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From the Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain.
| Correspondence to: Dr Jordi Roca, Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, Campus de Espinardo, University of Murcia, E-30071 Murcia, Spain (e-mail: roca{at}um.es). |
| Received for publication May 13, 2004; accepted for publication July 16, 2004. |
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Abstract |
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Key words: Antioxidants, cryopreservation, sperm, porcine
Cryopreservation of spermatozoa affects primarily the plasma membrane, with the damage varying in magnitude among species, owing to the structural complexity it presents. Species differences in the susceptibility of their spermatozoa to cooling, freezing, and thawing process are thus largely attributable to compositional variations of the sperm plasma membrane. The major susceptibility is related, in part, to the content of polyunsaturated fatty acids (White, 1993), which are susceptible to undergoing lipid peroxidation in the presence of the reactive oxygen species (ROS). The lipid peroxidation cascade is initiated when spermatozoa are attacked by ROS, which results in a loss of unsaturated fatty acids from the plasma membrane and a corresponding decline in the survival and fertilizing ability of these spermatozoa (Aitken, 1995). Boar spermatozoa are particularly susceptible to oxidative attack by ROS because they contain large amounts of polyunsaturated fatty acids (White, 1993).
Sperm oxidative damage is the result of an improper balance between ROS generation and scavenging activities. The scavenging potential of the ejaculate is normally maintained by adequate levels of antioxidants present in the seminal plasma (SP). When boar ejaculates are cryopreserved, spermatozoa are concentrated so that they can be adequately extended in cryoprotecting fluids, and removal of SP is customary. Spermatozoa lose, in consequence, the antioxidant protection that SP provides, leaving them vulnerable to oxidative attack. In addition, it is now generally accepted that the cryopreservation process induces the formation of ROS (Watson, 2000). In domestic animals, defective and dead spermatozoa have been identified as a major source of ROS generation during cryopreservation (Bailey et al, 2000). Consequently, during cryopreservation, boar spermatozoa are especially open to oxidative damage by the concomitant actions of their relatively high proportion of polyunsaturated fatty acids, the removal of the scavenging capability of SP before freezing, and the generation of ROS by defective and dead spermatozoa, ultimately leading to membrane lipid peroxidation and membrane damage.
One way of overcoming this cascade could be the incorporation of
antioxidants into the freezing extenders. To date, limited information is
available on the application of antioxidants to prevent cryodamage of boar
spermatozoa. Only 2 recent reports addressed the successful use of
-tocopherol (Peña et al,
2003) and butylated hydroxytoluene
(Roca et al, 2004) as
additives to extenders for freezing boar spermatozoa.
Seminal plasma is a powerful source of antioxidants (Saleh and Agarwal, 2002), and evidence suggests that as long as spermatozoa are suspended in SP, they are protected from oxidative damage. The most common ROS with potential implications in sperm oxidative damage include the superoxide anion (O2-) and hydrogen peroxide (H2O2). In order to counteract the toxic effect of these ROS, the SP contains the enzymes superoxide dismutase (SOD) and peroxidases, such as catalase (CAT). The SOD converts the O2- to H2O2, and the CAT then converts H2O2 to H2O and O2, eliminating the potential ROS toxicity (Aitken, 1995).
In view of the above, the present study evaluated the potential protective effect of SOD, CAT, or both, 2 oxygen species-scavenging enzymes normally present in boar SP, against cryopreservation-induced damage to boar spermatozoa. Spermatozoa were monitored postthaw for their survival and ability to produce blastocysts in vitro.
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Materials and Methods |
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·cm; Elgastat UHQPS, Elga Ltd, England). The basic medium used for sperm extension was Beltsville thawing solution (BTS, composed of 205 mM glucose, 20.39 mM NaCl, 5.4 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA; Johnson et al, 1988), containing kanamycin sulfate (50 µg/ml). The basic medium used for sperm cryopreservation was a lactose-egg yolk (LEY) extender composed of 80% (vol/vol) ß-lactose solution (310 mM in water), 20% (vol/vol) egg yolk, and 100 µg/mL kanamycin sulfate. The medium used for the collection and washing of oocyte-cumulus complexes was Dulbecco phosphate-buffered saline (DPBS) medium composed of 136.89 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.46 mM CaCl2-2H2O. The oocyte maturation medium was the bovine serum albumin (BSA)-free North Carolina State University-23 (NCSU-23) composed of 108.73 mM NaCl, 4.78 mM KCl, 1.7 mM CaCl2-2H2O, 1.19 mM KH2PO4, 1.19 mM MgSO4-7H2O, 25.07 mM NaHCO3, 5.55 mM D-glucose, 1 mM L-glutamine, 7 mM taurine, 5 mM hypotaurine, 75 µg/mL potassium penicillin G, and 50 µg/mL streptomycin sulfate (NCSU-23; Petters and Wells, 1993) and supplemented with 10% (vol/vol) porcine follicular fluid, 0.1 mg/mL cysteine, 10 ng/mL epidermal growth factor (EGF), 10 IU/mL eCG (Folligon, Intervet International BV, Boxmeer, the Netherlands), and 10 IU/mL of hCG (Chorulon, Intervet International). The fertilization medium was a modified Tris-buffered medium (TBM) composed of 20 mM Tris (Trizma Base), 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2-2H2O, 11 mM D-glucose, and 5 mM sodium pyruvate (TBM; Abeydeera and Day, 1997), supplemented with 2 mM caffeine and 0.2% BSA (fraction V; A-7888, initial fractionation by cold alcohol precipitation). The embryo culture medium was NCSU-23 with 0.4% BSA.
Stock solutions of SOD (S-5395; 1.5 x 105 IU/mL) and CAT (C-9322; 2 x 105 IU/mL) were prepared according to the instructions provided by the manufacturer (Sigma). After preparation, stock solutions were preserved at -80°C until used.
Semen Processing and Sperm Cryopreservation
Ejaculates were collected weekly from 3 fertile Pietrain boars. Sperm-rich
ejaculate fractions were obtained using the gloved-hand method and extended
(1:1, vol/vol) in BTS. After collection, semen characteristics (total sperm
numbers per ejaculate, subjective sperm motility, and acrosome integrity) were
microscopically evaluated, using standard laboratory techniques
(Martin Rillo et al, 1996), and only ejaculates with >75% motile spermatozoa and >80% normal
acrosomes were used.
Spermatozoa were cryopreserved using the straw freezing procedure described by Westendorf et al (1975) as modified by Thurston et al (1999). Briefly, extended semen (1:1, vol/vol in BTS) was cooled to 17°C over 3 h. After centrifugation at 800 x g for 10 minutes (Megafuge 1.0 R, Heraeus, Germany), the pellets were diluted in LEY (pH 6.2 and 330 ± 5 mOsm/Kg) to a concentration of 1.5 x 109 cells/mL. After further cooling to 5°C in 90 minutes, the spermatozoa were resuspended with LEY-glycerol-orvus ES paste (LEYGO) extender (92.5% LEY + 1.5% Equex STM [Nova Chemical Sales Inc, Scituate, Mass] and 6% glycerol, vol/vol; pH 6.2 and 1650 ± 15 mOsm/Kg) to a final concentration of 1 x 109/mL. The resuspended, cooled spermatozoa were packed into 0.5 polyvinyl chloride-French straws (Minitüb, Tiefenbach, Germany), and frozen using a controlled-rate freezer (IceCube 1810, Minitüb) as follows: cooled to -5°C at 6°C/min, from -5°C to -80°C at 40°C/min, held for 30 seconds at -80°C, then cooled at 70°C/min to -150°C, and finally plunged into liquid nitrogen.
Thawing and Postthaw Sperm Survival Evaluation
Thawing of the frozen spermatozoa was done in a circulating water bath at
37°C for 20 seconds. Thawed spermatozoa from 2 straws were extended with
BTS (1:2, vol/vol; 37°C) and incubated in a water bath at 37°C for 150
minutes. Spermatozoa were assessed for motility, kinematic parameters, and
viability.
Sperm motility and kinematic parameters were objectively evaluated using a computer-aided sperm analysis (CASA) system (sperm class analyzer [SCA]; Microptic, Barcelona, Spain). The incubated frozen-thawed spermatozoa were re-extended in BTS to a concentration of 20 x 106/mL. For each evaluation, a 4 µL sperm sample was placed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) and 3 fields were analyzed at 39°C to assess a minimum of 100 spermatozoa per sample. The proportion of total motile spermatozoa (% TMS), of rapidly progressive motility spermatozoa (% RPMS, >50 µm/s), average path velocity (VAP, µm/s), linearity (% LIN), straightness of the average path (% STR), amplitude of lateral head displacement (ALH, µm), and beat cross frequency (BCF, Hz) were determined.
Sperm viability was evaluated in terms of plasma membrane integrity, mitochondrial membrane potential, and acrosomal integrity. These characteristics were analyzed simultaneously using a modification of a triple fluorescent procedure described by Graham et al (1990), which includes DNA-specific fluorochrome propidium iodide (PI), the mitochondria-specific fluorochrome rhodamine-123 (R123), and the acrosome-specific fluorochrome fluorescein isothiocyanate-labeled peanut (Arachis hypogaea) agglutinin (FITC-PNA). Three hundred spermatozoa were counted under 1000x magnification (Eclipse E800; Nikon, Tokyo, Japan), using BV-2A filter (400 to 440 nm excitation filter, 455 nm emission, 470 nm barrier filter). Four staining patterns could be discerned: viable sperm with intact acrosome (no staining of the head, the tail midpiece stained green), viable sperm with acrosome reacted (both the tail midpiece and the acrosome region stained green), nonviable sperm with intact acrosome (sperm head stained red), and nonviable sperm with acrosome reacted (sperm head stained red and the acrosome region stained green). Values were expressed as percentage (%).
Measurement of Membrane Lipid Peroxidation
Membrane lipid peroxidation was estimated by the end-point generation of
malondialdehyde (MDA) determined by the thiobarbituric acid (TBA) test
(Esterbauer and Cheeseman,
1990). Briefly, extended spermatozoa (250 x 106
cells in 1 mL) were mixed with 1 mL of cold 20% (wt/vol) trichloroacetic acid
to precipitate proteins. The precipitate was pelleted by centrifugation (1500
x g for 10 minutes), and 1 mL of the supernatant was incubated
with 1 mL of 0.67% (wt/vol) TBA in a boiling water bath at 100°C for 10
minutes. After cooling, the absorbance was determined by spectrophotometry
(UNICAM PU 8610 Kinetics spectrophotometer; Philips, Eindhoven, Holland) at
534 nm. The results were expressed as a simple concentration of MDA
(pmol/108 cells).
Measurement of ROS in Thawed Sperm Suspensions
The chemiluminescence methods using luminol
(amino-2,3-dihidro-1,4-phthalazinedione; A-8511) and lucigenin
(bis-N-methylacridinium nitrate; M-8010) described by Aitken et al
(1992) were used with
modifications. Briefly, 250 µM of luminol (4 µL of 25 mM aqueous
solution, Na salt, freshly prepared) or lucigenin (4 µL of 25 mM aqueous
solution) were added to each tube containing 400 µL of frozen-thawed
spermatozoa (30 x 106 spermatozoa/mL) and mixed. Horseradish
peroxide (7.5 µL, 19 IU/mL; type VI, P-8375) was used to enhance
interaction of luminol and hydrogen peroxide. Levels of ROS were assessed by
measuring the luminol- and lucigenin-dependent chemiluminescence with a
luminometer (Multi-Biolumat LB 9505 C; Berthold Technologies, Bad Wildbad,
Germany) in the integrated mode at 37°C for 30 minutes. A positive control
of luminol assay was prepared by adding hydrogen peroxide (0.5-50 µM) to
400 µL of LEYGO plus BTS (1:36, vol/vol). The sensitivity and specificity
of the lucigenin assay were determined by using xanthine (0.05-0.2 mM) and
xanthine oxidase (0.05 U/mL). A negative control was prepared by adding
luminol or lucigenin to 400 µL of LEYGO plus BTS (1:36, vol/vol). Mean ROS
generation was calculated and expressed as the logarithm of photon counts
integrated for 30 minutes.
In Vitro Embryo Production
Ovaries were obtained from prepubertal gilts at a local slaughterhouse and
transported to the laboratory in 0.9% NaCl containing 75 µg/mL potassium
penicillin G and 50 µg/mL streptomycin sulfate maintained at 25°C to
30°C. Oocytes were aspirated from medium-sized follicles (3 to 6 mm in
diameter) with an 18-gauge needle fixed to a 10-mL disposable syringe. Oocytes
surrounded by a compact cumulus mass and having evenly granulated cytoplasm
were washed 3 times in maturation medium, and 45-50 oocytes were transferred
into each well of a Nunc 4-well multidish containing 500 µL of
pre-equilibrated maturation medium previously covered with warm mineral oil
and cultured for 22 hours with added hormones and then for another 22 hours
without hormones in 5% CO2 in air at 39°C. After the completion
of in vitro maturation, cumulus cells were removed with 0.1% hyaluronidase in
NCSU-23 and washed 3 times with fertilization medium. After washing, batches
of 30 oocytes were placed in 50-µL drops of the same medium in a 35 x
10-mm Petri dish. The dishes were kept in the incubator for about 30 minutes
until spermatozoa were added for fertilization. One hundred microliters of
extended spermatozoa from 1 pool of 3 frozen-thawed straws were washed 3 times
by centrifugation at 1900 x g for 3 minutes in DPBS medium
supplemented with 0.1% BSA, 75 µg/mL potassium penicillin G, and 50
µg/mL streptomycin sulfate (pH 7.2). At the end of the washing procedure,
the sperm pellet was resuspended in fertilization medium, and then 50 µL of
this sperm suspension was added to the medium that contained oocytes so that
each oocyte was exposed to 2000 sperm cells
(Gil et al, 2003). At 6 hours
after insemination, oocytes were washed and transferred (30 oocytes per well)
to a Nunc 4-well multidish containing 500 µL of embryo culture medium,
covered with mineral oil and cultured at 39°C, 5% CO2 in air
for 7 days. At 48 and 168 hours of culture, cleavage rate (taken as evidence
of fertilization) and blastocyst formation, respectively, were evaluated under
a stereomicroscope.
Experimental Design
Three separate experiments, each using a completely randomized design, were
performed to evaluate the effect of adding different concentrations of SOD
and/or CAT on freezer extender to the postthaw boar sperm performance, as
follows:
The concentrations of SOD and/or CAT used in the present experiments were the lowest concentrations that improved the postthaw sperm survival in a preliminary experiment (unpublished data). In all experiments, treatments were compared to a control sample, without SOD or CAT additives.
Each experimental day, stock solutions of the 2 oxygen species-scavenging enzymes were thawed and added to LEY and LEYGO extenders to get a final concentration of 600 and 400 IU/mL to SOD and CAT, respectively. After the centrifugation step during the cryopreservation process, the supernatant was removed and the sperm pellet was split into aliquots. Each aliquot was extended with different proportions of LEY and LEYGO supplemented or not with SOD and/or CAT to get the appropriate final enzyme concentrations. The extended aliquots were then cryopreserved according to the protocol described above.
Effects were reflected by data on sperm cryosurvival, recorded as sperm motility and viability, at 30 and 150 minutes postthawing, and by data on in vitro fertility and embryo development. In addition, data of ROS generation levels and of membrane lipid peroxidation (as MDA production) were also recorded at 30 and 150 minutes postthaw.
Statistical Analyses
All data editing and statistical analyses were performed in SPSS, version
11.5 (SPSS Inc, Chicago, Ill). Data from 7 replicates from each experiment
were analyzed by analysis of variance (ANOVA) using the mixed-procedure. Data
of postthaw sperm survival, ROS generation, and MDA levels were examined
according to a statistical model that includes the fixed effects of
antioxidant concentration and postthaw evaluation times, and the random effect
of semen pool. There were no interactions (P > .05) between
antioxidant concentration and postthaw evaluation times for any of all
parameters evaluated. Therefore, means were averaged throughout postthaw
evaluation times (30 and 150 minutes) to evaluate the main effect of
antioxidant concentration. Percentages of postthaw sperm survival were
subjected to arcsine transformation before analysis. Data of embryo
development (cleaved zygotes, blastocyst rate, and proportion of cleaved
zygotes that developed to the blastocyst stage) were examined according to a
statistical model that includes the fixed effects of antioxidant
concentrations and the random effect of semen pool. Embryo development data
were modeled according to the binomial model of parameters, as described by
Fisz (1980), before analysis.
When ANOVA revealed a significant effect, values were compared using the
Bonferroni test and were considered to be significant when P was less
than .05. Results are presented as least-squares means ± SEM.
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Results |
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Data from in vitro embryo production (Table 1) clearly showed that the addition of SOD in the freezing extender had no significant (P > .05) effects either on rates of oocyte cleavage or on blastocyst formation, or in the proportion of cleaved zygotes that reached the blastocyst stage.
Levels of ROS generation and MDA production are provided in Figure 1. Luminol-mediated chemiluminescence showed significantly lower (P < .05) levels of ROS in thawed spermatozoa that were frozen with SOD than in controls. Lucigenin-mediated chemiluminescence was unaffected (P > .05); all postthaw sperm samples showed low ROS levels. Production of MDA on frozen-thawed spermatozoa was unaffected (P > .05) by the presence of SOD on the freezing extender.
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Experiment 2: Effect of Addition of CAT
The addition of CAT (200 and 400 IU/mL) to the freezing extender
significantly (P < .01) increased postthaw rates of total sperm
motility and rapid progressive motility compared with controls. In relation to
sperm motion kinetics, only VAP was affected by the presence of CAT in the
freezing extender, being significantly (P < .05) higher in
spermatozoa frozen in the presence of CAT. The addition of CAT to the freezing
extender also significantly improved (P < .01) the postthaw
percentage of viable spermatozoa with intact acrosome
(Table 2).
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Data from in vitro embryo production (Table 2) revealed an improvement (P < .01) of cleavage rates and blastocyst formation when spermatozoa were frozen in the presence of 400 IU/mL of CAT compared with controls or the 200 IU/mL CAT groups. However, addition of CAT to the freezing extender did not affect (P > .05) the proportion of cleaved zygotes that developed to blastocyst stage.
Levels of ROS generation and MDA production are provided in Figure 2. Luminol-mediated chemiluminescence showed a significantly lower (P < .05) level of ROS in thawed spermatozoa frozen with CAT compared with controls. Levels of MDA on thawed spermatozoa were unaffected (P > .05) by the presence of CAT in the freezing extender.
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Experiment 3: Effect of the Combined Addition of SOD and CAT
The combined addition of SOD and CAT in the sperm freezing extender
affected (P < .01) all the postthaw sperm survival parameters
evaluated, irrespective the dose (Table
3). The SOD plus CAT combinations improved (P < .01)
the percentages of postthaw total sperm motility and rapid progressive
motility spermatozoa. In relation to the sperm kinematic parameters, VAP, ALH,
and BCF were higher and LIN lower in thaw spermatozoa frozen in the presence
of SOD plus CAT compared with controls. The addition of SOD plus CAT also
increased (P < .01) the postthaw rate of viable sperm with intact
acrosome compared with controls.
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Data from in vitro embryo production (Table 3) showed, when compared with controls, a significantly (P < .01) higher proportion of zygotes cleaved and of blastocysts developed when SOD and CAT were added in combination to the sperm freezing extender. However, the presence of SOD plus CAT did not exert any beneficial effect (P > .05) in the proportion of cleaved zygotes that reached the blastocyst stage.
Levels of ROS generation and MDA production are provided in Figure 3. Luminol-mediated chemiluminescence showed significantly lower (P < .05) levels of ROS in thawed spermatozoa frozen in the presence of SOD plus CAT than in controls. Lucigenin-mediated chemiluminescence was unaffected (P > .05) by the presence of SOD plus CAT in the sperm freezing extender, showing all postthaw sperm samples with low ROS levels. Production of MDA on thawed spermatozoa was unaffected (P > .05) by the presence of SOD plus CAT on the freezing extender.
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Discussion |
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Our finding that the addition of SOD or CAT—alone or in combination—improved postthaw boar sperm survival provides indirect evidence that an oxidative stress, involving the presence of O2- and H2O2, occurs during the freezing and thawing process. This suggestion is in agreement with that of Cerolini et al (2001), who, evaluating the changes in lipid composition undergone by boar spermatozoa during cryopreservation, suggested that cryopreservation enhanced lipid peroxidation and concluded that there is a need for O2- and H2O2 scavengers. In this way, it is well established that oxidative stress plays an important role in the cryoinjury damage of mammalian spermatozoa. Alvarez and Storey (1992) demonstrated that the freeze-thaw process enhanced lipid peroxidation in human sperm. Likewise, Chatterjee and Gagnon (2001) showed evidence of ROS generation by bovine spermatozoa undergoing cooling, freezing, and thawing processes. Although the present experiments were not designed to provide evidence of whether either ROS generation or lipid peroxidation develops during cryopreservation, the levels of chemiluminescence detected by luminol in control samples of thawed spermatozoa clearly show that oxidative stress takes place during cooling, freezing, and thawing of boar spermatozoa. Moreover, the fact that luminol-dependent chemiluminescence was significantly reduced by the addition of CAT and SOD alone or in combination to the sperm freezing extender reflects that these enzymes are capable of scavenging the ROS generated during the cryopreservation process, which reduces the potential damaging effects of oxidative stress and subsequently improves the postthaw functional competence of the thawed boar spermatozoa.
In contrast to the recognition of the damaging effects of oxidative stress, it is established that low concentrations of ROS, specifically O2- and H2O2, mediate the process of capacitation, hyperactivation, and acrosome reaction (human: de Lamirande et al, 1998; bovine: O'Flaherty et al, 1999). Although a drastic fall in ROS generation was shown, no reduction in fertilization rates (taken as the proportion of zygotes that cleaved) was found when CAT and SOD—alone or in combination—were used, indicating that the physiological events involved in the capacitation and fertilization processes were unaffected by the addition of these ROS-scavenging enzymes to the freezing extender at the concentration tested. On the contrary, cleavage rates were highest when CAT, alone (400 IU/mL) or in combination (200 and 400 IU/mL) with SOD, was added to the freezing extender. The data suggest a detrimental effect of excessive H2O2 on the fertilizing potential of thawed boar spermatozoa, which can be reversed by adding CAT to the freezing extender. This concept may be supported by the results of Blondin et al (1997), who demonstrated a reduction in oocyte penetration rates by bovine spermatozoa exposed to artificially produced ROS, generated through the use of the hypoxanthine-xanthine oxidase system, which was reversed by the presence of CAT. Similarly, Kuribayashi and Gagnon (1996), in a study in which murine sperm suspensions were preincubated with CAT prior to in vitro fertilization, indicated that CAT maintains the sperm potential to promote embryo development.
Although a significant improvement in fertilization rates (taken as the proportion of zygotes that cleaved) was observed in spermatozoa frozen in the presence of CAT, alone or in combination with SOD, no differences were found when SOD alone was used. Because the SOD enzyme in the spermatozoon catalyzes the O2- radicals and produces H2O2 (Aitken, 1995), the ineffectiveness of SOD alone might be explained by the fact that its positive effect in removing excessive levels of O2- could be counteracted by the negative effect of the corresponding excessive production of H2O2. The above results also suggest that H2O2—rather than O2-—is more harmful to the fertilizing capability of thawed boar spermatozoa.
In addition to the negative effects on motility and viability, ROS can also damage the DNA in the sperm nucleus (Aitken, 1999; Baumber et al, 2003). Ahmadi and Ng (1999) demonstrated, in research with homologous (mouse) and heterologous (human spermatozoa, hamster oocytes) models, a similar fertilization rate between DNA-damaged and live, intact spermatozoa. The ability to develop to the blastocyst stage was, however, reduced in oocytes fertilized with DNA-damaged spermatozoa. Extrapolating the above findings to boar spermatozoa, the lack of influence of the ROS-scavenger enzymes SOD and/or CAT on the proportion of cleaved zygotes that reached the blastocyst stage might suggest that the addition of these enzymes in the sperm freezing extender does not provide any additional protection to subsequent in vitro embryo development. Although the results of in vitro embryo production should be taken with caution because the blastocyst rate depends much more on others factors than the fertilizing capacity of spermatozoon, the ineffective addition of SOD and/or CAT might be explained by an inefficiency of these ROS-scavenging enzymes to protect sperm DNA. The protective action of CAT and SOD on sperm DNA is still controversial. Lopes et al (1998) demonstrated that CAT was ineffective in preventing human sperm DNA damage and suggested that H2O2 has little effect on sperm chromatin. In contrast, CAT, and not SOD, had protective effects on the DNA of cryopreserved equine spermatozoa. Moreover, Baumber et al (2003) suggested that H2O2 and not O2- is the ROS responsible for DNA damage in equine spermatozoa, at least during the cryopreservation process. The implications of H2O2 and O2-, and the protective role of CAT and SOD, on sperm DNA integrity and its consequences on subsequent sperm ability to develop embryos in vitro or in vivo of thawed boar spermatozoa requires further investigation.
Luminol- and lucigenin-dependent chemiluminescences are the most common methods used for measuring ROS in sperm samples (Sikka, 2004). Differences in the pattern of ROS generation were evident between luminol and lucigenin in the present study. Luminol does not discriminate between intracellular and extracellular ROS generation. In contrast, the lucigenin technique detects only extracellular ROS release (McKinney et al, 1996). Although interpretation of luminol and lucigenin data is complicated by the fact that a variety of biochemical pathways can lead to the generation of the chemiluminescence signal (Aitken et al, 1992), the results obtained in the present study showed that a significantly higher level of ROS was detected with luminol compared with lucigenin in thawed boar sperm suspensions. The high levels of chemiluminescence detected by luminol suggest that a substantial component of ROS generation during the cryopreservation process occurs inside the spermatozoa. Furthermore, because lucigenin detects mainly O2-, the low levels of lucigenin-dependent chemiluminescence detected suggest that extracellular O2- is not an important source of ROS in thawed boar spermatozoa.
It is well established that an excess of ROS causes peroxidation of plasma membrane lipids, boar spermatozoa being especially susceptible because of their high degree of polyunsaturated acids. In the present study, the levels of lipid peroxidation were determined by the measurement of MDA, a degradation product—of small molecular mass—of the peroxidative process (Aitken et al, 1993). The MDA test, assayed by the TBA reaction, remains one of the most efficacious methods for assessing levels of lipid peroxidation in sperm suspensions (Sikka, 2004). In the present study, unlike with human sperm (Gomez et al, 1998), it was not necessary to promote a lipid peroxidation chain reaction in order to generate results that fell within the detectable range of the spectrophotometer. In all thawed sperm samples, the levels of MDA were always high and not different from those previously assessed in thawed boar sperm samples (Roca et al, 2004). In addition, nonsignificant differences in MDA levels between control and CAT and/or SOD supplemented samples were evident. The lack of influence of these ROS-scavenger enzymes on MDA production together with the fact that luminol-dependent chemiluminescence was significantly reduced in samples containing these enzymes may reflect that the addition of CAT and SOD alone or in combination in the sperm freezing extender provides good protection against oxidative stress involved in the cryopreservation process, via mechanisms that do not implicate the production of MDA as a degradation product of the lipid peroxidation process. In this way, it is known that MDA is only one of the several degradation products generated during the lipid peroxidation process of sperm membranes (Gomez et al, 1998).
In conclusion, the present data suggest that the damage undergone by boar spermatozoa during the cryopreservation process is due, in part, to oxidative stress. The addition of SOD or CAT, alone or in combination, to the freezing extender reduces postthaw ROS generation, improving sperm motility and viability. Moreover, the supplementation with CAT, alone or in combination with SOD, also improves the in vitro fertilizing potential (taken as the proportion of zygotes that cleaved) of thawed spermatozoa. For these reasons, CAT, alone or in combination with SOD, could be used as a component in boar cryopreservation extenders.
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Acknowledgments |
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Footnotes |
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References |
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Ahmadi A, Ng S-C. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool. 1999; 284:696-704.[Medline]
Aitken RJ. Free radicals, lipid peroxidation and sperm function. Reprod Fertil Dev. 1995; 7:659-668.[Medline]
Aitken RJ. The human spermatozoon—a cell in crisis? J Reprod Fertil. 1999; 115:1-7.
Aitken RJ, Buckingham, DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol. 1992; 154:466-477.
Aitken RJ, Harkiss D, Buckingham, DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev. 1993; 35:302-315.[Medline]
Alvarez JG, Storey BT. Evidence for increased lipid peroxidative
damage and loss of superoxide dismutase activity as a mode of sublethal
cryodamage to human sperm during cryopreservation. J
Androl. 1992; 13:232-241.
Aurich JE, Schönherr U, Hoppe H, Aurich C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology. 1997; 48:185-192.
Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl. 2000; 21:1-7.[Medline]
Ball BA, Medina V, Gravance CG, Baumber J. Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5°C. Theriogenology. 2001; 56:577-589.[Medline]
Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species
and cryopreservation promote DNA fragmentation in equine spermatozoa.
J Androl. 2003; 24:621-628.
Blondin P, Coenen K, Sirard MA. The impact of reactive oxygen
species on bovine sperm fertilizing ability and oocyte maturation.
J Androl. 1997; 18:454-460.
Cerolini S, Maldjian A, Pizzi F, Gliozzi TM. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction. 2001; 121:395-401.[Abstract]
Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing and thawing. Mol Reprod Dev. 2001; 59:451-458.[Medline]
de Lamirande E, Tsai C, Harakat A, Gagnon C. Involvement of
reactive oxygen species in human sperm acrosome reaction induced by A23187,
lysophosphatidylcholine, and biological fluid ultrafiltrates. J
Androl. 1998; 19:585-594.
Eriksson BM, Petersson H, Rodríguez-Martínez H. Field fertility with exported boar semen frozen in the new FlatPack container. Theriogenology. 2002; 58:1065-1079.[Medline]
Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol. 1990; 186:407-421.[Medline]
Fisz M. Some probability distributions. In: Krieger De RE, ed. Probability Theory and Mathematical Statistics. Malabar, Fla: John Wiley & Sons Inc; 1980 :129-174.
Foote RH. Survival of bull spermatozoa in milk and yolk extenders
with added catalase. J Dairy Sci. 1962; 45:907-910.
Gil MA, Abeydeera LR, Day BN, Vazquez JM, Roca J, Martinez EA. Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs. Theriogenology. 2003; 60:767-776.[Medline]
Gomez E, Irvine S, Aitken RJ. Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4-hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function. Int J Androl. 1998; 21:81-94.[Medline]
Graham JK, Kunze E, Hammerstedt RH. Analysis of sperm cell viability, acrosomal integrity and mitochondrial function using flow cytometry. Biol Reprod. 1990; 43:55-64.[Abstract]
Johnson LA, Aalbers JG, Grooten HJG. Artificial insemination of swine: fecundity of boar semen stored in Beltsville TS (BTS), modified modena (MM) or MR-A and inseminated on one, three and four days after collection. Zuchthygyene. 1988; 23:49-55.
Kuribayashi Y, Gagnon C. Effect of catalase and thioredoxin addition to sperm incubation medium before in vitro fertilization on sperm capacity to support embryo development. Fertil Steril. 1996; 66:1012-1017.[Medline]
Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Andrologia. 1998; 23:171-175.
Martin Rillo S, Martinez E, Garcia Artiga C, De Alba C. Boar semen evaluation in practise. Reprod Dom Anim. 1996; 31:519-526.
Maxwell WMC, Stojanov T. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod Fertil Dev. 1996; 8:1013-1020.[Medline]
McKinney KA, Lewis SEM, Thompson W. Reactive oxygen species generation in human sperm: luminol and lucigenin chemiluminescence probes. Arch Androl. 1996; 36:119-125.[Medline]
O'Flaherty CM, Beorlegui NB, Beconi MT. Reactive oxygen species requirement for bovine sperm capacitation and acrosome reaction. Theriogenology. 1999; 52:289-301.[Medline]
Peña FJ, Johannisson A, Wallgren M, Rodríguez-Martinez H. Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci. 2003; 78:85-98.[Medline]
Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil. 1993; 48:61-73.
Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa. Theriogenology. 2003; 60:77-87.[Medline]
Roca J, Gil MA, Hernandez M, Parrilla I, Vazquez JM, Martinez EA.
Survival and fertility of boar spermatozoa after freeze-thawing in extender
supplemented with butylated hydroxytoluene. J Androl. 2004; 25:397-405.
Saleh RA, Agarwal A. Oxidative stress and male infertility: from
research bench to clinical practice. J Androl. 2002; 23:737-752.
Sikka SC. Role of oxidative stress and antioxidants in andrology
and assisted reproductive technology. J Androl. 2004; 25:5-18.
Thurston LM, Watson PF, Holt WV. Sources of variation in the morphological characteristics of sperm sub-populations objectively assessed by a novel automated sperm morphology analysis system. J Reprod Fertil. 1999; 117:271-280.
Watson PF. The cause of reduced fertility with cryopreserved semen. Anim Reprod Sci. 2000; 60-61:481-492.[Medline]
Westendorf P, Ritcher L, Treu H. Zur Tiefgefrierung von Ebersperma. Labor-und Besamungsergebnisse mit dem Hülsenberger Paillettenverfahren. Dtsch Tierärztl Wschr. 1975; 82:261-267.
White IG. Lipids and calcium uptake of sperm in relation to cold shock and preservation. Reprod Fertil Dev. 1993; 5:639-658.[Medline]
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