Semen preservation involves the collection, processing, and storage of stallion semen for future use in artificial insemination. This practice enables the extension of genetic material across geographical boundaries and temporal constraints. The preservation process typically includes semen evaluation, dilution with extenders, cooling, and sometimes cryopreservation. Factors such as semen quality, extender composition, and storage conditions influence the success of preservation. This page compiles peer-reviewed research studies and scholarly articles that explore techniques, challenges, and advancements in the field of equine semen preservation, focusing on optimizing fertility outcomes and extending the reproductive lifespan of stallions.
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...
Blach EL, Amann RP, Bowen RA, Sawyer HR, Hermenet MJ.Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when view...
Bize I, Driscoll DM.Preservation of stallion sperm forward motility was studied using a video recording system in semen diluted with media of different ionic strength and sodium content. After 8 hr of incubation at room temperature, semen diluted in a low ionic strength media containing sucrose displayed 65 +/- 9% motility with 68 +/- 3% of the motile sperm showing forward motility (diameter of head trajectory greater than or equal to 80 microns). In contrast, sperm populations diluted and incubated with a normal ionic strength media containing sodium had 56 +/- 7% motile sperm of which only 36 +/- 7% displayed f...
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. ...
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...
Berghuis GA.The conception rates of semen intended for shipment and those of recently obtained semen are compared in the present paper. Conception rates using recently obtained semen were significantly superior to those obtained with semen intended for shipment. A number of factors to which this difference could be due are briefly discussed.
Varner DD, Blanchard TL, Love CL, Garcia MC, Kenney RM.Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fract...
Francl AT, Amann RP, Squires EL, Pickett BW.The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with inseminatio...
Hansen PJ, Hoggard MP, Rathwell AC.The ability of stallion seminal plasma to modify phagocytosis of spermatozoa and Streptococcus zooepidemicus was examined. Phagocytosis was monitored indirectly as the H2O2 produced by peripheral blood leukocytes after addition of spermatozoa or bacteria. Hydrogen peroxide production after addition of ejaculated spermatozoa was greater (P less than 0.01) than after addition of epididymal sperm. Furthermore, pre-incubation of epididymal sperm with 6.25-50% seminal plasma caused a dose-dependent increase in subsequent H2O2 production by leukocytes (P less than 0.05). In addition, equine serum wa...
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...
Amann RP, Cristanelli MJ, Squires EL.Motility and fertility of frozen-thawed semen differs greatly amongst stallions. Differences in seminal plasma might be one cause of this variation. For 8 ejaculates from each of 17 stallions, seminal plasma was saved at -20 degrees C and spermatozoa were cryopreserved. Based on post-thaw sperm motility, seminal plasma samples from 7 stallions (2 good, 3 variable, 2 poor sperm motility) were selected for measurement of electrolytes, protein content and analysis by sodium dodecylsulphate gel electrophoresis (10% gel, Coomassie blue stain). Variation in seminal plasma was significant (P less tha...
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...
Rath D, Leiding C, Klug E, Krebs HC.Ejaculates were collected at 3-day intervals before, during and after a washing procedure with chlorhexidine (2%). Semen motility and pathology were determined before and after deep-freezing. Blood samples were taken before and within 1 h after washing procedures and then extracted in ether. This was followed by HPL chromatography. Chlorhexidine concentrations in blood and seminal plasma were distinctly higher in the treated stallions than in control groups. Concentrations in the control groups were below the detection limit of the column. Significant correlations between decreasing semen qual...
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 ...
Heiskanen ML, Pirhonen A, Koskinen E, Mäenpää PH.The role of various environmental conditions on sperm motility and ATP content was investigated by incubating raw and washed spermatozoa collected with an open-ended artificial vagina from 10 stallions in various biological and artificial media under different atmospheric conditions. Spermatozoa did not survive for more than 12 h when kept unextended in the original seminal fluid in any circumstances. The most favourable media tested for long-term sperm survival were Kenney's medium or Kenney's medium supplemented with 10 mM-theophylline and 10 mM-Hepes, pH 7.2. Centrifugation and slow cooling...
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...
Timoney PJ, McCollum WH, Roberts AW, Murphy TW.The chronic carrier state was virologically confirmed in 15 thoroughbred stallions naturally infected with equine arteritis virus based on the results of test matings and, or, isolations of the virus from semen. Carrier stallions were shown to shed equine arteritis virus in the semen for at least one to two years. Existence of a short-term or convalescent carrier state was also demonstrated in five additional stallions. The frequency of the long-term carrier state in stallions naturally infected with equine arteritis virus was 35 per cent; it varied considerably between groups of stallions on ...
Garner DL, Pinkel D, Johnson LA, Pace MM.Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained...
Province CA, Amann RP, Pickett BW, Squires EL.Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of can...
Douglas-Hamilton DH, Osol R, Osol G, Driscoll D, Noble H.A field trial of artificial insemination in horses with transproted, chilled semen was conducted using a specially designed container which permitted a controlled, slow initial rate of cooling (-0.3 degrees C/min) and maintenance of a final temperature of 4 degrees -6 degrees C for more than 36 hrs. Forty-six mares in 23 states were inseminated with semen from three German Warmblood stallions standing at stud in Hamilton, Massachusetts. A third-cycle conception rate of 91% was obtained.
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...
Froman DP, Amann RP.The effects of four vaginal lubricants on progressive spermatozoal motility were evaluated. Neat semen was exposed to 0, 5, or 10% (w/v) of H-R, sterile K-Y, nonsterile K-Y or Maxilube lubricating jellies for 10 min at 37 degrees C and then extended to 10x10(6) spermatozoa/ml. Spermatozoal motility was evaluated after 0, 1, 2, 4 and 6 or 8 h of incubation at 37 degrees C. For bovine spermatozoa, sterile K-Y jelly at 10% suppressed motility (P<0.05), but nonsterile K-Y, H-R and Maxilube jellies had no effect. Maxilube was toxic (P<0.01) to canine spermatozoa and is not recommended for use...
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...
Heiskanen ML, Pirhonen A, Koskinen E, Mäenpää PH.The role of various environmental conditions on sperm motility and ATP content was investigated by incubating raw and washed spermatozoa collected with an open-ended artificial vagina from 10 stallions in various biological and artificial media under different atmospheric conditions. Spermatozoa did not survive for more than 12 h when kept unextended in the original seminal fluid in any circumstances. The most favourable media tested for long-term sperm survival were Kenney's medium or Kenney's medium supplemented with 10 mM-theophylline and 10 mM-Hepes, pH 7.2. Centrifugation and slow cooling...
Elkhawagah AR, Nervo T, Poletto M, Martino NA, Gallo D, Bertero A, Vincenti L.The aim of the study was to ascertain effects of different concentrations of relaxin added to extender medium during the pre-freezing incubation periods on quality variables of stallion frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio freezing medium to a final concentration of 50 × 10 sperm/mL. The diluted semen was divided into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin. The semen samples were transferre...
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...
Meuffels-Barkas J, Wilsher S, Allen WRT, Ververs C, Lueders I.Recent loss of rhinoceros subspecies has renewed interest in using more advanced assisted reproductive technologies (ART) in rhinoceroses and elephants. Currently, only semen collection, semen preservation and artificial insemination (AI) have been used repeatedly with success in these species. Although ovum pick-up (OPU) and intra-cytoplasmic sperm injection (ICSI) have been reported in rhinoceroses, the techniques are not yet optimised. In contrast, multiple ART applications are routinely used in the horse. Since elephant and rhinoceroses share some reproductive features with equids, we post...
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...
Douglas-Hamilton DH, Osol R, Osol G, Driscoll D, Noble H.A field trial of artificial insemination in horses with transproted, chilled semen was conducted using a specially designed container which permitted a controlled, slow initial rate of cooling (-0.3 degrees C/min) and maintenance of a final temperature of 4 degrees -6 degrees C for more than 36 hrs. Forty-six mares in 23 states were inseminated with semen from three German Warmblood stallions standing at stud in Hamilton, Massachusetts. A third-cycle conception rate of 91% was obtained.
Cook NL, Masterson KR, Battaglia D, Beck R, Metcalf ES.Numerous variables affect invitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine invitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1M dimethyl sulfoxid...
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...
Kareskoski M, Venhoranta H, Virtala AM, Katila T.Artificial insemination (AI) with cooled stallion semen has increased markedly during the last decades in all countries, but fertility is often lower than when fresh semen or natural mating is used. The objective of this study was to examine field data (1634 ,cycles 523 Standardbred (SB) mares, 575 Finnhorse (FH) mares, and 90 stallions) using multivariable logistic regression for factors influencing the pregnancy rate (PR) after AI with cooled transported semen from SB and FH stallions. The PR per cycle for the material was 47%: Finnhorses 42% and Standardbreds 53%. When assessed with multiva...
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 ...
Garner DL, Evans KM, Seidel GE.The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some specie...
Province CA, Amann RP, Pickett BW, Squires EL.Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of can...
Delerue M, Breuil MF, Duquesne F, Bayon-Auboyer MH, Amenna-Bernard N, Petry S.Taylorella equigenitalis can be transmitted during artificial insemination. This report describes clinical T. equigenitalis transmission by cryopreserved stallion semen. T. equigenitalis isolates from a mare's vaginal discharge and semen from the same batch of the cryopreserved semen used for the insemination gave identical API ZYM, antibiotic susceptibility, and multilocus sequence typing results (ST-46); furthermore, the multilocus sequence typing lineage ST-46 is known to circulate in the country of semen collection. These results support the need for strict contagious equine metritis scre...
Cuervo-Arango J, Claes AN, Stout TAE.The diameter of embryos recovered from mares on Day 8 after ovulation varies greatly, from as little as 130 μm to as much as 2500 μm. Several factors have been proposed to affect embryo size at recovery, one of which is the type of semen (frozen vs fresh or cooled-transported) used to inseminate the mare. In addition, it has been shown that smaller embryos (<300 μm) recovered on Day 8 are less likely than larger embryos to result in successful pregnancy after transfer. However, whether the actual age of the embryo (interval from fertilization to flushing) in relation to its size also ...
Cocchia N, Pasolini MP, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G, Mancini A.New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: ...
Graham JK.The main advantage to using frozen semen in any breeding program is faster genetic gain for the inherited trait desired. Milk production of dairy cows doubled (from 26,000 to 52,000 kg of milk/cow per year) between 1950 and 1980, because the dairy industry was using semen only from bulls with the greatest genetic potential for milk production. This genetic gain could have been achieved without the use of frozen semen; however, the time required to achieve that same genetic progress would have been lengthened exceedingly. Fertility rates using frozen stallion spermatozoa are not equal to that o...
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...
Padilla AW, Foote RH.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...
Umair M, Claes A, Buijtendorp M, Cuervo-Arango J, Stout TAE, Henning H.Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was we...
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...
Lyle SK, Ferrer MS.The typical dose for insemination into the uterine body of the mare is > 300 x 10(6) progressively motile spermatozoa (PMS) and an insemination dose of > 200 x 10(6) PMS is recommended for frozen-thawed semen. Low-dose insemination techniques allow for a drastic reduction in the numbers of spermatozoa required to achieve pregnancy. Acceptable pregnancy rates can be achieved with doses ranging from 1 to 25 x 10(6) PMS in volumes ranging from 20 to 1000 microL. Two techniques have been described: hysteroscopic insemination and transrectally guided deep horn insemination using a pipette. Similar ...
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...
Morrell JM, Rodriguez-Martinez H, Johannisson A.This study compared the effect on semen quality of different handling methods used in the preparation of stallion semen doses for artificial insemination. The three methods were (i) extending the ejaculate to 30-50 x 10(6)/ml, (ii) single layer centrifugation (SLC) and (iii) sperm washing (centrifugation without a colloid). An additional treatment was to add seminal plasma (SP) in various proportions to some SLC preparations. The resulting samples were evaluated for sperm motility by computer assisted sperm analysis, membrane integrity using the Nucleocounter SP-100 and chromatin integrity by ...
Oliveira SN, Andrade LRP, Silva LFMC, Araujo EAB, Rayashi RM, Segabinazzi LGTM, Alvarenga MA, Dell'Aqua CP, Dell'Aqua JA, Papa FO.Treatments for seminal vesiculitis have poor outcomes in stallions; thus, the development of alternative strategies is warranted. This study aimed to evaluate fractionated semen collection as a method to restore the fertility of stallions diagnosed with seminal vesiculitis. Eighteen ejaculates from six stallions (three ejaculates/stallion) diagnosed with seminal vesiculitis were harvested in fractions, as follows: Fraction A (FA), the first two jets; Fraction B (FB), the third and fourth jets; and Fraction C (FC), the fifth and remaining jets of the ejaculate. All fractions were subject to sta...
Allen WR, Bowen JM, Frank CJ, Jeffcott LB, Rossdale PD.This short review article describes the various techniques currently available for artificial insemination in the horse. The collection and use of raw and extended semen is discussed together with the more recent developments in freezing semen. The expected conception rates with both fresh and frozen semen are quoted. The possible benefits in disease control and stud management are discussed, as well as the difficulties in controlling the use of A.I. from the Breed Registration Authorities point of view.
