Equine Sperm Research Articles

Presence and distribution pattern(s) of sperm protein at 22 kDa (SP22) on ejaculated and caudal epididymal equine spermatozoa prior to and following heat-induced testicular degeneration

SP22 is highly correlated with fertility in rats (Klinfelter et al., J Androl 1997;18:139-150) and has been identified on equine spermatozoa (Miller et al., Biol Reprod 2003;68:166-167). Environmental damage to spermatozoa affects localization pattern(s) of SP22, but it is unknown if testicular degeneration (TD) alters expression of SP22. We hypothesized that expression and localization of SP22 on ejaculated and epididymal spermatozoa are altered by heat induced TD. To test this hypothesis, a total of three ejaculates were collected every other day from each of five stallions. Eighty-four days following hemi-castration to recover epididymal sperm, the remaining testis was insulated for 14 days. The insulation was achieved using diapers, cotton, and elastikon in a sling-like fashion. Semen was collected on days 6, 8, 10, and 13 of insulation. The remaining testes were removed on day 14, and epididymal spermatozoa were recovered. Ejaculates were evaluated by spectrophotometer and computer assisted semen analyzer (Sperm Vision, Minitube of America, Verona, WI). The cauda epididymides were flushed with air to collect spermatozoa. The samples underwent centrifugation (600xg), before resuspension in 1X PBS. Ejaculated and epididymal spermatozoa were prepared for Western blot analysis and immunocytochemistry (ICC). Tissues from the castrated testes (preand post-insulation) were sectioned, placed in Zamboni’s fixative, embedded, and stained with hematoxylin and eosin. SP22 staining patterns were determined on 100 spermatozoa from each

Annual variation of DNA fragmentation assessed by SCSA™ in equine sperm

Rating agencies are information providers specializing in offering standardized evaluation on an issue or an issuer and they can take advantage of economies-of-scale principles. The specific characteristics of a rating business can affect the economic and financial equilibrium of the raters and determine some differences with respect to other information providers.This chapter considers a sample of raters and computes some standard indexes on the balance sheet, income statement and cash flow statement in order to evaluate if there is any difference in the indexes computed for the raters with respect to other information providers. Results demonstrate that the rating sector has a unique economic and financial equilibrium that only occasionally can be considered comparable with other information providers.

The effect of calcium, pH and other regulators of motility on the axoneme of demembranated stallion spermatozoa

Despite being a standard procedure in most other domestic species, in vitro fertilization (IVF) has had poor success in the horse. This appears to be due to the failure of equine sperm to spontaneously initiate hyperactivated motility in vitro, as pharmacological stimulation of hyperactivation has been shown to increase equine IVF rates. To investigate the control of hyperactivated motility in equine sperm, we evaluated factors affecting motility in a demembranated sperm model. Exposure of sperm to 0.02% Triton X-100 for 30 seconds maximized membrane permeability as shown by transmission electron microscopy and staining with propidium iodide, while maintaining motility as determined by computer-assisted sperm motility analysis. Using this model, we evaluated the effects of medium pH, free calcium, ATP and dibutyryl-cAMP on motility of demembranated sperm. We also assessed the effects of known physiological stimulators of hyperactivation, 4-aminopyridine and procaine, as well as motility inhibitors nickel and cadmium. A minimum of three replicates was performed for each experiment, and data was analyzed by analysis of variance, utilizing the Holm-Sidak method for multiple comparisons. Increasing concentrations of ATP were associated with an increase in both curvilinear velocity and amplitude of lateral head movement. Cadmiumwas also associated with increases in these parameters, in contrast to the significant decline in motility seen in other species; however, nickel decreased motility as expected. Sperm motility was not affected by dibutyryl-cAMP. At a standard free-calcium concentration of 100 nM, maximal motility was induced at pH 6.5, a

Protein content of equine seminal plasma and sperm plasma membrane in subfertile stallions

Protein content of equine seminal plasma and sperm plasma membrane in subfertile stallions

Influence of cholesterol-loaded cyclodextrin incorporation on the acrosome reaction of cooled equine spermatozoa

Sperm capacitation with subsequent acrosome reaction is an indispensable event for oocyte fertilization. To trigger such events, cholesterol efflux from the membrane is necessary. Although cholesterol-loaded cyclodextrin (CLC) incorporation to the membrane increases sperm resistance to cooling, it might also delay or inhibit sperm capacitation and acrosome reaction. The goal of this study was to assess CLC addition on the acrosome reaction of cooled equine spermatozoa after challenge with calcium ionophore. For this purpose, two ejaculates from five stallions were collected. Each ejaculate was diluted to 50 x 106 sperm/mL with skim milk-based extender and divided into two 5-mL aliquots. For each ejaculate, one aliquot was not treated

The effect of zinc on stallion sperm motility

Understanding the control of sperm motility in the horse may help to improve the success of equine in vitro fertilization. In humans, zinc (Zn) is found in high concentrations in the seminal plasma, and has been shown to block the sperm proton channel HVCN1. This effect prevents intracellular alkalinization and is thought to help regulate sperm motility. The aim of our study was to measure Zn levels in stallion reproductive fluids and tissues and to evaluate the effect of different concentrations of Zn on equine sperm motility parameters. Zinc levels were measured through flame atomic absorption spectroscopy. Average Zn concentrations (n 1⁄4 5 stallions) in serum, seminal plasma and epididymal fluidwere 8 mM, 24 mMand 87 mM respectively. Within tissues of the internal reproductive tract (n 1⁄4 2 stallions), the highest values for Zn were found in the bulbourethral glands (841 mM) and the lowest in the prostate (261 mM). The various tissue components of the testis contained 214 to 229 mM Zn. In a fractionated ejaculate (n 1⁄4 1 stallion), Zn concentrations were lowest in the pre-sperm fraction (1.8 mM) and highest in the sperm-rich fraction (35 mM). To assess the effect of Zn on sperm motility, ejaculates were collected from 3 fertile stallions, and sperm were washed and incubated in modified Whitten’s media at pH 7.1 to 7.4, with 0, 10, 50, 100 or 500 mM of ZnCl2, at 38 C in humidified air for 30 min. Five motility parameters (total motility (TMOT), progressive motility (PMOT), average-path velocity (VAP), curvilinear

Characterisation of the stallion sperm proteome

1. IntroductionThe quest for improved understanding of stallionfertilityanditsbiomarkershasresultedintheidentificationofahandfuloffunctionalspermproteinsinrecentyears[1-5];however,thegreaterpartoftheequinespermproteomehas not been previously characterised. This represents avaluable yet untapped resource in light of the recentcharacterisation of the mouse, rat, and human sperm pro-teomes, which documented w1000 non-redundant geneproducts present in the sperm of each species [5] andprovided new insights into the specialised processes thatregulate spermatozoa. Such information bears particularpertinence to spermatozoa when one considers that thesecells possess no capacity for transcription or translation ofgenes, making genomic approaches far less useful in theinvestigation of the significant structural and functionalchanges experienced by sperm in their lifetime. In thiscontext, mass spectrometry and bioinformatics wereemployed to document the proteome of equine sperma-tozoa, revealing a large number of gene products andproviding a solid foundation for further in-depth investi-gation of the molecular mechanisms that regulate spermfunction.2. Materials and methodsSpermatozoa were isolated from undiluted, gel-freesemen, collected from fertile stallions (n¼2), by discon-tinuousPercollgradientandwashinginprotein-freemedia.Soluble protein fraction was obtained by addition of lysisbuffer, then reduced and alkylated before methanol/chlo-roform precipitation.Samples were fractionated by 1D SDS gel electropho-resis and sectioning of gel lanes. After trypsin digestion,peptides were extracted, and analysed by liquid chroma-tography and tandem mass spectrometry.Peptide sequence data obtained were used to searchproteinand gene sequencedatabases toacquireprotein IDsand respective encoding genes. Interrogation of DAVIDBioinformatics database allowed investigation of geneontology of the detected peptides with respect to inferredcellular localisation, molecular function and biologicalprocesses pertaining to each identified protein.3. ResultsMass spectrometry and extensive bioinformatics anal-ysis allowed detection of 4262 peptides leading to identi-fication of 942 proteins present in stallion spermatozoa.Theseweremappedto810genesymbolsthatcouldthenbeused to investigate the potential functional and locationcharacteristics attributable to each gene product.Gene ontology analysis for inferred cellular localisationindicated that 90.7% of annotatable proteins (555 of 612)are intracellular, with 26.4% of the proteins localised tomitochondria.Thisisconsistentwithanalysesofrat,mouseand human sperm proteomes, where mitochondria are themost highly represented intracellular organelle.Molecular function analysis indicated that the vastmajority of proteins (538 of 623 annotated) participate invarious binding roles. Catalytic activity was attributed to56.5% of annotated proteins (352 of 623). Antioxidant ac-tivity and roles in electron transport were attributed to 1%and 4.2% of proteins, respectively.Categorization according to biological process indicatedmetabolism as the dominant process, with 64.8% of anno-tated proteins (333 of 514) involved in primary metabolicprocesses. A category of significant interest is that of pro-teins involved in response tochemical stimuli, highlighting67proteinsaspotentialplayersintheinteractionsofspermwith the extracellular environment. Among these are anumber of receptor/signalling proteins, chaperonins,phosphorylation candidates and proposed decapacitationfactor proteins; selected proteins of interest are listed in

Impact of using a fast-freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n=25) were frozen by conventional or a fast-freezing technique. Frozen semen was thawed by two thawing protocols (37°C 30s(-1) or 75°C 7s(-1) ). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n=25) were inseminated with 300 (n=11) or 150 (n=14) million spermatozoa. A greater (P<0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20min post-thawing in the fast-freezing technique than in the conventional one. Plasma membrane integrity was also greater (P<0.05) in semen frozen with the fast-freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast-freezing technique was 76% (19/25), and did not differ (P>0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep-horn insemination maximises the use of equine semen. The fast-freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.

027 Temperature-dependent tolerance to hypotonic stress and cryosurvival of stallion sperm

Success rates for cryopreservation of equine sperm show a large variation among individual stallions. Factors that have been implicated with this inter-male variation in cryosurvival are the ability of sperm to tolerate osmotic stress, and temperature-induced changes in membrane phase state and organization. Cells are exposed to osmotic stress both upon the addition of cryoprotective agents as well as during freezing and thawing. Moreover membranes undergo phase changes during cooling and warming causing transport of molecules that normally are membrane-impermeable. The aim of this study was to evaluate intra-individual variability in osmotic properties of stallion sperm and its correlation with cryosurvival. Furthermore, the temperature dependency of hypo-osmotic tolerance was studied and correlated with membrane phase behavior. Pre- and post-freeze sperm motility and plasma membrane integrity were assessed using computer assisted sperm analysis and flow cytometry. The critical osmolality, at which half of the sperm population survived exposure to hypotonic saline solution, was taken as a measure for osmotic tolerance. Temperature-dependent changes in membrane conformational disorder were evaluated using Fourier transform infrared spectroscopy. Sperm membranes display a main phase transition in the 10–30 °C temperature range, with two distinct melting events. The melting event around 30 °C becomes more pronounced with removal of cholesterol. Stallion sperm membranes exhibited good resistance towards hypotonic stress in the 15–30 °C temperature range, whereas membrane stability was found to be decreased at 4 and 37 °C. The decreased tolerance towards exposure to hypotonic stress at 4 °C could be caused by the membrane phase transition which the membranes undergo during cooling. The decreased hypo-osmotic tolerance at 37 °C might be due to the fluidity of stallion sperm membranes at this temperature. The critical osmolality at 22 °C, was found to vary between 55 and 170 mOsm kg −1 among sperm from different stallions. Clear correlations were found for pre- versus post-freeze sperm motility as well as membrane integrity, assessed in either freezing extender or after dilution in isotonic medium. Pre-freeze percentages of membrane intact sperm after exposure to hypotonic stress showed a correlation with sperm motility after cryopreservation. This correlation, however, disappeared when data were corrected for initial numbers of membrane intact sperm in the sample. It is concluded that studies on pre-freeze tolerance towards hypotonic stress cannot be used to predict sperm cryosurvival rates for individual stallions. Furthermore, membrane phase behavior and the overall membrane fluidity at a given temperature are involved in osmotic stability. Source of funding: This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy). Conflict of interest: None declared. harriette.oldenhof@tiho-hannover.de

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro.

CatSper and the Relationship of Hyperactivated Motility to Intracellular Calcium and pH Kinetics in Equine Sperm1

In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx.

Short‐Term Storage and Swim‐Up Selection Do Not Affect the X/Y Ratio in Equine Spermatozoa

The standard procedure of artificial insemination with fresh equine spermatozoa involves short-term storage (to 48h at 5°C). This procedure is accompanied by a gradual loss of sperm viability. The aim of this study was to investigate whether the X/Y ratio of equine spermatozoa is affected by short-term storage and the swim-up procedure. We used a standard protocol, for short-term storage (0, 24 and 48h at 5°C) of stallion semen diluted in the commercial extender EquiPro™ (Minitüb GmbH, Tiefenbach, Germany). After each set-up storage period, the motile fraction of sperm cells was selected by the swim-up method. The X/Y ratio was evaluated by fluorescence in situ hybridization (FISH) in the fresh, non-selected sperm, and in motile spermatozoa selected after each of the storage periods. Molecular probes for the equine chromosomes X and Y were used. The X/Y ratio in all sperm samples analysed in this study (fresh and stored) was not different from the theoretical 1:1 value. The incidence of chromosomally abnormal sperm cells in the fresh (0.28%) and motile (0.13%) sperm samples was not significantly different. The two approaches (sperm storage up to 48h and the swim-up procedure) applied to this study did not affect the X/Y ratio in the motile fraction of equine spermatozoa. This finding does not conform to phenomena described for human and cattle. For this reason, the finding may imply species-related differences.

Cooling of ejaculated and epididymal stallion sperm

After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P&lt;0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.

Preliminary Results: The Advantages of Low-Density Lipoproteins for the Cryopreservation of Equine Semen

The aim of this study was to determine the best concentration of low-density lipoproteins (LDL) in a semen extender to improve the percentage of motile spermatozoa in equine sperm after freezing and thawing in comparison with standard extenders. Ten extenders were compared: 1 with 2% egg yolk (EY), 8 with different concentrations of LDL (0.25%, 0.50%, 0.75%, 1%, 2%, 3%, 4%, and 5%), and INRA 96; all of the extenders contained 2.5% glycerol. Fourteen ejaculates were collected from four different stallions. The first dilution was made with equal parts at +37°C, centrifuged (600 × g/10 min), and resuspended in the corresponding extenders to obtain a final concentration of 100 × 106 spermatozoa/ml. The resulting mixture was cooled to 4°C over 1 hour, packed into four 0.5-ml straws, and left for a further 30 minutes at +4°C. Finally, the straws were frozen in nitrogen vapors 4 cm over liquid nitrogen for 10 minutes before being immersed in liquid nitrogen at −196°C and stored. Two straws per extender and per ejaculate were thawed in a water bath at +37°C for 30 seconds. The contents of each straw were recovered into a cryotube and placed in a water bath at +37°C for 10 minutes before being examined with an image analyzer. The best post-thaw motility results were obtained with the extenders made with 0.5%, 2%, and 3% LDL and with the control extender made with egg yolk; no significant difference was observed between these extenders. The last two straws were thawed to perform four sperm function tests. The hypo-osmotic test was used to assess the integrity of the plasma membrane; the 2% and 3% LDL treatments were the most suitable and were comparable to that with whole egg yolk for protecting stallion sperm during cryopreservation (32.3%, 32.4%, and 31.3%, respectively). The Pisum sativum agglutinin-fluorescein isothiocyanate test was used to verify the integrity of the acrosomes; the best results were obtained with the 0.5%, 0.75%, and 3% LDL and INRA96 extenders; no significant differences were observed among the 85.8%, 85.0%, 84.7%, and 84.8% extenders. The acridine orange test was used to assess DNA integrity; there were no significant differences among the various extenders: the DNA was preserved in 98% of the spermatozoa. Finally, spermatozoal morphology was examined using Spermac stain; 78% of the spermatozoa did not present any anomalies in the 0.25% and 2% LDL extenders. In conclusion, the 2% LDL extender gave the best post-thaw percentage of motile spermatozoa. The results of the sperm function test were also superior for this extender.

Effects of ambient heat stress of equine sperm chromatin by evaluation of two differing techniques

Effects of ambient heat stress of equine sperm chromatin by evaluation of two differing techniques

Small-dose sulpiride injections in horses: Repeatability of prolactin responses and use for assessment of dopaminergic suppressive activity by pergolide and cabergoline formulations

s / Journal of Equine Veterinary Science 33 (2013) 321-399 370 gradient would in theory increase the concentration of higher quality sperm in the insemination dose. With stallions whose semen parameters are less than optimal, such a separation would be desirable for eliminating the abnormal sperm in the insemination dose and from potentially competing with normal sperm within the mare’s reproductive tract. The process for preparing for the centrifugation is costly from the standpoint of time but the result is a more viable insemination dose. On a commercial breeding operation during the summer of 2012, this process was employed to attempt to improve pregnancy rates in mares bred to a stallionwith documented subfertile semen parameters. A commercially-available equine sperm purification protocol, EquiPure, was used as the treatment in 92 the stallion’s ejaculate. One hundred ninety seven mares were inseminated with either treated (EquiPure) or nontreated semen. Thirty-two of the 63 mares bred with the treated insemination doses were pregnant (51%). Of the 134 mares bred with the non-treated semen, 75 were determined to be in foal (56%). There was no difference in pregnancy rates between themares inseminatedwith treated or untreated semen. For the 67 mares bred on the farm, 15 became pregnant when the ejaculates had been treated with EquiPure and 23 were pregnant when bred with nontreated semen resulting in 68% vs 51% pregnant, respectively. There were 130 mares bred with cooled transported semen; only 17 bred with EquiPure treated semen were pregnant (41%) vs 52with non-treated shipped semen 58%). For this stallion, the treatment with EquiPure did not significantly improve the pregnancy rate in the mares. Therefore, it was determined, for this stallion, that the process would not be employed in future breeding. Unfortunately, because of the many variables of breeding in the commercial setting, the positive results observed in a controlled laboratory setting did not carry over to the

Efeito da adição de glutationa peroxidase e cisteína ao diluidor de congelação do sêmen equino

Foram utilizados ejaculados (n=25) de garanhões para avaliar o efeito de glutationa peroxidase (GPx) e cisteína na viabilidade de espermatozoides congelados. O sêmen foi diluído em Botu Crio, com antioxidantes, e foram formados os grupos: G1, Controle; G2, 1U GPx ; G3, 5U GPx; G4, 0,5mM cisteína; G5, 1mM cisteína. Depois foi envasado em palhetas (0,5mL) e congelado. Após descongelação, 37°C por 30 segundos, alíquotas foram analisadas quanto à integridade de membrana plasmática (IMP) e acrossoma (IAc), potencial de membrana mitocondrial (PMM) e cinética, nos tempos zero (T0) e 60 minutos (T60). GPx 5U e cisteína 0,5mM determinaram maior (P&lt;0,05) IAc em T0 do que em T60. Cisteína 1mM resultou em maior (P&lt;0,05) IAc em T60 do que GPx 1 e 5U e cisteína 0,5mM. O PMM de um garanhão no T60 foi mais alto (P&lt;0,05) do que o de dois garanhões. VCL e VAP foram maiores (P&lt;0,05) no T0 do que no T60 do grupo controle, e um garanhão apresentou, em geral, valores cinéticos mais altos (P&lt;0,05) do que os demais. Conclui-se que a adição de glutationa peroxidase, nas concentrações de 1U e 5U, e de cisteína, nas concentrações de 0,5mM e 1mM, não interferem na integridade de espermatozoides criopreservados de equinos, mas preservam os parâmetros cinéticos de VCL e VAP após 60 minutos de incubação. Ressalta-se, ainda, que o garanhão tem uma forte influência nas características espermáticas pós-congelação.

Effects of α-tocopherol and Ascorbic Acid on Equine Semen Quality after Cryopreservation

This study investigated the effects of ascorbic acid and α-tocopherol supplementation on semen quality parameters of equine thawed-frozen semen. Semen was divided in seven different treatments in a final concentration of 100 × 106 sperm/mL by using Gent extender containing no supplements (control) and the following supplements with three different concentrations: α-tocopherol (0.5, 1, and 2 mM) and ascorbic acid (0.45, 0.9, and 1.8 g/L). After thawing, all samples were maintained at 37°C, while analyses were performed at 0, 60, and 120 minutes. Evaluation of viability and acrosome status (using Pisum sativum agglutinin conjugated to fluorescein isothiocyanate and propidium iodide), mitochondrial membrane potential (5,5′,6,6′-tetrachloro-1,1′,3,3′tetraethylbenzimidazolyl carbocyanine iodine [JC-1]), membrane lipid peroxidation (LPO; C11-BODIPY581/591), and stability of the plasmatic membrane (merocyanine 540 and Yo-Pro-1) of each sample was determined by flow cytometry. Relative to the control group, supplementation with α-tocopherol improved (P ≤ .05) postthaw membrane LPO, yet the higher concentrations of ascorbic acid (0.9 and 1.8 g/L, respectively) showed a negative effect on membrane LPO. Neither antioxidant significantly increased (P > .05) the acrosome integrity and mitochondrial membrane potential of frozen-thawed spermatozoa, although supplementation with α-tocopherol and ascorbic acid (0.9 and 1.8 g/L, respectively) had a positive effect on membrane integrity and stability (P ≤ .05). For all semen parameters, the lower concentration of ascorbic acid (0.45 g/L) did not show significant differences (P > .05) compared with the control. In conclusion, α-tocopherol seems to be an efficient antioxidant for reducing the oxidative stress provoked by cryopreservation, decreasing lipid peroxidation on equine spermatozoa.

Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT)

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene.Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein.

246 EFFECT OF NITRIC OXIDE INHIBITION (N-ω-NITRO-L-ARGININE METHYL ESTER) AND SCAVENGER (METHYLENE BLUE) ON PLASMA MEMBRANE PEROXIDATION OF EQUINE CRYOPRESERVED SPERM

The peroxidation of plasma membrane lipids has been claimed to be a major factor involved in sublethal cryodamage. Some authors have reported that reactive oxygen species (ROS) accumulation could be an important factor leading to further damage in post-thaw sperm. Oxidative stress occurring in the sperm is a phenomenon associated with increased rate of oxidation of cellular components and excessive production of ROS and nitrogen. The objective of this study was to evaluate the effect of NO inhibition [N-ω-nitro-l-arginine methyl ester (l-NAME)] and scavenger (methylene blue) on the plasma membrane peroxidation of equine cryopreserved capacitated sperm. Three ejaculates were obtained from each of three stallions (n = 9). Semen was packaged into 0.5-mL straws to a concentration of 200 × 106 sperm mL–1 in BotuCrio® extender and frozen using an automated technique with a programmed machine. Four straws were thawed in a water bath at 37°C for 30 s and centrifuged in bovine IVF media supplemented with sodium bicarbonate and BSA for the capacitation of equine sperm. The supernatant was withdrawn, and semen was then incubated in the same media with l-arginine, with or without the NO synthase inhibitor, l-NAME, and in media with l-arginine with or without the NO scavenger, methylene blue: treatment (T)1 = control; T2 = 10 mM l-arginine (based on previous experiments); T3 = 1 mM l-NAME; T4 = 100 mM methylene blue; T5 = 10 mM l-arginine + 1 mM l-NAME; and T6 = 10 mM l-arginine + 100 mM methylene blue for 60, 120, and 300 min at 38°C under 5% CO2. After incubation, cells presenting membrane peroxidation were identified using an association of the fluorescent probes C11-BODIPY581/591 (1 mg mL–1; D-3861) and PI (0.5 mg mL–1; L0770). Nitric oxide production was identified using a 4,5-diaminofluorescein-2/diacetate (10 µM) probe associated with PI. The H33342 probe was used to avoid those particles presenting the same size and granularity as sperm cells being included in the counting. Both evaluations were performed using flow cytometry. Data were analysed by ANOVA, and the means were compared within each time with Tukey’s test, with a level of significance of 5%, using SAS software (SAS Institute Inc., Cary, NC, USA). Lipid peroxidation of the membrane was not influenced by the treatments at the different times (P &gt; 0.05). This characteristic was reduced for the groups treated with methylene blue (T4 and T6) at times 60, 120, and 300 min compared with the other treated groups. Nitric oxide produced by sperm was not influenced by treatment at different times (P &gt; 0.05). However, methylene blue addition was able to decrease NO production in treatments T4 and T6 at all the incubation times evaluated compared with the other treatments. Therefore, methylene blue removed NO and decreased plasma membrane lipid peroxidation, suggesting an antioxidant role of this scavenger. On the other hand, more research is needed to elucidate the mechanism of action of methylene blue on the physiology of cryopreserved equine sperm. Supported by FAPESP (grant 2009/54906-5).