Equine Sperm Research Articles

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the \u201czombie sperm\u201d dilemma

PurposeTo define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect.MethodsSperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions.ResultsUsing PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results (“zombie sperm”). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0.ConclusionFlow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

In Stallion Spermatozoa, Superoxide Dismutase (Cu-Zn) (SOD1) and the Aldo-Keto-Reductase Family 1 Member b (AKR1B1) Are the Proteins Most Significantly Reduced by Cryopreservation.

Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a split sample design, 30 ejaculates from 10 different stallions were analyzed as fresh spermatozoa, and another aliquot from the same ejaculate was analyzed as a frozen thawed sample. The proteome was studied under both conditions using UHPLC-MS/MS and bioinformatic analysis conducted to identify discriminant variables between both conditions. Data are available through the ProteomeXchange Consortium with identifier PXD022236. The proteins most significantly reduced were Aldo-keto reductase family 1 member B (p = 2.2 × 10–17) and Superoxide dismutase (Cu–Zn) (p = 4.7 × 10–14). This is the first time that SOD1 has been identified as a discriminating variable using bioinformatic analysis, where it was one of the most highly significantly different proteins seen between fresh and frozen thawed semen. This finding strongly supports the theory that alteration in redox regulation and oxidative stress is a major factor involved in cryodamage and suggests that control of redox regulation should be a major target to improve current cryopreservation procedures.

Effects of In Vitro Interactions of Oviduct Epithelial Cells with Frozen-Thawed Stallion Spermatozoa on Their Motility, Viability and Capacitation Status.

Simple SummaryThe use of assisted reproductive techniques, which involve the manipulation of sperm and oocytes in the laboratory, support owner production of valuable animals’ offspring. However, several limitations remain underlining the need to further optimize existing protocols as well as to develop new strategies. For example, the required conditions to make equine spermatozoa competent to fertilize an oocyte in vitro (IVF) have not been established. Therefore, our initial goal was to optimize different conditions associated with frozen equine sperm manipulations in order to improve their quality. We observed that simple factors such as sample concentration, incubation period and centrifugation time affect the sperm motility. Since in vivo fertilization involves the interaction between spermatozoa and epithelial cells in the mare’s oviductal tract, our next goal was to mimic this environment by establishing primary cultures of oviductal cells. Using this in vitro system, we were able to select a sperm population capable of fertilization. In short, this study provides a novel protocol that improves the yield of fertilization-capable sperm obtained from equine frozen spermatozoa.Cryopreservation by negatively affecting sperm quality decreases the efficiency of assisted reproduction techniques (ARTs). Thus, we first evaluated sperm motility at different conditions for the manipulation of equine cryopreserved spermatozoa. Higher motility was observed when spermatozoa were incubated for 30 min at 30 × 106/mL compared to lower concentrations (p < 0.05) and when a short centrifugation at 200× g was performed (p < 0.05). Moreover, because sperm suitable for oocyte fertilization is released from oviduct epithelial cells (OECs), in response to the capacitation process, we established an in vitro OEC culture model to select a sperm population with potential fertilizing capacity in this species. We demonstrated E-cadherin and cytokeratin expression in cultures of OECs obtained. When sperm–OEC cocultures were performed, the attached spermatozoa were motile and presented an intact acrosome, suggesting a selection by the oviductal model. When co-cultures were incubated in capacitating conditions a greater number of alive (p < 0.05), capacitated (p < 0.05), with progressive motility (p < 0.05) and with the intact acrosome sperm population was observed (p < 0.05) suggesting that the sperm population released from OECs in vitro presents potential fertilizing capacity. Improvements in handling and selection of cryopreserved sperm would improve efficiencies in ARTs allowing the use of a population of higher-quality sperm.

103 Characterization of the receptors of the endocannabinoid system in equine sperm: Possible role of anandamide in sperm function

Conventional IVF in horses remains challenging. In particular, stallion sperm fails to penetrate the zona pellucida, possibly due to incomplete invitro sperm capacitation. Therefore, there is a need to elucidate, in horses, molecules with a proven role during capacitation in other mammals. Our laboratory has described the relevance of the endocannabinoid system in capacitation of bovine and murine sperm. We reported that anandamide (AEA), an endocannabinoid present in follicular and oviducal fluids, induced capacitation-associated events. The aims of this work were to characterise the localization of cannabinoid receptors in equine sperm and to evaluate the effects of AEA on levels of tyrosine-phosphorylated proteins (pY) and substrates phosphorylated by protein kinase A (pPKA). Both cannabinoid receptors (CB1, CB2, TRPV1) and pPKA and pY were localised in sperm by indirect immunofluorescence. Sperm (15×106mL−1) were incubated, at 38.5°C in air, in modified Tyrode’s-albumin-lactate-pyruvate (TALP) with 25mM NaHCO3, 5mM dextrose and 1mgmL−1 polyvinyl alcohol (PVA; TALP-Bic-PVA) or TALP-Bic-PVA supplemented with AEA (0.1, 1, 10, 100nM, and 1µM) for 4h. After incubation, Western blot was used to determine levels of pY and pPKA in 4.5×106 sperm. Cryopreserved sperm samples from three stallions were evaluated. The normality of data distributions and homoscedasticity were verified with the Shapiro-Wilk and Levene tests, respectively. Data were analysed by one-way ANOVA and Bonferroni post hoc test, with P&amp;lt;0.05 considered significant. Based on immunofluorescence, CB1 was mainly localised in the post-acrosomal region and flagellum (93.4%±5.5, mean±s.d.), CB2 in the post-acrosomal region and middle piece (89.9%±28.3), and TRPV1 in the post-acrosomal region and flagellum (89.3%±9). Sperm positive for pPKA had fluorescence in the middle piece and principal piece of the flagellum. Incubation with 1nM AEA for 4h induced a 61% increase in pPKA levels compared with TALP-Bic-PVA medium alone, with no induction of pY levels in any treatment. In conclusion, cannabinoid receptors were present in equine sperm, and incubation with AEA induced an increase in PKA activity, an essential event associated with sperm capacitation. To our knowledge, this was the first report describing the presence of receptors of the endocannabinoid system in equine sperm and the potential role of AEA in the acquisition of sperm fertilizing ability.

131 In&amp;nbsp;vitro nuclear maturation and blastocyst developmental rates after intracytoplasmic sperm injection of equine oocytes held for 24&amp;thinsp;h at room temperature in Tyrode&amp;rsquo;s albumin lactate pyruvate-Hepes (TALP-h) or in a commercial embryo holding medium

The interest in equine intracytoplasmic sperm injection (ICSI) for commercial and research applications has rapidly increased. Shipping immature oocytes at room temperature has been proven successful, and to identify the optimal conditions for holding oocytes, several mediums are being tested. The aim of this study was to compare the effect of holding equine oocytes in Tyrode’s albumin lactate pyruvate-Hepes (TALP-h, Bavister and Yanagimachi 1977 Biol. Reprod. 16, 228-237) medium or in commercial embryo holding medium (EHM, Syngro® Holding) on invitro nuclear maturation rates and pre-implantation embryo development after ICSI. Cumulus–oocyte complexes (COCs) were recovered from ovaries of slaughtered mares and assigned randomly in 2-mL cryovials with TALP-h or EHM, with a maximum of 30 oocytes per cryovial. COCs were shipped to the ICSI laboratory at 20 to 25°C for 24 to 28h followed by IVM for 24h in a humidified atmosphere of 5% CO2 in air at 38.5°C. Maturation medium was TCM-199 with 10% fetal bovine serum, 1μL mL−1 insulin-transferrin-selenium, 1mM sodium pyruvate, 100mM cysteamine, and 0.1mg mL−1 FSH. After mechanical cumulus cell removal, nuclear maturation rate was assessed using a stereomicroscope. Oocytes with an intact oolemma and extrusion of the first polar body (PB) were classified as mature, oocytes without a visible PB were considered immature, and oocytes without an intact oolemma were considered degenerate. Matured oocytes were subjected to ICSI without piezo-drill system (one proved stallion) in 20-μL droplets of TALP-h with a 7-μm glass sharp micropipette in an inverted microscope (Nikon Eclipse TE-300 microscope) using hydraulic micromanipulators (Narishige, Medical Systems). Presumptive ICSI zygotes were cultured in DMEM F12/Global Total® with 6% fetal bovine serum for 9 days at 38.5°C in a humidified atmosphere of 5% O2 and 5% CO2 in air. On Day 5 of culture, cleavage was recorded and medium was refreshed. Blastocysts rates were recorded on Day 7 and 9 of culture. Invitro nuclear maturation rates are shown in Table 1. We observed a significantly higher proportion of immature oocytes in the EHM group compared with the TALP-h group. After ICSI of some matured oocytes of each group, no significant differences were observed in cleavage or blastocyst rate (Table 1). Our results suggest that either TALP-h or commercial embryo holding medium are suitable for oocyte shipping and to support blastocyst development after ICSI. Table 1. Invitro nuclear maturation rates and pre-implantation embryo development after intracytoplasmic sperm injection (ICSI) Maturation rates Medium Oocytes Mature [n (%)] Immature [n (%)] Degenerate [n (%)] TALP-h 315 173 (54.9) 26 (8.3)a 116 (36.8) EHM 273 132 (48.4) 55 (20.1)b 86 (31.5) Total 588 305 (51.9) 81 (13.8) 202 (34.4) Embryo development ICSI (n) Cleaved [n (%)] Day 7 Blastocyst [n (%)] Day 9 Blastocyst [n (%)] TALP-h 35 23 (65.7) 7 (20) 9 (25.7) EHM 26 19 (73.1) 3 (11.5) 5 (19.2) Total 61 42 (68.9) 10 (16.4) 14 (23) a,bDifferent superscript letters indicate statistical significance (Fisher’s exact test, P&amp;lt;0.05).

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation.

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration.

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5mM caffeine-treated groups). Compared to that in the control, the addition of 5mM caffeine induced an increase in sperm motility (38.9±2.8 versus 32.6±3.4%), and a decrease in nitrite concentration (11.4±2.1 versus 12.8±2.9µM/µg protein, p<.05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.

The Relationship of Mitochondrial Membrane Potential, Reactive Oxygen Species, Adenosine Triphosphate Content, Sperm Plasma Membrane Integrity, and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.

Patterns of MTT reduction in mammalian spermatozoa.

MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

Effect of MnTBAP on in vitro capacitation of frozen-thawed stallion sperm

In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 106 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm.

Heat SHOCK proteins in equine spermatozoa: Expression and correlation to kinetic and environmental parameters

Heat Shock Proteins are chaperones primary involved in the repair of cellular damages induced by temperature. The harmful effect of temperature on the male gonad is well known, on the contrary knowledge on the effects of the environment on semen quality are still insufficient. The aim of this paper was to learn more about the role of HSPs and the environment in modulating the physiology of equine male gonads. We showed a detailed analysis of equine semen characteristic and the expression level of three HSPs (60-70-90) over a one-year period analyzing the effects of temperature and humidity and the correlation among the different variables. We showed also that the interpretation of results depends strongly on the way in which data are assembled and analyzed, therefore we compared results obtained from three different ways of grouping: according to single months, to weather seasons and to mare reproductive periods. Results showed that the expression of the three HSPs is correlated to the environment through temperature and humidity and that it reaches the highest level in the breeding season and in summer. We found also that HSPs expression is correlated to some variables describing the quality of equine semen (concentration) and the kinetic of spermatozoa (total motility-MOT, %, average path velocity -VAP, μm/s- and lateral head displacement -ALH, μm). No correlation was found between HSPs expression and the mitochondrial membrane potential; while viability and HSP90 expression resulted positively correlated. The month-by-month analysis evidenced that in February equine semen has the highest kinetic characteristics (increased linearity –LIN, %-, straightness –STR, % -and average path velocity -VAP, μm/s) with the highest number of motile, progressive motile and rapid cells.These results may have a great impact in the comprehension of functional aspects of the physiology of equine semen and may have potential implications for breeders who want to understand the period (and/or month) of the year in which equine semen reaches the best characteristics with increased chances for better results in reproductive practice.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Does Coenzyme Q10 Exert Antioxidant Effect on Frozen Equine Sperm?

During semen cryopreservation, the sensitivity of equine sperm to oxidative stress is increased by the eliminated seminal plasma. Thus, antioxidant addition to the semen extender can be helpful to the sperm survival after freezing and thawing. This work aimed to test whether coenzyme Q10 (CoQ10) added in different concentrations to the INRA 82 freezing extender has antioxidant function on equine sperm to improve its fertilizing ability. Semen samples from five stallions were frozen with the extenders: (T1) INRA 82, control, (T2) T1+ 5 μM CoQ10, (T3) T1+ 25 μM CoQ10, and (T4) T1+ 50 μM CoQ10. After sample thawing, sperm motility and kinetics characteristics were evaluated using a computer-assisted sperm analysis and sperm membrane functionality and integrity were evaluated with a hypo-osmotic swelling test and an epifluorescence microscopy, respectively. The nitrite (NO2-) and hydrogen peroxide (H2O2) concentrations of the semen samples were measured with spectrophotometry. There was no difference on the sperm characteristics among all treatments (P > .05). However, the 25 μM CoQ10 (T3) decreased NO2− concentration (6.7 ± 2.2 μM/μg protein) compared with the treatments T1, T2, and T4 (64.3 ± 3.7, 59.4 ± 5.3, 45.1 ± 8.6 μM/μg protein), respectively, as well H2O2 concentration (1.8 ± 0.3 μM/μg protein) compared with the control (4.6 ± 0.4 μM/μg protein) and 5 μM CoQ10 treatments (4.8 ± 0.2 μM/μg protein, P < .05). In conclusion, 25 μM CoQ10 plays a significant role as antioxidant to the frozen equine sperm, decreasing NO2− and H2O2 concentrations. Thus, its addition to the INRA 82 freezing extender may be beneficial to the fertilizing ability of equine semen.

Centrifugation Force and Time Alter CASA Parameters and Oxidative Status of Cryopreserved Stallion Sperm.

Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300× g for 5′, 300× g for 10′, 1500× g for 5′ and 1500× g for 10′ vs. no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.

Effects of the addition of docosahexaenoic acid and ?-tocopherol on quality of equine spermatozoa stored at 5°C

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and ?-tocopherol (?-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; ?-Toh; DHA/?-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of ?-Toh; : 10 ng mL-1 of DHA + 2 mM of ?-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p &lt; 0.05) when compared to control (62.1 ± 16.2), DHA/?-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p &lt; 0.05) than the groups treated with DHA (2,036.0 ± 687.0), ?-Toh (1,890.8 ± 749.5) and DHA/?-Toh (1,821.1 ± 627.2). In conclusion, ?-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes.

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 sulfoxide (DMSO) or 3.5% glycerol. Immature equine oocytes were obtained from ovarian follicles of mares during diestrus by transvaginal aspiration (n=128). After invitro maturation, MII oocytes (n=90) were fertilised by ICSI with thawed stallion spermatozoa (n=45 in both the DMSO and glycerol groups). The embryo cleavage rate was greater in the DMSO than glycerol group (73.3% vs 46.7% respectively; P=0.0098), but the blastocyst development rate per fertilised oocyte was similar between the two groups (28.9% vs 15.6% respectively; P=0.128), as was the blastocyst production rate per cleaved embryo (39.4% vs 33.3% respectively; P=0.653). In this study, cryopreservation of equine spermatozoa in 1M DMSO was correlated with significantly higher cleavage rates in IVM oocytes fertilised by ICSI compared with spermatozoa cryopreserved using 3.5% glycerol. Although not statistically significant in this small number of stallions, increased blastocyst production and individual stallion variability was observed among CPA treatments. This warrants further critical examination of cryoprotectants used in equine sperm subpopulations used for ICSI in a larger number of stallions.

116 Effect of different energy sources on motility and calcium ionophore-induced acrosome reaction in equine sperm

Equine sperm appears to differ from sperm of other species, preferring oxidative phosphorylation over glycolysis for energy production. However, there is little information about the effects of different energy sources on capacitation-related events in equine sperm. We evaluated stallion sperm incubated in the presence of different energy sources at a pH (~8.3) known to support some aspects of capacitation. Fresh stallion semen (n=9 ejaculates) was washed, then diluted to 30×106 spermmL−1 with modified Whitten's medium containing 25mM bicarbonate, 7mgmL−1 bovine serum albumin (BSA), and only one energy source (5mM glucose, 10mM lactate, or 10mM pyruvate). The diluted sperm were incubated in air for 0.5, 2, or 4h, then exposed to 5 µM calcium ionophore A23187 (CaI) or vehicle (V) for 10min. Sperm were washed and incubated in the assigned medium for 30min, then evaluated by computer-assisted sperm motility analysis and by live/dead staining (Fixable Live/Dead Red, Molecular Probes) followed by fixation (2% v/v paraformaldehyde) and staining for acrosome reaction, using Pisum sativum agglutinin (FITC-PSA, Sigma Aldrich). Stained sperm were evaluated via flow cytometry to detect live acrosome-reacted sperm. Data were analysed using ANOVA with Tukey post hoc comparison (SAS 9.4, SAS Institute). Medium pH at 4h was 8.37 to 8.48. There were no significant differences between CaI and V treatments for any experimental endpoint (P&amp;gt;0.05). At 4h, pyruvate values for total motile sperm (46±5%) and curvilinear velocity (113±8µm s−1) were significantly higher than those for lactate (32±5% and 86±5µm s−1) or glucose (27±4% and 90±5µm s−1). The percentage of live sperm that were acrosome-reacted (%L-AR) did not increase significantly until 4 h; at this time,%L-AR was higher for lactate (46±3%) than for pyruvate (21±3%) or glucose (18±3%; P&amp;lt;0.05). To the best of our knowledge, this is the first critical evaluation of the effect of different energy sources on motility and acrosome reactivity of equine sperm. The higher motility in pyruvate supports the concept that stallion sperm utilises oxidative phosphorylation more effectively than glycolysis. The apparent lack of effect of CaI on%L-AR may be related to the 1- to 2-h delay typically seen after its use in equine sperm; the present data show that incubation even for 4h in potentially capacitating conditions does not prepare equine sperm to respond quickly to CaI. Surprisingly, a high rate of spontaneous acrosome reaction was achieved in the lactate treatment. The notably higher%L-AR with lactate than with pyruvate suggests that this effect is not due to utilisation of substrate for oxidative phosphorylation but may be associated with conversion of lactate to pyruvate via lactate dehydrogenase, or possibly with effects of lactate through other pathways. Although pyruvate was associated with greater motility, this could be related to higher oxidative stress, which could have influenced the%L-AR. Further work is needed to determine whether the acrosomal changes observed are related to functional capacitation.

120 Use of fixable dyes to analyze equine sperm membrane integrity and acrosome reaction after A23187 treatment

Conventional IVF is not successful in the horse, and current work is focused on factors affecting sperm capacitation in this species. Challenges arise in assessing equine sperm incubated in media containing capacitation promotors, as some of these factors cause sperm head-to-head binding (aggregation). Our preliminary microscopic findings showed that sperm aggregates are largely of viable sperm, whereas nonviable sperm individualize. Thus, data obtained using technologies that analyse only individual cells and gate out aggregates, such as flow cytometry, may not accurately represent the study population. We developed a fixable live/dead/acrosome staining protocol (LD-PSA) that minimizes sperm aggregation. The aim of this study was to evaluate the percentage of viable and acrosome-reacted equine sperm after A23187 treatment, using either LD-PSA or a standard staining protocol (PI-PSA). Sperm from 9 ejaculates were suspended in Hanks’ balanced salt solution (HBSS) and exposed for 10min at 37°C to vehicle (V) or to 10 µM A23187 (C10). The sperm were washed, resuspended in HBSS medium with added lactate and pyruvate and containing 7mgmL−1 of bovine serum albumin (BSA), and assessed immediately (0h) or incubated at 37°C for 2h (the period needed for equine sperm to respond to A23187). Motility was analysed using computer-assisted semen analysis. Each treatment was stained by PI-PSA: propidium iodide and fluorescein isothiocyanate-Pisum sativum agglutinin (PSA) in DPBS; and by LD-PSA: Live/Dead Fixable Red, paraformaldehyde 2%, Triton×1%, and FITC-PSA in Dulbecco's phosphate-buffered saline with Accumax (Stem Cell Technologies), a commercial proprietary cell agglutination inhibitor, before flow cytometric analysis. Differences were analysed using repeated-measures two-way ANOVA. The% total motile sperm (TMOT) for V and C10 treatments were 76.3±3.0 and 71.2±4.7 at 0h (P&amp;gt;0.05), and 70.5±14.8 and 2.4±0.8 at 2h (P&amp;lt;0.05). On flow cytometry, the percentage of events outside the gate for V sperm (0h and 2h combined) was 31.9% in PI-PSA and 21.9% in LD-PSA samples (P&amp;lt;0.01). Measured viability in V samples was significantly lower when stained with PI-PSA than with LD-PSA at 0h (49.2±4.6 vs. 67.1±4.9) and tended to be lower (P=0.07) at 2h (44.0±4.9 vs. 55.1±2.8). Notably, the viability recorded in PI-PSA was 26 percentage points lower than was the TMOT at both 0h and 2h, indicating nonrepresentative results, as nonviable sperm should not be motile. By LD-PSA, this difference was 9 points at 0h and 15 points at 2h. Vehicle sperm showed significantly higher AR values in PI-PSA than in LD-PSA at 0h (30.4±4.0 vs. 17.7±2.4) and 2h (41.9±4.5 vs. 24.0±1.8), as did C10 sperm at 0h (28.9±2.7 vs. 18.0±2.5). The lower values for viability than total motility likely reflect agglutination of viable sperm and thus their exclusion from analysis on flow cytometry. The anti-clumping measures employed in the LD-PSA protocol were associated with increased correspondence of measured viability with TMOT. Thus, LD-PSA may offer a more accurate technique to assess viability and acrosome status of equine sperm incubated in capacitating conditions.

121 Effect of pentoxifylline on motility of good- and poor-quality frozen-thawed equine sperm

Equine semen used for intracytoplasmic sperm injection (ICSI) is typically frozen-thawed and may be of poor quality. To prepare sperm for ICSI, semen is typically centrifuged to remove freezing extender. However, centrifugation can cause damage to sperm, which is especially meaningful if sperm quality is already poor. We evaluated a method for selection of sperm without centrifugation, using a “swim-over” technique, and assessed the effect of pentoxifylline, a phosphodiesterase inhibitor that increases sperm motility in other species. To mimic poor-quality semen, we thawed frozen semen (1×) and re-froze it three additional times (4×). Aliquots (0.25 µL; 50,000 sperm) of 1× or 4× semen were placed at the bottom of the right leg of an “H,” made using 15µL of medium by tracing a template placed below a Petri dish. The medium used (Hanks’ balanced salt solution with 40mg mL BSA and added lactate and pyruvate) contained different concentrations of pentoxifylline (0, 0.5, 1, 2 or 4mgmL−1). One µL of medium was removed from the tip of the left arm of the H after 15 and 30min incubation, and the number of sperm were counted. In a second study, we evaluated the effect of pentoxifylline on sperm motility parameters using computer-assisted sperm motility analysis. After thawing, 1× and 4× semen was washed to remove freezing extender and resuspended in the same medium but with 7mgmL−1 bovine serum albumin (BSA), containing the different pentoxifylline concentrations. In Study 1, the number of collected sperm did not differ significantly for 1× sperm exposed to 0 to 4mgmL−1 pentoxifylline (means of 15 to 23 sperm at 15min, and 18 to 25 sperm at 30min). Similarly, in 4× frozen semen, there was no significant difference in number of collected sperm between 0mgmL−1 and 2 or 4mgmL−1 pentoxifylline concentrations (&amp;lt;1 to 6 at 15 min; 5 to 6 at 30min). In Study 2, at 0min,% total motility was significantly higher in 1 and 2mgmL−1 pentoxifylline than in 0mgmL−1 for 1× sperm (47.8±1.7 and 49.3±1.9, vs. 32.1±3.9, respectively; P=0.018) and significantly higher for 1, 2, and 4mgmL−1 pentoxifylline than for 0mgmL−1 for 4× sperm (3.9±0.9, 5.7±0.4, and 8.2±0.5, vs. 1.2±0.4; P=0.0001). Similar results were found at 15 and 30min for 1×, and at 15min for 4×. Pentoxifylline at 1 to 4mgmL−1 significantly increased the percentage of progressive motility in 1× sperm at 30min (17.8±1.3, 21.8±2.7, and 20.3±1.2, vs. 10.0±0.4; P=0.002) and, at 4mgmL−1, increased the percentage of progressive motility in 4× sperm at 0min (1.43±0.1 vs. 0.2±0.1; P=0.005) and 15min (1.4±0.2 vs. 0.1±0.0; P=0.0001). Exposure of poor-quality semen to pentoxifylline at 4mgmL−1 improved total and progressive motility but did not increase the recovery of motile sperm in a swim-over collection preparation.

Specific Activity of Superoxide Dismutase in Stallion Seminal Plasma Is Related to Sperm Cryotolerance.

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability.