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From the * Department of Medicine and Animal
Surgery, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain;
Institute of Biomedicine, Spanish National
Research Council, CSIC, Valencia, Spain; and
Department of Biochemistry and Molecular and
Cell Biology, Faculty of Veterinary Medicine, University of Zaragoza,
Spain.
| Correspondence to: Jordi Roca, Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain (e-mail: roca{at}um.es). |
| Received for publication February 20, 2007; accepted for publication April 5, 2007. |
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Abstract |
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Key words: Cryopreservation, pig
The SP composition varies among species as well as among males within the same species (reviewed by Strzezek et al, 2005). Thus, different SP profiles have been found between boars of different in vivo fertility (Flowers and Turner, 2001). Moreover, it has been suggested that the important variability among boars to sustain sperm cryopreservation could be related to differences in SP composition (Roca et al, 2006a), which may have a genetic origin (Thurston et al, 2002). In this way, as seen in other species, differences in SP protein profiles have been related to low and high sperm freezability in bulls (Jobim et al, 2004) and stallions (Zahn et al, 2005). Then, a logical hypothesis is that the supplementation of freezing extender with SP from boars with good sperm freezability could eventually improve the ability of boar spermatozoa to sustain freezing-thawing processes.
Therefore, the present study examined the possible protective effects of the addition of homologous SP to freezing extender on boar sperm cryosurvival. In addition, because the SPs were collected from ejaculates of boars with different sperm freezability, the amounts of the most relevant SP components were quantified to relate these to the encountered effect.
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Materials and Methods |
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-cm; Elgastat UHQPS; Elga Ltd, Lane End, Buckinghamshire, United
Kingdom).
Boars and Ejaculate Collection and Processing
Procedures involving animals were in accordance with the recommendation of
the Bioethics Committee of Murcia University. Healthy and fertile mature boars
of various commercial hybrid lines undergoing regular semen collection for
commercial artificial insemination and included in a sperm cryobank program
were used. Boars were housed in individual pens in environmentally controlled
buildings (commercial insemination station of PROINSERGA SA, Segovia, Spain).
They were given ad libitum access to water and were fed commercial diets
according to the nutritional requirements for adult boars.
Sperm-rich fractions were collected by the gloved-hand method, extended (1:1, vol/vol) in Beltsville Thawing Solution ([BTS] 205 mM glucose, 20.39 mM NaCl, 5.4 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA), and evaluated by standard laboratory techniques (subjective analysis). Only ejaculates with at least 200 x 106 sperm per milliliter, at least 85% sperm with normal morphology, and at least 75% and at least 80% of motile and viable sperm, respectively, were selected for cryopreservation. The selected ejaculates were packed for shipping at 17°C and delivered 14–16 hours later to the Laboratory of Andrology of the Veterinary Teaching Hospital of the University of Murcia, Spain.
Collection and Storage of SP Samples
In a preliminary study in which 116 ejaculates from 29 mature fertile
hybrid boars (4 ejaculates per boar) were cryopreserved, the boars were
classified into 3 groups according to the postthaw sperm survival assessed at
30 and 150 minutes after thawing as good, moderate, and poor freezers
(Roca et al, 2006a). Four
boars were selected among them as SP donors: SP1 was a poor sperm freezer
(less than 40% of motile and viable spermatozoa), SP2 was a moderate sperm
freezer (40%–60%), and SP3 and SP4 were good sperm freezers (more than
60%). SP was obtained during the cryopreservation process. After
centrifugation (2400 x g for 3 minutes) of sperm-rich fractions
diluted 1:1 (vol/vol) in BTS, the supernatant was collected and recentrifuged,
filtered through a 10-µm nylon mesh filter to remove debris or clumped
spermatozoa, and examined using phase microscopy to ensure no spermatozoa
remained. Samples containing spermatozoa were refiltered. The supernatant (SP
diluted in BTS) was stored frozen at –20°C until further use. Before
using, SP was thawed at room temperature.
Sperm Cryopreservation
Ejaculates were cryopreserved using the straw freezing procedure described
by Westendorf et al (1975) as
modified by Thurston et al
(2001) and Carvajal et al
(2004). Briefly, pooled
extended sperm-rich fractions were centrifuged at 2400 x g for
3 minutes at 17°C, and sperm pellet was re-extended in lactose egg yolk
(LEY) extender (80% [vol/vol] 310 mM ß-lactose, 20% [vol/vol] egg yolk,
100 µg/mL kanamycin sulphate [pH 6.2], and 330 ± 5 mOsmol/kg) to
yield to a concentration of 1.5 x 109 cells per milliliter.
After further cooling to 5°C in 90–120 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], 6% glycerol
[vol/vol] [pH 6.2], and 1145 ± 17 mOsmol/kg) to a final concentration
of 1 x 109/mL. The resuspended and cooled spermatozoa were
packed into 0.5-mL PVC-French straws (Minitüb, Tiefenbach, Germany) and
frozen using a controlled-rate freezing instrument (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, cooled at 70°C/min to –150°C, and finally
plunged into liquid nitrogen (LN2). The straws remained in the
LN2 tank for at least 2 weeks before thawing, which was done in
circulating water at 37°C for 20 seconds. Thawed spermatozoa from 2 straws
per ejaculate were resuspended in BTS (1:2, vol/vol; 37°C) and incubated
in a water bath at 37°C for 150 minutes.
Evaluation of Postthaw Sperm Quality
Measurements were done 30 minutes and 150 minutes after thawing. Postthaw
spermatozoa were assessed for motility and viability (plasma membrane and
acrosome integrity). Sperm motility was objectively evaluated using a
computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer;
Microptic, Barcelona, Spain) following the procedure described by Cremades et
al (2005). Briefly,
BTS-diluted thawed spermatozoa were further resuspended in BTS at a
concentration of 30 x 106/mL. For each evaluation, a 4-µL
sperm sample was placed in a prewarmed (39°C) Makler counting chamber
(Sefi Medical Instruments, Haifa, Israel), and 3 fields were analyzed to
assess a minimum of 100 spermatozoa. The analysis yielded the following
motility parameters: TSM (total motile spermatozoa, %), VCL (curvilinear
velocity, µm/s), VSL (straight linear velocity, µm/s), VAP (average path
velocity, µm/s); LIN (linearity, %), and ALH (amplitude of lateral head
displacement, µm).
Plasma membrane and acrosome integrity was evaluated simultaneously using a triple fluorescent procedure described by Nagy et al (2003). Aliquots (100 µL each, about 3 x 106 cells) were transferred into culture tubes, and dyes were added at a final concentration of 25 nM SYBR-14 (100 µM stock solution in dimethyl sulfoxide [DMSO]; Component A of LIVE/DEAD Sperm Viability Kit, L-7011; Molecular Probes, Europe BV, Leiden, The Netherlands), 1 µg/mL of peanut agglutinin conjugated with phycoerythrin solution (1 mg/mL stock solution Phycoprobe R-PE-PNA, P44; Biomeda Corp, Foster City, Calif), and 12 µM propidium iodide (PI) (1.5 mM in phosphate buffer [PBS]; Component B of Sperm Viability Kit). Samples were mixed and incubated at room temperature (20°C–22°C) in the dark for 10 minutes. Just before analysis, 400-µL PBS was added to each sample and remixed before run through a flow cytometer. Flow cytometer analysis was performed by using a Coulter Epics XLTM (Coulter Corp, Miami, Fla). All dyes were excited by an argon ion 488-nm laser. SYBR-14 fluorescence (particles containing DNA, living cells) was detected with a 525 nm band-pass (BP) filter, PI signal (nonviable cells) was detected by a 620 nm BP filter, and PE-PNA fluorescence (cells with damaged acrosomes) was detected using a 575 BP filter. Acquisition and analysis were done using the EXPO 2000 (Coulter) software. Debris was gated out based on scatter properties and double gated out based on SYBR-14 or PI fluorescence. Subpopulations were divided by quadrants, and the frequency of each subpopulation was quantified. Acquisition was stopped after recording 10 000 gated events, and event rates were kept around 800 cells per second. Only the percentage of live spermatozoa with intact acrosome was considered in results.
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
following the procedure described by Carvajal et al
(2004). 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 (picomoles per
108 cells).
In Vitro Oocyte Maturation and Fertilization
The protocol used was that previously described by Gil et al
(2004). Briefly, oocytes were
obtained from ovaries of prepubertal gilts collected at a local
slaughterhouse. The oocytes, surrounded by a compact cumulus mass and having
evenly granulated cytoplasm, were matured (45–50 oocytes in 500 µL
per well) in bovine serum albumin (BSA)-free NCSU (North Carolina State
University)-23 medium supplemented with 10% (vol/vol) porcine follicular
fluid, 0.1 mg/mL cysteine, and 10 ng/mL epidermal growth factor for 44 hours
in 5% CO2 in air at 39°C (22 hours with added hormones, 10
IU/mL equine chorionic gonadotropin [eCG] and 10 IU/mL human chorionic
gonadotropin [hCG] [Intervet International BV, Boxmeer, The Netherlands], and
then for another 22 hours without hormones). After the completion of in vitro
maturation, batches of 30 denudated oocytes (0.1% hyaluronidase) were placed
in 50-µL drops of preequilibrated in vitro fertilization (IVF) medium
(modified tris-buffered medium [mTBM];
Abeydeera and Day, 1997). The
dishes with the oocytes were kept in the incubator for about 30 minutes until
spermatozoa were added for fertilization. A total of 100 µL of extended
spermatozoa from 1 pool of 3 frozen-thawed (FT) straws was washed 3 times by
centrifugation at 1900 x g for 3 minutes in Dulbecco's
Phosphate-Buffered Saline (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
IVF 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 spermatozoa. At
6 hours after insemination, oocytes were washed and transferred (30–35
oocytes per well) to a Nunc 4-well multidish containing 500 µL of NCSU-23
with 0.4% BSA and cultured at 39°C, 5% CO2 in air for
10–12 hours. Then oocytes were mounted on slides, fixed in 25% (vol/vol)
acetic acid in ethanol (24 hours at least), stained with 1% lacmoid in 45%
(vol/vol) acetic acid, and examined under a phase contrast microscope at
magnifications of 400x. The fertilization parameters evaluated were
percentage of penetrated ([number of oocytes penetrated/total inseminated
oocytes] x 100) and monospermic oocytes [(number of monospermic
oocytes/total penetrated oocytes) x 100] and the number of spermatozoa
per oocytes (mean number of spermatozoa in penetrated oocytes). Four
replicates were done, and at least 120 oocytes per each FT sperm sample were
evaluated.
Determination of SP Protein Profiles
Total protein concentration of SPs was estimated following the procedure
described by Bradford (1976).
SP proteins were isolated by reverse-phase high-performance liquid
chromatography (RP-HPLC). Briefly, SP proteins were separated using an ETTAN
LC HPLC system (Amersham Biosciences) and a Lichrospher RP100 C18 column (250
x 4 mm, 5-µm particle size; Merck, Darmstadt, Germany) eluted at 1
mL/min with a linear gradient of 0.1% trifluoroacetic acid in water (solution
A) and acetonitrile (solution B), isocratically (5% solution B) for 5 minutes,
followed by 5%–25% B for 10 minutes, 25%–60% B for 50 minutes, and
60%–70% B for 10 minutes). Protein detection was at 215 nm.
Assessment of the Antioxidant Components of SP
Total Nonenzymatic Antioxidant Capacity—
Measurements were performed on deproteinized SP (adding 5% trichloroacetic
acid on ice). Samples were subsequently centrifuged at 10 000 x
g, the pellet was discarded, and measurements were performed on the
supernatant. Total nonenzymatic antioxidant capacity (TAC) was measured using
the ABTS (2,2'-Azino-di-(3-ethylbenzthiazoline sulphonate)/horseradish
peroxidase [HRP] decoloration method (Cano
et al, 1998). This method is based on the capacity of different
components to scavenge the ABTS radical cations (ABTS·+)
compared with a standard antioxidant (Trolox) in a dose-response curve.
Preformed ABTS·+ was obtained by mixing 1 mmol/L ATBS, 60
µmol/L H2O2, and 0.25 µmol/L HRP in 50 mM sodium
phosphate buffer (pH 7.5). Then, 10 µL of SP was added to the reaction
medium (200 µL) and the decrease in absorbance, directly proportional to
the ABTS·+ quenched, was determined at 730 nm at 5 and 30
minutes in a 96-well microplate reader (Anthos 2010, Rosys Anthos, Germany)
Data were expressed as micromoles of Trolox equivalents.
Enzymatic Antioxidant Activity— For estimation of the enzymatic antioxidant activity, SP samples (1 mL) were centrifuged at 10 000 x g for 5 minutes. Then the supernatant was separated and kept in ice until the analysis. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) activities were measured as described by Martí et al (2003) for ram spermatozoa. The absorbance changes were monitored with an UV-Vis Hitachi spectrophotometer (U-2000; Hitachi Ltd, Tokyo, Japan) measuring the NADPH oxidation or (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) XTT sodium salt reduction at 340 or 470 nm, respectively. One enzyme unit (IU) was defined for GPx and GR as the oxidation of 1.0 µmol/min of NADPH at 25°C at pH 7.5 or as the amount of SOD capable of transforming 1.0 µmol/min of O2·–. The enzyme activity was expressed in units per milliliter per milligram (U/mL/mg) of protein.
Experimental Design
Extended sperm-rich fractions (1:1, vol/vol in BTS) from 9 boars were
pooled and centrifuged. After centrifugation, the pooled sperm pellet was
split into 5 aliquots and then extended with LEY alone (control) or LEY
supplemented with 5% (vol/vol) of SP from poor (SP1), moderate, (SP2), and
good sperm freezers (SP3–SP4). Eight replicates were done. The SP
inclusion level was chosen based on its demonstrated efficacy in a preliminary
experiment where other concentrations were also tested (unpublished data).
Statistical Analysis
Statistical analyses were performed by SPSS version 14 (SPSS Inc, Chicago,
Ill). Data were analyzed as a split plot design using a mixed-model analysis
of variance (ANOVA). To fulfill the assumption of a normal distribution,
percentage data of postthaw sperm quality were log-transformed before
statistical analysis. The effect of SP on the postthaw sperm survival was
examined according to a statistical model that includes the fixed effects of
postthaw evaluation time (30 and 150 minutes), SP donor (0 to 4), and the
random effect of replicate. There were no interactions between evaluation time
and SP donor. Therefore, data of the postthaw evaluation times were combined
and analyzed as a complete data set. The antioxidant composition of SP was
analyzed by 1-way ANOVA. Data of IVF were analyzed according to statistical
model, including the fixed effect of SP donor and the random effect of
replicate. Data in percentages of penetrated and monospermic oocytes were
modeled according to the binomial model of parameters before analysis. When
ANOVA revealed a significant effect, values were compared using the Bonferroni
or Turkey's post-hoc test and were considered to be significant when the
P value was less than .05. Results are presented as mean ±
SEM.
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Results |
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The addition of SP on the freezing extender had no effect (P > .05) on MDA production of FT spermatozoa (Figure 1). Furthermore, the maximum and minimum levels of MDA were observed in sperm samples frozen with SP from good freezers (119.4 and 86.0 pmol MDA per 108 for SP3 and SP4, respectively).
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The total protein content varied greatly irrespective of the SP origin (Table 3). Among individual proteins, PSP-I and PSP-II were, in this order, the more abundant proteins in the 4 SPs evaluated. The reverse-phase separated PSP proteins followed the same pattern as the total protein profile, showing the highest (PSP-I) and lowest (PSP-II) quantity of these proteins in the SPs from the boars considered good sperm freezers (Figure 3).
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Discussion |
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It is clear that the composition of SP varies among species, among males within the same species, and among ejaculates within the male (reviewed by Strzezek et al, 2005). Thus, an adequate knowledge of the composition of SPs used in the present study should help to understand the different response of spermatozoa to cryopreservation. Proteins are among the major components of SP, and they affect sperm functionality. For instance, Barrios et al (2005) have recently shown that SP proteins are capable of protecting ram spermatozoa against cold shock. On the other hand, heparin-binding SP proteins (HBPs) have shown a dose-dependent adverse effect on buffalo epididymal spermatozoa during cryopreservation (Harshan et al, 2006). In the boar, spermadhesins represent about 75% of the total protein content of SP, with PSP-I and PSP-II being the most abundant proteins, amounting to up to 50% of the total proteins (Calvete et al, 1995). We have reported that these proteins can preserve sperm quality of highly diluted fresh and FT spermatozoa (Centurión et al, 2003; Caballero et al, 2004; García et al, 2006). However, although the SP proteinogram differs among SPs, those differences seemed unrelated to the different protective effects observed. Moreover, this approach had been assessed in a previous experiment in which we showed that the supplementation of the freezing extender with PSP-I/PSP-II or HBP did not affect postthaw sperm survival positively or negatively (Cremades et al, 2004). Confounding results were also found by Peña et al (2006) in relation to portions of the ejaculate with clear differences in SP contents. The lack of any effect should be due to either the presence of other substances in SP or in the freezing extender, which limit or mask the action of those proteins, avoiding its adsorption onto the sperm membranes or suppressing its activity (Vadnais et al, 2005a). In fact, it has been shown that the activity of bovine SP proteins on sperm membranes is inhibited by the formation of stable complexes with egg yolk lipoproteins (Manjunath et al, 2002; Bergeron and Manjunath, 2006). In this respect, it should be taken into account that freezing extenders used for cryopreserving boar spermatozoa contain 20% egg yolk.
Besides the above-mentioned effects of certain proteins, boar SP contains a wide variety of organic and inorganic components capable of modulating different sperm functions, including protection from the negative effects of reactive oxygen species (Strzezek, 1999; Strzezek et al, 2005). Lipid peroxidation, originated by raeactive oxygen species, has been claimed as one of the main causes of sperm damage during freezing and thawing processes (Bilodeau et al, 2000). In addition, supplementation of freezing extenders with antioxidant components has been demonstrated to be effective for the improvement of postthaw boar sperm quality (ie, vitamin E [Peña et al, 2003] and catalase, SOD, and butylated hydroxytoluene [Roca et al, 2004; 2005]). Thus, qualitative and/or quantitative differences in SP components with antioxidant properties could explain the distinct effectiveness of different SPs. To test this possibility, we have determined the concentrations of the more relevant antioxidants components present in boar SP (Strzezek et al, 2005). As expected, the concentration of the different antioxidants showed significant differences among the 4 boars providing SP, although always within the normal range of boar SP according to the values previously showed by Strzezek (2002). However, it is noteworthy that the antioxidant properties of the SPs sources were not related to postthaw sperm quality. Moreover, no differences in lipid peroxidation, indirectly measured as MDA production, were found among sperm samples. Therefore, we conclude that the antioxidant property of SPs was not a major factor responsible for the distinct influence of SP on postthaw sperm survival.
Among the major protective effects attributed to SP are preventing spermatozoa from undergoing capacitation and to reverse capacitation in a sperm population that has apparently already undergone this process (Vadnais et al, 2005a,b; Kaneto et al, 2002). Fresh or FT boar spermatozoa, incubated with whole SP or with specific SP proteins, have a limited ability to penetrate oocytes under in vitro conditions (Suzuki et al, 2002; Caballero et al, 2004). In the present study, although without a postthaw sperm quality-related pattern, SP added to the freezing extender did not decrease the ability of thawed spermatozoa to penetrate oocytes under in vitro conditions. Moreover, the highest rates of penetrated oocytes were obtained from sperm samples frozen in the presence of SP. This observation could be related to the contact time of SP with the spermatozoa. In the present experiments, SP was added to the freezing extender and FT spermatozoa were thoroughly washed before IVF. Thus, the IVF results achieved seem to be related primarily to the higher proportion of spermatozoa surviving the cryopreservation process, in the presence of some sources of SP, rather than to the possible role of SP on the sperm capacitation process. Further studies are needed to clarify any other eventual relation.
In conclusion, the addition of SP to the freezing extender can improve boar sperm cryosurvival. However, the beneficial effect depends on SP source, being only significant when SP was collected from boar with good sperm freezability. Looking at the SP composition, it is likely that neither protein profiles nor the antioxidant capacity of SP can explain per se the differences on effectiveness among SPs. Therefore, further investigations are necessary to elucidate which components of SP and mechanisms linked to the SP are responsible for the potential protective effect on boar sperm freezability.
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Acknowledgments |
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Footnotes |
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