmore RG, Bretzlaff KN, Shull JW.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. ...
Ecot P, Vidament M, de Mornac A, Perigault K, Clément F, Palmer E.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...
Hatami M, Qasemi-Panahi B, Daghigh Kia H, Moghaddam G, Janmohammadi H.Equine semen cryopreservation is one of the major procedures for the genetic conservation of rare and endangered genotypes. The current study was conducted to evaluate the effect of egg yolk plasma (EYP) enriched with β-carotene as an antioxidant supplement on INRA-96 extender regarding freezing Arabic stallion sperm. For this purpose, β-carotene various concentrations were utilized as a supplementary ingredient in formulating the diets of laying hens. Birds were randomly divided into four groups, fed with 0 (control), 500, 1000 and 2000 mg/kg in a supplemented diet with β-carotene. Subse...
Schulman ML, Harper CK, Bell E, Nel A, Guthrie AJ.The aim of this study was to establish and validate a method to permit microsatellite analysis of DNA profiles obtained from frozen-thawed stallion sperm cells. This would provide reliable and accurate verification of the identification of a semen donor. Ejaculates from 5 pony stallions were collected, processed and frozen in 0.5 ml plastic straws. Aliquots of 100 microl of the frozen-thawed semen thus obtained were either placed directly, or diluted (1:10; 1:100; and 1:1000) and placed on slides of FTA paper. Similarly, blood samples obtained from each of the stallions were placed onto slides...
Miller CD.This manuscript is a review of current protocols, advantages, and disadvantages of breeding mares with frozen-thawed equine semen. Issues affecting pregnancy rates are discussed, including proper mare selection, induction of ovulation, insemination dose, timing of insemination (single-dose versus multiple-dose insemination), methods of insemination (transrectal-guided deep-horn versus hysteroscopic insemination), and post-insemination mare management procedures. In a retrospective analysis of breeding records, a single-dose of frozen-thawed semen was inseminated within 6h post-ovulation; the p...
Rieger D, Bruyas JF, Lagneaux D, Bézard J, Palmer E.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...
Lagneaux D, Pomarici AM, Sattler M, Bruneau B, Duchamp G, Camillo F, Palmer E.Day 6.5 equine embryos (n=30) were frozen in a medium containing glycerol (2.5-10.0%) supplemented with 0, 20 or 100 mmol L-glutamine 1(-1). After thawing, the embryos were tested individually, using 4',6'-diamidino-2-phenylindole (DAPI) staining to evaluate cell death. Three embryos (one frozen at each L-glutamine concentration) were transferred together into individual recipient mares. Pregnancy diagnosis was performed at day 12 (age of embryo). Embryos were collected at day 14 (age of embryo) and were identified by PCR amplified microsatellite analysis. Nine of ten recipient mares that rece...
Bogard F, Bouchet B, Murer S, Filliard JR, Beaumont F, Polidori G.Cold therapy is commonly used to relieve pain and inflammation and to aid in muscle recovery after exercise in human medicine. A number of applications have also been observed in veterinary practice. In this article, a critical evaluation of equine protocol applied with a new commercial concept of equine whole-body cryostimulation (WBC) was made. With this new concept of WBC, the protocol usually utilized for relieving pain and discomfort in humans has been extended to horses. The investigations described herein focus on the reduction of horse skin temperature when applying human WBC protocols...
Gloria A, Carluccio A, Petrizzi L, Noto F, Contri A.Equine spermatozoa from the cauda epididymis were previously collected and frozen, and the fertility was assessed. Most studies were performed on healthy stallions that had undergone routine castration or on the epididymis collected at the abattoir, but there are no studies on the quality of epididymal semen in subjects which have died from colic or which underwent intensive care. The present study was designed to verify whether a severe illness could affect epididymal semen quality and freezability in the stallion. Therefore, epididymal semen characteristics during the freezing process in sta...
Ruiz L, Echegaray A, Lafuente A.The aim of this study was the optimization of the sperm freezing protocols for the Pure Breed Andalusian Horse (AH) stallions. The study was performed in 84 ejaculates from 14 stallions (6 ejaculates per stallion). We examined the effect of individual stallion, centrifugal force and centrifugation extender on post-thaw sperm quality. Neither centrifugal force nor centrifugal extender had any significant effect on post-centrifugation or post-thawing sperm quality. Stallion was the principal source of variation in our experiments, showing individual significant differences (p < 0.05) in all para...
Cunha Dos Santos FC, Morrell JM, Nunes MM, Nogueira CE, Curcio BR, Malschitzky E.Epididymal sperm cryopreservation represents the ultimate option to preserve spermatozoa of valuable stallions. Objective: The study aims to evalute whether single layer centrifugation (SLC) prior to cryopreservation or after post-thawing improves the quality of stallion epididymal sperm. Methods: Epididymal sperms of stallions were harvested (N=20). Sperm samples were subjected to treatments: conventional centrifugation, SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). All samples were cryopreserved, thawed and evaluated. SLC+ were thawed, single layer cenrifuged and resuspende...
Schneider RK, Mayhew IG, Clarke GL.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...
Alvarenga MA, Papa FO, Ramires Neto C.The use of stallion frozen semen minimizes the spread of disease, eliminates geographic barriers, and preserves the genetic material of the animal for an unlimited time. Significant progress on the frozen thawed stallion semen process and consequently fertility has been achieved over the last decade. These improvements not only increased fertility rates but also allowed cryopreservation of semen from "poor freezers." This article reviews traditional steps and new strategies for stallion semen handling and processing that are performed to overcome the deleterious effects of semen preservation a...
Poitras P, Guay P, Vaillancourt D, Zidane N, Bigras-Poulin M.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...
Guimarães T, Lopes G, Pinto M, Silva E, Miranda C, Correia MJ, Damásio L, Thompson G, Rocha A.Freezability of equine semen may be influenced by microorganism population of semen. The objective of this study was to verify the effect of single-layer density gradient centrifugation (SLC) of fresh semen before cryopreservation on semen's microbial load (ML) and sperm cells kinetics after freezing-thawing. For that, one ejaculate was collected from 20 healthy stallions and split into control (C) samples (cryopreserved without previous SLC) and SLC samples (subjected to SLC). Semen cryopreservation was performed according to the same protocol in both groups. Microbial load of each microorgan...
Watson ED, Barbacini S, Berrocal B, Sheerin O, Marchi V, Zavaglia G, Necchi D.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...
Klinc P, Kosec M, Majdic G.The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. Objective: To investigate the influence of glass bead column separation on the freezability of stallion semen. Objective: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. Methods: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard proc...
O'Rielly JL.The IgG concentration of plasma from 13 mares was measured by radial immunodiffusion when fresh and after storage at -4 degrees C and thawing by 3 methods. There was no significant (P > 0.05) reduction in the IgG concentration when plasma was thawed over 6 hours at 22 degrees C (1352.9 +/- 101.6 mg/dL) (mean +/- SEM) compared with the fresh sample (1369.5 +/- 88.1 mg/dL). In contrast, there was a significant (P < 0.05) reduction in the IgG concentration of plasma that was rapidly thawed at 57 degrees C over 50 minutes (1142.9 +/- 66.2 mg/dL), or placed in a microwave oven for 20 to 25 minutes ...
Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM.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...
Hinrichs K, Choi YH.Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embr...
Loomis PR.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...
Ponthier J, Franck T, Detilleux J, Mottart E, Serteyn D, Deleuze S.Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on the impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent, which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty-five straws from different s...
Fernández-Hernández P, García-Marín LJ, Bragado MJ, Domingo A, González-Fernández L, Macías-García B.In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), β-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three s...
Ghallab AM, Shahat AM, Fadl AM, Ayoub MM, Moawad AR.The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5-ml plastic straws. After cooli...
Alghamdi AS, Troedsson MH, Xue JL, Crabo BG.To compare the effect of semen extender and seminal plasma on postthaw motility and filtration through a glass wool-Sephadex (GWS) filter for frozen stallion semen. Methods: 7 stallions from which we collected > or = 3 ejaculates/stallion. Methods: 4 experiments were conducted to evaluate postthaw quality of frozen stallion semen. Kenney extender was compared with glucose-EDTA extender by use of various dilution rates that resulted in differing concentrations of seminal plasma. Stallions known to produce semen with poor postthaw quality were used to investigate whether a particular extender or...
Smits K, Hoogewijs M, Woelders H, Daels P, Van Soom A.Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity...
Squires EL, McCue PM, Vanderwall D.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...
Snoeck PPDN, Pessoa THO, Pereira MGS, Bastos ICL, de Melo MIV.The objective of this study was to compare the BotuCrio extender with the Merk - egg yolk and the INRA 82 modified by the inclusion of acetamide, methyl cellulose and trehalose in substitution of glycerol for freezing equine semen. The semen was diluted after centrifugation to obtain 100 x 10 of sperm/ml in: BotuCrio (control); Merk - egg yolk or INRA 82 modified (Experiment 1). The extended semen was packaged in 0.5 ml straws, cooled and frozen in a freezing machine. The control extender was superior in preserving the motility, VCL, VSL, VAP, LIN, STR and the BCF when compared to the Merk - e...
Volkmann DH, van Zyl D.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 ...
Crowe CA, Ravenhill PJ, Hepburn RJ, Shepherd CH.Historically, artificial insemination (AI) using frozen semen has been perceived to have poorer success rates and be more labour intensive than using chilled semen. A retrospective study was therefore conducted to compare the conception rate achieved by AI between chilled and frozen semen, using fixed time insemination protocols over 2 breeding seasons. Objective: Artificial insemination using chilled semen produces a higher conception rate than that achieved with frozen semen. Methods: Mares (n = 251) were inseminated with either chilled (n = 112) or frozen (n = 139) semen in the 2006 and 200...
Hinrichs K.Methods for holding of oocytes and embryos during shipment as well as for their cryopreservation can greatly aid equine reproductive management. Oocytes can be held at room temperature overnight or at cooler temperatures for two nights without affecting maturation or embryo development after intracytoplasmic sperm injection. In contrast, methods for cryopreservation of equine oocytes that support high rates of embryo development have not yet been established. Equine embryos may be held overnight at temperatures from 5°C to 19°C without reduction in viability, but longer holding periods, or h...
