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.
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. ...
Bermudez V, Miller RB, Johnson W, Rosendal S, Ruhnke L.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.
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...
Welsch U, Buchheim W, Schumacher U, Schinko I, Patton S.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...
Kosiniak K, Bittmar A.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...
Müller Z.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...
Wilson JM, Caceci T, Potter GD, Kraemer DC.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...
Magistrini M, Chanteloube P, Palmer E.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...
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 ...
Arns MJ, Webb GW, Kreider JL, Potter GD, Evans JW.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...
Czlonkowska M, Boyle MS, Allen WR.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.
McKibbin LS, Paraschak DM.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...
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...
Eicker SW, Ainsworth DM.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.
Cochran JD, Amann RP, Froman DP, Pickett BW.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...
Valeri CR, Valeri DA, Gray A, Contreras TJ, Lindberg JR.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.
Müller Z.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...
Bader H.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...
Guay P, Rondeau M, Boucher S.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...
Thomas KW, Pemberton DH.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.
Rifkind JM.The oxidation of horse hemoglobin by Cu(II) has been followed by the changes in the electron spin resonance spectra of copper. By stopped-flow and freeze-quenching techniques, it is shown that the second-order rate constant for the binding of Cu(II) to hemoglobin is greater than 5 X 10(5) mol-1 s-1 and the apparent first-order rate for the reduction of Cu(II) to Cu(I) is 0.051 s-1. It is also shown that the binding of Cu(II) to hemoglobin is followed by an alteration of the Cu(II) spectrum, decreasing the g values. This process has an apparent rate constant of 17 s-1 and presumably involves a ...
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...
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. ...
Matsukawa K, Akagi S, Adachi N, Sato F, Hasegawa T, Takahashi S.In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates...
van Heule M, Verstraete M, Blockx Z, De Blende P, Dini P, Daels P.Cooled storage of semen after thawing can expand the use of frozen semen, providing the possibility of thawing and evaluating the semen at the storage site and subsequently shipping the semen. Our objectives were (1) to examine the motility and viability of frozen-thawed semen after cooled storage and (2) to compare two cooled-storage protocols for frozen-thawed semen. The samples (n = 31) were either placed immediately in a passive cooling box for 8 or 24 hours (CB) or placed in a refrigerator at 4°C for 30 minutes and then transferred to a passive cooling box (REF). Total and progressive ...
Álvarez C, Luño V, González N, Guerra P, Gil L.This study aimed to evaluate the addition of mare colostrum in stallion freezing extenders to improve sperm quality. First, colostrum samples were collected from four mares after the foal's birth and their composition was determined. Ejaculates were collected from nine fertile stallions. Sperm samples were pooled, diluted, and cryopreserved into three experimental extender groups: Lactose-based extender supplemented with mare colostrum (20%), lactose-based extender supplemented with egg yolk (20%), and BotuCrio. The quality of the post-thaw semen samples were evaluated assessing sperm motility...
Catalán J, Llavanera M, Bonilla-Correal S, Papas M, Gacem S, Rodríguez-Gil JE, Yeste M, Miró J.The aim of this study was to evaluate whether red-light stimulation increases the longevity and resilience of cryopreserved stallion sperm to withstand post-thaw incubation for 120 min. Sixteen frozen straws of 0.5 mL from eight stallions were used. Samples were cryopreserved, thawed through incubation at 38 °C for 30 s and divided into the control and samples exposed to red-light using a triple LED photo-activation system (wavelength: 620-630 nm). Three irradiation protocols consisting of different light-dark-light intervals (1-1-1, 2-2-2 and 3-3-3 min) were tested. Sperm quality param...
Alamaary MS, Haron AW, Ali M, Hiew MWH, Adamu L, Peter ID.Different types of extenders have a variety of components which show the tolerance effect on sperm protection during freezing procedures. In the present study, we have examined the impact of the extenders HF-20 and Tris, which were locally manufactured, and they are competing with commercial extenders INRA Freeze® (IMV Technologies, France) and EquiPlus Freeze® (Minitube, Germany) on the quality of horses frozen semen. Methods: A total of 15 ejaculates from three healthy stallions were collected and cryopreserved in the same environment. Each semen sample collected was divided into four equa...
Hernández-Avilés C, Ramírez-Agámez L, Varner DD, Love CC.The current study determined the effect of the egg-yolk (phospholipid source) level (egg yolk [20% EY] vs. skim-milk + egg yolk [SM + 2% EY]), cryoprotectant (glycerol [Gly] vs. glycerol + methylformamide [Gly + MF]), and pre-freeze cooling rate (-0.1 vs. -1 vs. -5 °C/min) on post-thaw stallion sperm quality. In Experiment 1, ejaculates (n = 27) from 9 stallions (3 ejaculates each) with varied sperm quality (High, Average, or Low) were frozen in EY-Gly, SMEY-Gly, EY-Gly + MF, or SMEY-Gly + MF extenders. Sperm in each group were cooled from 22° to 5°C using either -0.1 °C/min or -1 ...
Bahrami A, Divar MR, Azari M, Kafi M.In the present study, we aimed to evaluate the possible protective effects of the nicotinic acid (NA) at three concentrations (10, 20, and 40 mM) on the equine cooled and frozen-thawed spermatozoa quality markers including viability, plasma membrane or acrosome integrity, DNA fragmentation, lipid peroxidation, and total oxidant levels. We also evaluated the effects of NA on preservation of the post-thaw sperm quality after 6 hours of cold storage before freezing. Five stallions were used for semen collections. The current experiment was repeated six times using pooled semen samples from two ...
Fayrer-Hosken R, Stanley A, Hill N, Heusner G, Christian M, De La Fuente R, Baumann C, Jones L.The cellular effects of tall fescue grass-associated toxic ergot alkaloids on stallion sperm and colt testicular tissue were evaluated. This was a continuation of an initial experiment where the effects of toxic ergot alkaloids on the stallion spermiogram were investigated. The only spermiogram parameter in exposed stallions that was affected by the toxic ergot alkaloids was a decreased gel-free volume of the ejaculate. This study examined the effect of toxic ergot alkaloids on chilling and freezing of the stallion sperm cells. The effect of toxic ergot alkaloids on chilled extended sperm cell...
Hannan MA, Haneda S, Murata K, Takeuchi S, Cheong SH, Nambo Y.Until now, there have been no reports of foals born through embryo transfer after artificial insemination using frozen semen in Japan. The aims of this study were to develop a riding crossbred horse and evaluate the prospects of embryo transfer technology in multiplying horse population. In both donor and recipient mares, luteolysis was induced by the administration of 0.1 mg Cloprostenol to synchronize the onset of estrus, and ovulation was induced by administering 2000 IU human chorionic gonadotropin (hCG) or 0.75 mg Deslorelin. Frozen semen from an Irish Connemara pony stallion was used to ...
Ball BA, Vo A.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...
Güvenc K, Reilas T, Katila T.Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen co...
Scherzer J, Davis C, Hurley DJ.The major difficulty in providing the benefits of embryo cryopreservation for equine agriculture is the mismatch between the optimal embryo age for collection from the mare (7-8 days after ovulation was detected) and the optimal age for freezing under current methods (6.5 days after ovulation). To overcome this limitation, we tested a method to enhance penetration of cryopreservative across the capsule and trophoblast of day 7 and 8 embryos combined with rapid freezing by vitrification. Six small embryos (<300 μm in diameter) were collected on day 6-7 after ovulation and twelve larger embryos...
Huber D, Amsler E, Vidondo B, Kaeser R, Wespi B, Sieme H, Burger D.There exist differences in the reproductive behavior of stallions and mares under free-running and domestic in-hand breeding conditions. Contrary to artificial insemination programs, a stallion mates a mare multiple times per estrus under natural conditions. The objective of this study was to determine if multiple periovulatory artificial inseminations (MI), four times in two different time intervals instead of two, would result in increased pregnancy outcome or higher incidences of breeding induced endometritis. Methods: Eighty-two estrous mares were allocated randomly to one of three experim...
Consuegra C, Crespo F, Dorado J, Diaz-Jimenez M, Pereira B, Sánchez-Calabuig MJ, Beltrán-Breña P, Pérez-Cerezales S, Rizos D, Hidalgo M.The aim of this study was to evaluate the fertilizing capacity of frozen or vitrified stallion sperm after assessing different warming procedures. In Experiment 1, different warming procedures were compared after sperm vitrification: immersion in extender at 43 °C (C), or in a water bath at 37 °C/30 s (W37), 43 °C/10 s (W43) or 60 °C/5 s (W60). With the W60 treatment, there were greater values (P < 0.05) for VCL (83.93 ± 3.6 μm/s) and ALH (3.00 ± 0.2 μm) than freezing and with the C group, and greater values (P < 0.001) for PM (35.33 ± 2.5 %) than with the W43 treatment. In Experiment...
Blommaert D, Franck T, Donnay I, Lejeune JP, Detilleux J, Serteyn D.The aim of this work was to completely replace the egg yolk a classical diluent for freezing equine semen by a cyclodextrin-cholesterol complex. At the same time, the reduction in the glycerol content used for cryopreservation and the incubation time between sperm and the freezing media were evaluated. Horse ejaculates were frozen with four different freezing extenders: a frozen reference medium (IF) containing egg yolk and 2.5% glycerol and media without egg yolk but supplemented with 1.5 mg 2-hydroxypropyl-beta-cyclodextrin cholesterol (HPβCD-C) complex and containing either 1% (G1), 2% (G...
de Andrade AF, Zaffalon FG, Celeghini EC, Nascimento J, Tarragó OF, Martins SM, Alonso MA, Arruda RP.Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autol...
Vieira LA, Gadea J, García-Vázquez FA, Avilés-López K, Matás C.After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4°C up to 96h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing-tha...
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...
Gonzalez-Castro RA, Trentin JM, Carnevale EM, Graham JK.We examined the effects of different freezing extenders, cryoprotectant agents (CPA) and initial thawing temperatures for preparing doses of refrozen stallion sperm for intracytoplasmic sperm injection (ICSI). Single ejaculates, from twelve stallions, were frozen in lactose-EDTA-egg yolk extender (LE) with 5% glycerol. In experiment 1, sperm were initially thawed to 5 °C or 37 °C, before being diluted in LE or skim milk-egg yolk extender (SMEY) containing either 5% glycerol (GLY), 5% methylformamide (MF) or 5% of a combination of both (GMF). In experiment 2, frozen sperm were initially t...
Albrizio M, Moramarco AM, Nicassio M, Micera E, Zarrilli A, Lacalandra GM.It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level fo...
Ferreira-Silva JC, Basto SRL, Moura MT, Rocha JM, Freitas Neto LM, Santos Filho JP, Silva Filho ML, Oliveira MAL.The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes ...
Rosa MVD, Rosa M, Botteon PTL.This study aimed to evaluate the use of equine amniotic membrane (EAM), frozen indirectly using liquid nitrogen and stored between -10° and -24°C, in the treatment of equine skin lesions. Six healthy female horses, aged 3-10 years, were included in this study. EAM was collected from previously evaluated healthy parturient mares. Wounds were surgically created at the distal ends of the forelimbs. One limb was chosen for treatment, and the contralateral limb was chosen as the control. Pain sensitivity, presence of granulation tissue, secretions, and bleeding after debridement during cleaning w...
Arns MJ, Webb GW, Kreider JL, Potter GD, Evans JW.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...
Govaere JL, Hoogewijs MK, De Schauwer C, De Vliegher S, Van Soom A, Duchateau L, de Kruif A.Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen-thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly de...
Heiskanen ML, Hilden L, Hyyppä S, Kangasniemi A, Pirhonen A, Mäenpää PH.The first (1 to 3) sperm-rich fractions of the ejaculate were collected from 4 stallions using an open-ended vagina. The volume of the collected fractions was 12 +/- 8 ml with a density of 475 +/- 200 million spermatozoa/ml. Before freezing, the semen was diluted with a skim-milk based extender 1:1 to 1:8 (volume of semen: volume of extender), depending on the initial sperm concentration to achieve a final concentration of 100 million/ml. The total number of spermatozoa in an insemination dose ranged from 0.7 to 1 billion spermatozoa. Within 12 h after ovulation, 48 mares were inseminated in 7...
Scherzer J, Fayrer-Hosken RA, Ray L, Hurley DJ, Heusner GL.Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non-surgical embryo recovery and transfer are well-established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstratio...
Heise A, Kähn W, Volkmann DH, Thompson PN, Gerber D.The use of epididymal stallion spermatozoa for routine artificial insemination can secure easy future use of valuable genetics after unforeseen death or injury of a valuable stallion. The aims of this study were to (1) directly compare pregnancy rates for fresh and frozen-thawed stallion epididymal and ejaculated spermatozoa after conventional artificial insemination and (2) to investigate the effect of seminal plasma on the fertility of epididymal spermatozoa after insemination. Twenty-one mares were randomly assigned to three stallions. Mares were inseminated at five consecutive oestrous per...
Ebel F, Vallejos A, Gajardo G, Ulloa O, Clavel E, Rodríguez-Gil JE, Ramírez-Reveco A.The aim of this study was to evaluate seasonal changes in basic parameters of sperm quality and freezability behaviour of ejaculates from 10 fertile heavy draft stallions. A total of 140 ejaculates were collected, processed and evaluated during both the breeding (September-November) and non-breeding seasons (April-June). Fresh semen was evaluated for volume, concentration, total spermatozoa per ejaculate, plasma membrane integrity and total sperm motility. Cryopreserved samples were evaluated for plasma membrane integrity and sperm motility by the CASA system, and for the freezability index (F...
