Accepted Manuscript The Effect of Low Oxygen During The Early Phases of Sperm Freezing in Stallions with Low Progressive Motility: Can We Improve Post Thaw Motility of Stallion Sperm? Christa Darr, Kelly Martorana, Tawny Scanlan, Stuart Meyers PII:
S0737-0806(16)30030-2
DOI:
10.1016/j.jevs.2016.03.022
Reference:
YJEVS 2070
To appear in:
Journal of Equine Veterinary Science
Received Date: 4 February 2016 Revised Date:
30 March 2016
Accepted Date: 30 March 2016
Please cite this article as: Darr C, Martorana K, Scanlan T, Meyers S, The Effect of Low Oxygen During The Early Phases of Sperm Freezing in Stallions with Low Progressive Motility: Can We Improve Post Thaw Motility of Stallion Sperm?, Journal of Equine Veterinary Science (2016), doi: 10.1016/ j.jevs.2016.03.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The Effect of Low Oxygen During The Early Phases of Sperm Freezing in Stallions with Low Progressive Motility: Can We Improve Post Thaw Motility of Stallion Sperm?
*corresponding author,
[email protected]
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Christa Darr, Kelly Martorana, Tawny Scanlan, Stuart Meyers* Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, 1089 Veterinary Medicine Dr., Bldg VM3B Davis, CA 95616
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Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, 1089 Veterinary Medicine Dr., Bldg VM3B Davis, CA 95616
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Acknowledgement: This project was supported by the Center for Equine Health with funds provided by the State of California satellite wagering fund and contributions by
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private donors.
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ABSTRACT The objective of this study was to determine the effectiveness of oxygen removal in minimizing oxidative stress in order to improve fertile longevity of equine spermatozoa. Stallion ejaculates (n=6) were treated with increasing doses of the
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oxygen scavenger Oxyrase® (0.6 U/ml, 1.2 U/ml, 2.4 U/ml, and 5.0 U/ml). Samples were cryopreserved, thawed, and analyzed for motility over 24 hours at ambient temperature. These data suggest a role of excessive oxygen in the reduced motility of cryopreserved samples as 2.4 U/ml Oxyrase® minimized the loss of post thaw motility over the other
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doses and control treatments. Oxygen, a key component of reactive oxygen species (ROS) generation, is directly involved in oxidative damage and its removal may trigger
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metabolic changes that are more suited for post thaw motility maintenance. Removal of oxygen from semen prior to cryopreservation may promote increased post thaw fertility and may have the potential to overcome stallion variability in cryosurvival. Keywords: stallion, cryopreservation, oxidative stress, sperm, Oxyrase®
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INTRODUCTION
The use of frozen semen in most species is limited by inter- and intra-species morphology, membrane composition, biophysical traits, and metabolic differences between spermatozoa that prevent broad success and widespread standardization of
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cryopreservation protocols. This high degree of variation between ejaculates, males, and entire species, reflects the level of sperm sensitivity to the freeze–thaw process [1].
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The initial cooling steps of the cryopreservation process essentially set the stage in a biophysical sense for sperm survival upon thawing; cooling exposes these cells to osmotic fluctuation and membrane changes that eventually lead to a state of oxidative stress [2, 3]. Oxidative stress is associated with an upregulation of reactive oxygen species (ROS), in which mitochondria are both the main source and target of ROS [4]. This susceptibility of mitochondria to oxidative stress is not conducive to post thaw survival, as the integrity of these organelles are crucial to ATP production, Ca2+ homeostasis, and other cell functions. Successful low temperature survival requires sperm to survive numerous stressful events that are encountered including, as outlined
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by Mazur [5, 6], volume excursions, mechanical stresses, centrifugation, osmotic damage, and cryopreservative toxicity. Oxidative byproducts are believed to be the major contributing factor to sperm cell death and poor fertility of frozen sperm [7]. When sperm are exposed to low temperatures, cellular production of ROS increases and this
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results in increased susceptibility to DNA, membrane, and mitochondrial oxidative attack from metabolic byproducts compared to fresh sperm [8-10].
Plasma membranes, particularly high in polyunsaturated fatty acids (PUFA), are extremely sensitive to peroxidation by ROS and these reactive molecules are produced
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by membrane exposure to oxygen (O2). Consequently, peroxidation results in leaky membranes and eventually a total loss of motility [11]. A likely explanation for loss of
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motility could be due to the proximity of the damage in sperm flagellae to energygenerating mitochondria. The mitochondria are the main source of ROS in the spermatozoon [4] and are implicated in many of the pathological processes in sperm cells [12]. Further, mitochondria are more likely to sustain damage from oxidative attack because, unlike nuclear DNA, mitochondrial DNA (mtDNA) is not protected by protamines and mtDNA may not be transcriptionally and translationally functional in
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ejaculated sperm.
Due to the principal role of O2 in the formation of ROS and the significant involvement of ROS and O2 radicals in reduced post thaw sperm quality, sperm cell survivability has the potential to be improved if the cryopreservation procedure (CPA
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exposure, freezing, and thawing) was carried out under anaerobic conditions. Oxygen also has crucial roles in ATP production and there is evidence that stallion sperm
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utilizes oxygen primarily by oxidative phosphorylation [13]. The rate of lipid peroxidation (LPO) in sperm and the rate of consequent motility loss are linear functions of the partial pressure of O2 in the medium [11]. To achieve low O2 tension, incorporation of a commercially available E. coli membrane preparation, Oxyrase®, into the cryomedium has been utilized [14, 15]. This commercial product contains an extract of bacterial electron transport systems that in the presence of a suitable hydrogen donor substrate (i.e. lactate) can decrease O2 in solutions to effectively low levels [16]. We selected Oxyrase® for this study because it has been used for mouse and monkey sperm
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preservation as well as other cell systems and it has been shown to not be cytotoxic to sperm. The present study examines the effects of lowering the oxygen concentration using the Oxyrase® bacterial cell wall reagent on the motility of frozen-thawed equine
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sperm. To determine if anaerobic conditions positively influenced post thaw quality of sperm, cells were treated with increasing doses of Oxyrase® prior to cryopreservation.
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2. MATERIALS AND METHODS
2.1. Reagents
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INRA96 cryopreservation medium was purchased from Breeder’s Choice, Inc. (Rochester Hills, MI). Freshly laid chicken eggs were obtained from the Hopkins Avian Facility, Department of Animal Science, UC Davis. EC-Oxyrase® (product no. EC-0100) was obtained from Oxyrase Corp. (Mansfield, OH), is supplied as a sterile frozen stock solution at 30 units/ml (U/ml) in 20 mM Phosphate Buffer at a neutral pH, and will be termed “Oxyrase” henceforth throughout this manuscript. One unit of Oxyrase activity
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will reduce dissolved oxygen (in 1 mL of air saturated 40 mM phosphate Buffer, pH 8.4, at 37°C) at the rate of 1% per second. All other ch emicals were obtained from Sigma Chemical Company (St. Louis, MO).
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2.2. Animal Handling, Semen Collection, and Sperm Preparation Light horse breed stallions (n=6) were individually housed at the Center for
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Equine Health and Animal Science Horse Barn at the University of California, Davis. Stallions were maintained according to the Institutional Animal Care and Use Committee protocol approved by the University of California. Stallions were phantomtrained for semen collection into a Missouri model artificial vagina equipped with an inline nylon mesh filter to separate the gel fraction of the ejaculate. The gel-free semen fraction was extended in INRA96. Prior to semen collections for this study, stallions were collected daily for seven days to stabilize extragonadal sperm reserves. Sperm concentration was determined and semen was diluted with INRA96 extender without
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cryopreservative or Oxyrase® treatments to 50-100 million sperm/ml within 2-3 minutes of collection for transport to the laboratory.
2.3. Motility Evaluation
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All stallions selected for this study were noted for having low progressive motility (<50%) during summer months in order to have higher sensitivity for Oxyrase® treatment
differences in these studies. Fresh sperm (50 x 106/ml, washed, centrifuged) and prefreeze sperm (50 x 106/ml, washed and resuspended with cryopreservative and various
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levels of Oxyrase®) sperm motility characteristics were measured with a computer-
assisted sperm analyzer (CASA) using HTM Ceros (Version 12.2 g; Hamilton Thorne
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Biosciences, Beverly, MA). Sperm that were cryopreserved were analyzed at 0, 6, and 24 hours post thaw. At least 200 cells in a minimum of five fields were evaluated on a pre-warmed slide. Slides were maintained at 37oC through use of a heated slide holder (Hamilton Thorne Research). The following instrument settings were used for CASA analysis: frame rate, 60 Hz; frames acquired, 30; minimum contrast, 80; minimum cell size, 4 pixels; static VAP cutoff, 20 m/s; static VSL cutoff, 10 m/s; progressive VAP
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threshold, 25 μm/s; progressive STR threshold, 80%; static intensity limits, 0.6 - 1.4; static size limits, 0.6 - 2.31; and static elongation limits 0-80. Percent total motility (TM) and percent progressive (forward) motility (PM) were determined at all time points
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analyzed.
2.4. Oxyrase® Treatment of Fresh and Cryopreserved Sperm
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Oxyrase® (EC-Oxyrase® suspension), derived from a membrane fraction of Escherichia coli, removes the majority of oxygen from liquid media [5]. Extended semen samples were centrifuged at 300xg for 10 minutes (washed) and then pellets were resuspended into 15 ml Falcon tubes in the following Oxyrase® treatments at 400 x 106 sperm/ml in 5 ml cryopreservation media (INRA96): 0.6 U/ml (2.1% v/v), 1.2 U/ml (4.3% v/v), 2.4 U/ml (8.7% v/v) and 5.0 U/ml (25.0% v/v). The tubes were then placed in a 37°C water bath for 60 minutes along with a test sa mple containing 0.001% methylene blue for each concentration of Oxyrase® to confirm decreased oxygen in the control and cryopreserved sperm samples. In preliminary studies using methylene blue decoloration
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[17], we detected decreased O2 concentration at less than 15 min following incubation of washed sperm in INRA96 medium (FIGURE 1). Incubation of Oxyrase® cryopreservation medium at 37°C resulted in the mos t effective O2 removal in comparison to ambient temperature. Control fresh extended semen was incubated with
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or without Oxyrase for 30 min at ambient temperature prior to CASA analysis.
2.5. Cryopreservation
After incubation for 60 minutes in INRA96 containing four concentrations of
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Oxyrase®, sperm samples were removed from the 37°C water ba th and placed on the benchtop at ambient temperature and were supplemented with 3% egg yolk. Slow
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addition over a one minute period of glycerol cryoprotectant to the INRA96 milk-based medium with continuous gentle mixing was then performed at ambient temperature to achieve a final concentration of 3% glycerol. Semen was loaded into 0.5 mL clear polyethylene straws (IMV technologies, Maple Grove, MN, USA) at 400 x 106 cells/mL and allowed a 15 min room temperature equilibration period. Semen straws were cryopreserved using a liquid nitrogen programmable freezer (Planer PLC, Middlesex,
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UK) at an initial cooling rate of -0.5°C/min from a mbient to 5°C, 5°C to 0°C at a rate of 10°C/min, and lastly a rate of -100°C/min until rea ching -110°C. Once sample temperature reached -110°C, straws were plunged int o liquid nitrogen (LN2, -196°C) and stored in a liquid nitrogen semen tank for at least 24 hours before evaluation after
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transfer from the programmable freezer. The duration of this freezing curve was 45 min. Straws were thawed for 30 sec at 37°C, dilute d with 0.5 ml (1:1, vol:vol) INRA96
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containing no cryopreservative and assessed for total and progressive motility after a 5 min incubation immediately upon thawing (0 hours), at 6 hours, and at 24 hours post thaw at ambient temperature.
2.6. Flow Cytometric Detection of ROS Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with an argon laser (488nm). Green fluorescence was detected with a FL1 530/30 band pass filter and red fluorescence was measured using an FL3 >670 long pass filter. All data was acquired and analyzed using CellQuest Pro®
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software (Becton Dickinson) with a total of 10,000 events collected per sample. Forward scatter and side scatter measurements were taken to generate a scatter plot, which was used to gate for sperm cells only, excluding any larger contaminating cells. Post thaw spermatozoa were washed once with centrifugation at 300xg for 8 min to
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remove cryopreservation medium. Intracellular superoxide anion production was
detected with Dihydroethidium (DHE). Oxidation of DHE by superoxide anion produces DNA-sensitive ethidium and 2-hydroxy ethidium that together generate a red nuclear fluorescence in cells that excites at 488 nm, and emits at a wavelength of 630 nm. DHE
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was used in addition to a viability probe, SYTOX Green, to account for dead cells.
SYTOX Green is a nuclear chromosome counterstain that is impermeant to live cells
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and thus only stains non-viable cells. DHE (2 µM) and SYTOX Green (0.05 µM) were added together and allowed to incubate for 15 min prior to flow cytometric assessments. Data are presented as the percentage of live cells labeled with high relative levels of red fluorescence.
2.7. Statistical analysis
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This experiment was conducted six times, each time with sperm from a different male. Analysis was performed using R statistical software (R Foundation for Statistical Computing, Vienna, Austria) with a level of significance P < 0.05. The effects of oxygen removal were analyzed using a mixed effects model to account for the repeated
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measures of the design. In the model, stallion was used as a random effect to account for the well documented inter-stallion variability based on the assumption that these
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stallions are a representative sample from the larger population. Percentage data were transformed with the arcsin of the square root. Model fit was assessed visually with a Normal Probability Plot. Differences between treatments were compared with Tukey’s multiple comparison analysis in the multcomp package of R. Data are presented as untransformed sample means (mean ± SEM).
3.0 RESULTS AND DISCUSSION Effects from Oxyrase® in fresh stallion sperm
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Figure 1 demonstrates the effect on oxygen removal in sperm-free medium of Oxyrase® treatment at four concentrations when incubated at ambient temperature in closed 1 ml eppendorf tubes after Oxyrase® loading. Prior to freezing, fresh semen from six stallions demonstrated 81.9 ± 3.2% total motility and 38.1 ± 3.2% progressive
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motility. Total motility (Fig. 2A) in fresh extended semen was not affected by either
Oxyrase® treatment or incubation for 6 hr or 24 hr (not shown). Progressive motility (Fig. 3A) in fresh extended semen at ambient temperature declined with exposure to all
controls were evident at 0 and 6 hrs (P <0.05).
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Oxyrase® concentrations at ambient temperature for 30 min although differences from
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Post thaw motility of stallion sperm in response to Oxyrase® addition
Immediately upon thawing for 30 sec at 37°C, the u ntreated frozen-thawed sperm samples resulted in a post thaw total motility of 24.2 ± 5.0% and progressive motility of 15.7 ± 3.4% (Fig. 2B, 3B). Both total and progressive motility was decreased (P <0.05) in thawed sperm in comparison to fresh semen from the same stallions (Fig. 2B, 3B). Upon thawing (T0), the untreated (0 U/ml) Oxyrase® treatment group resulted
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in total and progressive motility no different than the 0.6 U/ml, but less than the 1.2 U/ml, 2.4 U/ml and 5 U/ml Oxyrase® treatment groups. However, sperm samples treated with 2.4 U/ml Oxyrase® prior to cryopreservation resulted in the highest average total and progressive motility after thawing (Fig. 3 and 4). These results indicate that
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69.2% of the total motility and 98% of the progressive motility were preserved after a freeze-thaw cycle in sperm cells frozen in a cryopreservation medium supplemented
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with 2.4 U/ml Oxyrase®.
Frozen-thawed sperm samples measured at 6 hours post thaw (Fig. 2C, 3C) with
Oxyrase® supplementation at 1.2 U/ml, 2.4 U/ml, and 5 U/ml concentrations demonstrated substantial motility benefits, as these treatments resulted in significantly greater total motility maintenance compared with the 0 U/ml Oxyrase® control group (P <0.05). Most notably, 2.4 U/ml Oxyrase® continued to maintain the greatest average total motility after 6 hours of incubation post thaw compared to the other Oxyrase® concentrations although not significant. In addition, progressive motility was best maintained in the 2.4 U/ml Oxyrase® groups compared with the 0 U/ml, 0.6 U/ml
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treatment groups (P <0.01), however not significantly different than the 1.2 U/ml and 5.0 U/ml treatment group (P =0.17). After 24 hours of incubation post thaw, the 1.2 U/ml, 2.4 U/ml, and 5 U/ml groups resulted in significantly greater total motility (Fig. 2D) compared with the 0 U/ml
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Oxyrase® control group (P <0.05). More specifically, sperm samples exposed to 2.4 U/ml Oxyrase® cryopreservation medium preserved significantly greater total motility at 24 hour measurement compared with the 0 U/ml and 0.6 U/ml Oxyrase® groups (P
<0.05); however, 2.4 U/ml Oxyrase® treatment did not significantly differ from the 1.2
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U/ml and 5 U/ml Oxyrase® treatment group (P =0.15). Progressive motility (Fig. 3D) was best maintained in the 2.4 U/ml Oxyrase® group compared to the 0 U/ml controls (P
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<0.05).
Sperm viability measured using flow cytometry and SYTOX green fluorescence clearly demonstrated that the cryopreservation process resulted in decreased cell viability, as was expected. However, the two highest levels of Oxyrase® preserved 33.0 ± 2.9% and 36.8 ± 3.1% of viable sperm which was greater than controls (P <0.05, Fig. 4). Further, we observed low levels of ROS production in fresh extended sperm in
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control and all Oxyrase® treatments. Following freezing and thawing, stallion sperm demonstrated a 5-fold overall increased ROS production by live sperm suggesting that oxidative stress due to low temperature storage is highly detrimental to equine sperm (Fig. 5). However, in this study we failed to demonstrate that ROS production could be
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decreased by Oxyrase® treatment, although motility was seen to improve, and this was supported by the significant decrease in sperm viability.
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Oxyrase® is an Escherichia coli membrane derivative containing the bacteria’s electron transport system and can reduce oxygen concentrations in solutions in the presence of cells with a hydrogen donor substrate. This product has been shown to be beneficial in creating an artificial anaerobic environment for the food industry [18], in antibiotic testing [19, 20], and has been for used in cryopreservation of mouse, and monkey sperm [5, 14, 21]. Interestingly, Oxyrase® has little or no effect (positive or negative) on the motility of unfrozen sperm as we have observed in our study and as previously reported [5, 21]. In addition to its electron transport machinery, Oxyrase® contains a penicillin binding protein that may inactivate penicillin and related antibiotics
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according to the company’s package insert. Microbiological studies have demonstrated that Oxyrase improves culture of numerous bacteria and has been used to optimize antibiotic susceptibility testing [19, 20, 22]. Although INRA96 cryopreservation medium contains sodium penicillin, gentamicin sulphate, and amphotericin B covering a broad
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spectrum, we observed no bacterial contamination in this study in any treatment group. Although oxidative insult is at the root of most, if not all, sublethal cell damage resulting from cryopreservation [8, 15, 23, 24], there have been few measures proposed to mitigate excessive oxygen processing. While numerous studies have used
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antioxidant compounds to restrict formation and to scavenge ROS in sperm from
various species, few studies have successfully demonstrated that this strategy is fully
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effective to ensure cell survival. Alternatively, Adler [16] and Mazur [5, 21] have proposed that removal of formed elemental O2, perhaps prior to ROS conversion from O2, during the cryopreservation process may confer substantial protection to cells undergoing cryopreservation. Mazur [5] described this anaerobic effect on mouse sperm in four major beneficial ways. First, it nearly eliminates the damage resulting from centrifugation. Second, it reduces the osmotic damage associated with the abrupt
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introduction and removal of hyperosmotic concentrations of glycerol. Third, because of the second point and in combination with short exposures to reduce glycerol toxicity, it enhances one’s ability to use concentrations of glycerol that are about three times higher than those generally used previously. Fourth, in combination with the higher
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glycerol concentrations, the beneficial anaerobic effect substantially increases the normalized motility of sperm frozen to ??-70°C from about 20 to ??39%.
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While we did not observe striking benefits from Oxyrase® treatment as were described in the mouse and monkey, we did demonstrate that this artificial anaerobic media prevents substantial motility loss resulting from cryopreservation in horse semen. We demonstrated maintenance of 70% and 98% of thawed total and progressive sperm motility at the higher levels of Oxyrase®, which could be a significant improvement for many stallions with resistance to cryopreservation. The enhancement of sperm total and progressive motility in frozen-thawed sperm and in sperm stored at ambient temperature for six hours at 2.4 U/ml Oxyrase® could be beneficial to stallions with a range of post-thaw sperm motility. Utilization of Oxyrase for transporting fresh extended
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or export of cryopreserved sperm should be studied further for any negative consequences for microbial growth for stallion semen with potential bacterial exposure since there could be a propensity, at least in theory, to potentiate bacterial growth in
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stored semen.
4.0 Conclusions
Supplementation of 2.4 U/ml Oxyrase® to cryopreservation medium prior to
cryopreservation consistently resulted in significantly greater total and progressive
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motility over 24 hours of measurement compared with the 0 U/ml Oxyrase® control
group. The ascending motility values at the 3 time points reflect a dose response that indicates a saturation effect at the 5 U/ml Oxyrase dose. Further studies are necessary
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motility as well as freezability.
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to determine the effects from Oxyrase® treatment in stallions with high and low levels of
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Figure Legends Figure 1: Methylene blue assay for dissolved oxygen in INRA96. Eppendorf tubes (1.5 ml) containing 0.001% methylene blue in 1 ml INRA96 for each concentration of Oxyrase® were placed in a 37°C water bath for 60 minutes to confirm decreased
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oxygen in the control and cryopreserved sperm samples. Significant clearing of
methylene blue indicating that scavenging of free oxygen is visible at the 2.4 and 5.0 U/ml levels of Oxyrase®.
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Figure 2: Total sperm motility in fresh extended and cryopreserved stallion sperm
treated with Oxyrase®. A. Sperm assessed after initial dilution, washing and adjustment to 50 x 106 sperm/ml and addition of Oxyrase, B. Sperm assessed immediately after
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thawing at 37oC for one minute, containing cryopreservatives and Oxyrase® prior to freezing, C. Post-thaw sperm assessed after 6 hrs incubation at ambient temperature, D. Post-thaw sperm assessed after 24 hr storage at ambient temperature. Different superscripts indicate significant differences (P <0.05) within total motility endpoints
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(n=6)
Figure 3: Progressive sperm motility in fresh extended and cryopreserved stallion sperm treated with Oxyrase®. A. Sperm assessed after initial dilution, washing and adjustment to 50 x 106 sperm/ml and addition of Oxyrase, B. Sperm assessed immediately after
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thawing at 37oC for one minute, containing cryopreservatives and Oxyrase® prior to freezing, C. Post-thaw sperm assessed after 6 hrs incubation at ambient temperature,
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D. Post-thaw sperm assessed after 24 hr storage at ambient temperature. Different superscripts indicate significant differences (P <0.05) within total motility endpoints (n=6)
Figure 4: Percentage of membrane-intact sperm labeled using SYTOX Green fluorescence detected by flow cytometry. A. Fresh, extended sperm at four levels of Oxyrase® treatment, B. Post-thaw sperm containing four levels of Oxyrase®. Different superscripts indicate significant differences (P <0.05) within total or progressive motility
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endpoints (n=6). All post-thaw sperm displayed membrane integrity lower than that of fresh sperm (P <0.05).
Figure 5: Percentage of ROS-producing (DHE-positive) sperm fluorescence detected by
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flow cytometry. A. Fresh, extended sperm at four levels of Oxyrase® treatment, B. Postthaw sperm containing four levels of Oxyrase®. Different superscripts indicate
significant differences (P <0.05) within total or progressive motility endpoints (n=6). All
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post-thaw sperm displayed DHE fluorescence higher than that of fresh sperm (P <0.05).
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Acknowledgement: This project was supported by the Center for Equine Health with funds provided by the State of California satellite wagering fund and contributions by
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private donors.
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Removal of oxygen from equine semen prior to cryopreservation may promote increased post thaw fertility. Bacterial cell wall extract Oxyrase had no effect on fresh sperm total or progressive motility of stallion sperm. Thawed cryopreserved equine sperm showed increased motility with the addition of Oxyrase in a dose-dependent manner in thawed sperm up to 24 hrs after thawing in comparison to thawed sperm containing no Oxyrase.
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