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.
Kavak A, Johannisson A, Lundeheim N, Rodriguez-Martinez H, Aidnik M, Einarsson S.Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ej...
Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Agüero A.Many theories have been postulated concerning the possible effects of cryopreservation on spermatozoa, including suggestions the freeze-thawing process produces membranes that have greater fluidity and are more fusogenic, thus inducing changes similar to those of capacitation. The main objectives of this study were to determine at what stage of the freeze-thaw process membrane changes occur and whether evaluation with chlortetracycline (CTC) stain could predict the freezability of stallion sperm. Sperm viability and state of capacitation were simultaneously evaluated using CTC and Hoechst 3325...
Brinsko SP, Blanchard TL, Rigby SL, Love CC, Varner DD.The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculat...
Rosati I, Berlinguer F, Bogliolo L, Leoni G, Ledda S, Naitana S.It is clear that, in the horse, there are many weak links in the process of in vitro embryo production; an optimal culture system for equine oocytes does not exist, and related data are conflicting. Therefore, the ability of 3 different culture systems to support embryonic development of ICSI horse oocytes was examined. Oocytes (n = 261) suitable for culture were collected from 55 ovaries and divided, according to cumulus morphology, into 2 categories: expanded cumulus and compacted cumulus. Oocytes with expanded and compacted cumulus were cultured for in vitro maturation in TCM 199 + 10% FCS ...
Devireddy RV, Swanlund DJ, Alghamdi AS, Duoos LA, Troedsson MH, Bischof JC, Roberts KP.The effects of extracellular ice and cryoprotective agents on the measured volumetric shrinkage response and the membrane permeability parameters of equine spermatozoa have been reported previously. The volumetric shrinkage data were obtained using a differential scanning calorimeter technique that was independent of cell shape. The aim of this study was to examine the effects of collection and cooling conditions on the motility and the water transport parameters at subzero temperatures of equine spermatozoa. Stallion semen samples were collected using either a commercial lubricating agent, wh...
Mantovani R, Rora A, Falomo ME, Bailoni L, Vincenti L.The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprote...
Pommer AC, Rutllant J, Meyers SA.Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditio...
Carver DA, Ball BA.Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during...
Schembri MA, Major DA, Suttie JJ, Maxwell WM, Evans G.Chlortetracycline (CTC) fluorescence staining analysis was used to investigate cryopreservation-induced capacitation-like changes in equine spermatozoa. Freshly ejaculated spermatozoa were found to display a high proportion of F pattern cells (uncapacitated; 93.6%) and a lower proportion of B pattern (capacitated; 5.4%) and AR pattern (acrosome-reacted; 1%) cells. Following cryopreservation in modified Kenney's medium, capacitation-like changes were observed. There was a significant increase in the proportion of spermatozoa displaying the B pattern (64.8%; P<0.001) and AR pattern (32.8%; P&...
Chopin JB, Chopin LK, Knott LM, de Kretser DM, Dowsett KF.An 8-year-old mare, with a foal at foot, was inseminated on foal heat with frozen semen, with the resultant pregnancy lost between days 34 and 41. The right ovary developed a large anovulatory follicle that was non-responsive to multiple doses of ovulating agents. The follicle eventually appeared to luteinise, although plasma progesterone concentrations did not reflect this. Another follicle developed, responded to GnRH and resulted in a pregnancy from frozen semen that went to term with a healthy foal. When the mare was examined after foaling, the structure on the right ovary appeared to be a...
Morris LH, Allen WR.The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex-sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex-sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 x 10(6) spe...
Harnal VK, Wildt DE, Bird DM, Monfort SL, Ballou JD.Genome resource banks (GRBs) and assisted reproductive techniques are increasingly recognized as useful tools for the management and conservation of biodiversity, including endangered species. Cryotechnology permits long-term storage of valuable genetic material. Although, the actual application to endangered species management requires technical knowledge about sperm freezing and thawing, a systematic understanding of the quantitative impacts of various germ plasm storage and use scenarios is also mandatory. In this study, various GRB strategies were analyzed using the historical data from th...
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...
Pommer AC, Linfor JJ, Meyers SA.Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong spe...
Lo CC, Thompson JA, Lowry VK, Varner DD.We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as sub...
Champion ZJ, Vickers MH, Gravance CG, Breier BH, Casey PJ.Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are both present in blood plasma and IGF-I has been measured in epididymal fluid and seminal plasma. This study was designed to investigate the direct effects of GH or IGF-I on the motility of mature equine spermatozoa in vitro. We compared the effects of one concentration (100 ng/ml) of recombinant bovine GH (rbGH) and recombinant human IGF-I (rhIGF-I) on motility and motion characteristics of equine spermatozoa over a 24 h period. Motility was maintained longer in spermatozoa treated with either rbGH or rhIGF-I during a 24 h period...
Lindsey AC, Schenk JL, Graham JK, Bruemmer JE, Squires EL.The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sp...
Choi YH, Love CC, Love LB, Varner DD, Brinsko S, Hinrichs K.This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment...
Ball BA, Vo A.The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescenc...
Inagaki M, Kikuchi M, Orino K, Ohnami Y, Watanabe K.Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations ...
Bruemmert JE, Coy RC, Squires EL, Graham JK.Stallion spermatozoa maintain high fertilizing capacity if cooled to 5 degrees C and inseminated within 24 h. However, if spermatozoa are stored for 48 h, fertilizing capacity declines. Therefore, multiple shipments of semen are often required to inseminate mares that remain in estrus for days. Therefore, experiments were designed to determine if adding antioxidants to stallion spermatozoa stored at 5 degrees C for 48 h could maintain motility and fertilizing ability. In the first experiment stallion spermatozoa were incubated in a skim milk (SM) or a skim milk-egg yolk medium in combination w...
Linfor JJ, Meyers SA.Single-cell gel electrophoresis (SCGE), or comet assay, has the ability to detect damage at the single cell level and has not been reported for equine sperm. The ability to detect nuclear damage at the single cell level could aid in the advancement of protocols for optimal semen preservation. The goals of these experiments were to adapt this assay for use with equine sperm and to utilize the assay for determining the integrity of equine sperm DNA following treatments with storage at various decreased temperatures (-20 degrees C and 5 degrees C). Results from experiments in which sperm were fro...
Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MH, Bischof JC, Roberts KP.Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5 degrees C/min and 20 degrees C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 microm a...
Vidament M, Yvon JM, Couty I, Arnaud G, Nguekam-Feugang J, Noue P, Cottron S, Le Tellier A, Noel F, Palmer E, Magistrini M.In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure ...
Batellier F, Vidament M, Fauquant J, Duchamp G, Arnaud G, Yvon JM, Magistrini M.In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in ord...
Metcalf ES.Despite the numerous benefits of having the capability to transport semen internationally, there are serious potential ramifications if that semen is contaminated with a communicable disease. Bacteria: Many commensal bacteria colonize the exterior of the stallion penis and are not regarded as pathogenic. They may be cultured from an ejaculate. Alterations of the normal bacterial flora on the exterior genitalia may cause the growth of opportunistic bacteria such as Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcus zooepidemicus, which, if inseminated, may cause infertility in suscepti...
Lindsey AC, Bruemmer JE, Squires EL.Mares are generally inseminated with 500 million progressively motile fresh sperm and approximately 1 billion total sperms that have been cooled or frozen. Development of techniques for low dose insemination would allow one to increase the number of mares that could be bred, utilize stallions with poor semen quality, extend the use of frozen semen, breed mares with sexed semen and perhaps reduce the incidence of post-breeding endometritis. Three low dose insemination techniques that have been reported include: surgical oviductal insemination, deep uterine insemination and hysteroscopic insemin...
Troedsson MH, Loset K, Alghamdi AM, Dahms B, Crabo BG.Insemination of mares with bacteria-free equine spermatozoa results in an influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen. In vitro studies have demonstrated that equine spermatozoa activate complement, resulting in cleavage of factors C5a and C3b. Since uterine secretion is rich in complement, it is likely that an interaction between spermatozoa and uterine secretion results in C5a-mediated chemotaxis and migration of PMNs into the uterine lumen. Once in the uterine lumen, the PMNs phagocytize bacteria and spermatozoa, which is an important part of sperm elimination from...
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...
Gibb Z, Grupen CG, Maxwell WM, Morris LH.The fertility of sex-sorted, cryopreserved stallion sperm must be improved for the sex-sorting technology to be applied commercially. Objective: To optimise the conditions used to liquid store stallion sperm prior to sex-sorting and assess the fertility of sperm following sex-sorting and cryopreservation. Methods: Both in vitro experiment and randomised controlled trial in healthy, client-owned mares. Methods: Stallion ejaculates (n = 9) were diluted in either a skimmed milk (KMT) or BSA (I-BSA) based media to 25 × 106 sperm/ml directly (+SP25) or washed to remove seminal plasma and diluted t...
Mittelholzer C, Johansson I, Olsson AK, Ronéus M, Klingeborn B, Belák S.Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-P...
Lançoni R, Celeghini ECC, Gonella-Diaza AM, Júnior VG, de Carvalho CPT, Zoca GB, Garcia-Oliveros LN, Batissaco L, Oliveira LZ, de Arruda RP.This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very l...
Kareskoski AM, Palviainen M, Johannisson A, Katila T.For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expressio...
Hernández-Avilés C, Ramírez-Agámez L, Love CC, Friedrich M, Pearson M, Kelley DE, Beckham AMN, Teague SR, LaCaze KA, Brinsko SP, Varner DD.Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were ...
Kareskoski M, Vakkamäki J, Laukkanen K, Palviainen M, Johannisson A, Katila T.Matrix metalloproteinase (MMP)-2 and MMP-9 are gelatinases that take part in several reproductive processes. The aim of this study was to measure levels of MMP-2 and MMP-9 in fractionated stallion ejaculates, and to evaluate the association between these components and semen quality, and sperm longevity during cooled storage. Semen quality were assessed separately for sperm-rich fractions (HIGH), sperm-poor fractions (LOW), and whole ejaculate samples (WE) from 33 stallions. After cooled storage with SP either present or removed, sperm motility and DFI were determined. The relative activity of...
Pérez-Marín CC, Vizuete G, Vazquez-Martinez R, Galisteo JJ.Few studies have been published about cryopreservation and embryo assessment in horses and donkeys. Objective: To evaluate the viability of embryos collected from mares and jennies that were cryopreserved by slow freezing or by vitrification. Methods: Randomised controlled experiment. Methods: Horse (n=19) and donkey (n=16) embryos (≤300 μm) were recovered on days 6.5-7.5 post-ovulation and assigned to control or cryopreservation protocols of slow freezing or vitrification. For slow freezing, 1.5 mol/L ethylene glycol (EG) was used. For vitrification, horse embryos were exposed to 1.4 mol/L...
Serafini R, Varner DD, Bissett W, Blanchard TL, Teague SR, Love CC.The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabil...
Pace MM, Sullivan JJ.Fertilization rate was highest in mares inseminated with frozen semen within 12 hr of ovulation. Foaling rate was improved (P less than 0-05) by increasing the number of motile spermatozoa inseminated from 40 X 10(6) to 80 X 10(6) but was not further improved by increasing the number to 160 X 10(6) or by increasing the frequency of insemination from once to twice daily. The fertilizing capacity of spermatozoa frozen in one of the hydrogen ion extenders studied was dependent upon relative osmotic pressure and method of freezing (ampoules or pellets). Adjusting glycerol concentration from 7% to ...
Mari G, Bucci D, Love CC, Mislei B, Rizzato G, Giaretta E, Merlo B, Spinaci M.The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (...
Álvarez C, Luño V, González N, Gil L.Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted...
Casey PJ, Robertson KR, Liu IK, Espinoza SB, Drobnis EZ.Subfertility in stallions is common, and methodologies are needed to increase the fertility in these animals. In other species, removal of the dead sperm from semen increases the quality and fertility of semen. With horse semen we evaluated 48 combinations of column separation techniques using micro-spin chromatography columns. The greatest improvement in motility was observed with glass wool, whereas glass beads exhibited the greatest recovery of motile sperm. Although centrifugation time did not influence recovery rate or percent motility, a column length of 2 cm was superior for recovery of...
Sieme H, Bonk A, Hamann H, Klug E, Katila T.The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto...
Miller L, Woodward EM, Campos JR, Squires EL, Troedsson M.The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, ...
Assumpção TI, Lançoni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Malmgren L.The storage and transport of cooled, liquid semen is an effective way of facilitating the use of desirable stallions for breeding mares located on distant farms. The Equitainer System is the most widely used transport container and it has been shown that it is possible to ship semen in this container and obtain good conception rates. However, the cost of Equitainers is high, and stud-farms that ship large quantities of semen have tended to rely on cheaper alternatives, even though little documentation exists concerning their reliability, especially under extreme temperature conditions. Two dif...
Squires EL.There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on t...
López ML, Olea N, Retamal CA.Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific d...
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...
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...
Consuegra C, Crespo F, Dorado J, Ortiz I, Diaz-Jimenez M, Pereira B, Hidalgo M.Vitrification of sperm is based on high-speed freezing by direct exposure to liquid nitrogen using non-permeable cryoprotectants, mainly disaccharides; yet, the concentration of cryoprotectants has a species-specific effect on the sperm cell. The aim of this study was to assess different sucrose concentrations for stallion sperm vitrification. Semen samples (n = 9) were collected from three stallions, centrifuged and resuspended to a concentration of 50 × 10 sperm/ml in a base extender (INRA96 + 1% of bovine serum albumin) with three different sucrose concentrations (Molar): 20 mM (S...
Guimarães T, Lopes G, Ferreira P, Leal I, Rocha A.Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material of valuable stallions after emergency castration or unexpected death. For that, testicles and epididymides are generally sent refrigerated to the laboratory. Collection of epididymal spermatozoa is a simple procedure that reduces the volume of the material to be shipped, and may improve the quality of the chilled epididymal sperm cells. In the present study we compared the characteristics of frozen/thawed epididymal spermatozoa after refrigeration of the epididymis or after direct refrigeration of the exte...
Morse-Wolfe B, Bleach E, Kershaw C.Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three f...
Loomis PR, Squires EL.Success with frozen semen requires attention to detail and a basic understanding of the techniques for properly handling and thawing and inseminating frozen semen. Practitioners should also be familiar with strategies used for managing mares for insemination with thawed semen. This manuscript will review those techniques and also present fertility data collected in a commercial setting. Factors that affect pregnancy rates for mares inseminated with frozen-thawed semen such as timing and frequency of insemination were examined for two separate data sets consisting of 332 and 536 mare cycles col...
Jung H, Kim N, Yoon M.The main objective of this study was to evaluate the efficacy of an additional cryoprotectant in 10% dimethyl sulfoxide (DMSO) on cryopreserving germ cells from stallions at different reproductive stages. Testicular samples were obtained from pre-pubertal (1-1.5 yr, n=6) and post-pubertal (3-7 yr, n=5) stallions. Germ cells were isolated using a two-enzyme digestion procedure and cryopreserved in minimal essential medium alpha containing 10% fetal bovine serum and 10% DMSO with or without addition of trehalose (50, 100, or 200mM) or polyethylene glycol (PEG, 2.5, 5, or 10%). Viability, cell po...
Usuga A, Gutiérrez V, López ME, Pérez LF, Jaramillo L, Rojano B, Restrepo G.Microbial growth in semen may cause a decline of sperm quality and fertility; however, the addition of antifungals to semen extender has been shown to impair the overall fertility of the sperm. The aim of this study was to evaluate the antifungal activity of conventional and natural compounds, and their effect on the motility and kinetics of cooled stallion semen. A total of 15 ejaculates from five stallions were collected using the artificial vagina. Each ejaculate was supplemented with: fluconazole at 12.5 (F1), 25 (F2) and 50 (F3) mg/ml; amphotericin-B at 6.5 (A1), 12.5 (A2) and 25 (A3) mg/...
Novello G, Segabinazzi LGTM, Lisboa FP, Canuto LE, Freitas-Dell'Aqua CP, Dell'Aqua JA, Canisso IF.This study compared the postthaw semen parameters of stallions with high and low body condition score (BCS) and evaluated associations between body morphometric parameters and postthaw semen parameters. Twenty stallions were split into Low BCS (BCS<7, n = 11) and High BCS (BCS ≥7, n = 9) groups, and underwent a complete morphometric analysis (e.g., neck scores and circumference, crest neck height, body weight, and height), and subcutaneous body fat thickness (SFT) at the tail head, withers, shoulders, and retroperitoneal space. A fasted oral sugar test (OST) was conducted on all stallio...
Brandon CI, Srivastava PN, Heusner GL, Fayrer-Hosken RA.Acrosin, Arysulfatase A, and beta-N-acetylglucosaminidase are three key enzymes localized within the mammalian acrosome that play a pivotal role in the penetration of the oocyte. The objectives of this study were to compare two methods of enzyme extraction based on the activities of these enzymes from equine spermatozoa. Method A utilized a 0.5 M Tris-maleate buffer containing 0.1% Triton X-100 and Hyamine 2389. Method B used 0.05 M Tris-HCl, 0.05 M MgCl2 in 0.05 M Tris-maleate, followed by 0.05 M Tris-maleate containing 0.1% Triton X-100. Results indicated that acrosin was initially bound in ...
Brito LFC, Loomis PR, Klohonatz KM, Althouse GC.Neat stallion semen can contain a variety of microorganisms, some of which may impair sperm quality and/or cause infection of the mares' reproductive tract. For this reason, antibiotics are commonly added to semen extenders. A combination of gentamicin, tylosin, lincomycin and spectinomycin (GTLS) has been recommended for use, but there are no reports on the use of this mixture in equine semen extender. Penicillin and amikacin (PA) are safe for preserving sperm quality while effectively controlling bacterial growth in equine cooled stored semen, but data on frozen semen are scarce. Therefore, ...
Backman T, Bruemmer JE, Graham JK, Squires EL.The ability to ship cooled stallion sperm for subsequent freezing at a facility specializing in cryopreservation would be beneficial to the equine industry. Stallion sperm has been centrifuged, cooled to 5 degrees C for 12 h, and frozen without a detrimental effect on motility in a previous study; however, no fertility data were available. Experiment 1 compared the post-thaw motility of sperm cooled for 18 h at 15 or 5 degrees C at either 400 or 200 x 10(6) sperm/mL and then frozen. Storage temperature, sperm concentration, or the interaction of temperature and concentration had no effect on t...
