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From the Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, Campus de Espinardo, 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 October 7, 2003; accepted for publication December 23, 2003. |
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Abstract |
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Key words: Cryopreservation, g-force, frozen-thawed sperm, semen quality
From the first protocols used (Pursel and Johnson, 1975; Westendorf et al, 1975), many papers have been published in which several steps of the cryopreservation process have been evaluated, such as cooling and warming rates, cold shock and holding time during cooling, cryoprotective agents and their concentration, and packaging systems (for reviews, see Bwanga, 1990; Holt, 2000; Johnson et al, 2000; Watson, 2000). However, very little attention has been paid to the centrifugation step, which the sperm are subjected to before being frozen. Centrifugation is a necessary step in boar sperm cryopreservation protocol that is used to concentrate the sperm population so that they can be rediluted with freezing extenders. Reviews of boar sperm cryopreservation research show that a variety of centrifugation regimes have been used: 300 x g for 10 minutes (Pursel and Johnson, 1975), 800 x g for 10 minutes (Westendorf et al, 1975), 800 x g for 15 minutes (Paquignon and Courot, 1976), and 1400 x g for 5 minutes (Larsson et al, 1977). Two of those freezing methods, the Beltsville (Pursel and Johnson, 1975) and the Hülsenberg (Westendorf et al, 1975), are the basis for boar sperm cryopreservation protocols currently in place for commercial use. In these protocols, the centrifugation step is performed with the centrifugation regime unchanged from the original.
On the other hand, it has been shown that for several species, such as the rat (Cardullo and Cone, 1986), human (Ng et al, 1990), and mouse (Katkov and Mazur, 1998), centrifugation is a potentially sperm-damaging step during semen processing. Specifically, it has been demonstrated (Aitken and Clarkson, 1988) that centrifugal pelleting of unselected human sperm populations caused the production of reactive oxygen species (ROS) within the pellet, which induced irreversible damage to spermatozoa and the impairment of their in vitro fertilizing ability. Studies on the effect of different centrifugation regimes on human sperm (Shekarriz et al, 1995) have concluded that the time of centrifugation is more critical than the g-force for inducing sperm damage; thus, the use of short-term centrifugation is recommended in the preparation of sperm for assisted reproductive techniques.
To our knowledge, studies on the effect of centrifugation on frozen-thawed boar spermatozoa have not been reported. Therefore, the purposes of this report were to evaluate 1) the effect of different centrifugation regimes on the quantity of sperm recovered after supernatant removal and their viability, and 2) the effect of different centrifugation regimes before freezing on the motility, viability, capacitation-like changes, oocyte penetrating ability, and lipid peroxidation of frozen-thawed spermatozoa.
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Materials and Methods |
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Animals and Sperm Cryopreservation
Ejaculates were collected weekly by the gloved-hand method from 3 mature
Pietrain boars (Agropor AI Center, Murcia, Spain) for 8 weeks. Sperm-rich
fractions were extended (1:1 [vol/vol]) in Beltsville Thawing Solution (BTS)
(Pursel and Johnson, 1975).
After collection, sperm characteristics (sperm concentration, subjective sperm
motility, acrosome integrity, and normal morphology) were microscopically
evaluated by standard laboratory techniques
(Martín Rillo et al,
1996), and only ejaculates with more than 75% motile spermatozoa
and more than 80% normal acrosomal ridges were used. Immediately after
evaluation, the diluted sperm-rich fractions were pooled and slowly cooled to
17°C for 240 minutes. At 17°C, diluted spermatozoa were centrifuged
(Megafuge 1.0 R, Heraeus, Germany; the centrifugation regimes used are
described in the experimental design), and all of the supernatants were
discarded.
Spermatozoa were cryopreserved using a modification of the straw-freezing procedure described by Westendorf et al (1975) adapted to 0.5-mL straws by Thurston et al (1999). After centrifugation, the pellets were diluted to a concentration of 1500 x 106 cells/mL with lactose-egg yolk (LEY) extender (pH 6.2, and 330 ± 5 mOsmol/kg), composed of 80% (v/v) ß-Lactose solution (310 mM in distilled water), 20% (v/v) egg yolk and 100 µg/mL kanamycin sulfate. After further cooling to 5°C for 120 minutes, diluted spermatozoa were resuspended to a final concentration of 1000 x 106 cells/mL with LEY-glycerol-Orvus-ES-Paste (LEYGO) extender (pH 6.2 and 1650 ± 15 mOsm/kg), consisting of 92.5% (v/v) LEY, 6% (v/v) glycerol and 1.5% (v/v) Equex STM (equivalent to Orvus ES Paste; Graham et al, 1971). French straws (0.5 mL; Minitüb, Landshut, Germany) were filled with diluted and cooled spermatozoa. Sperm packaged in this manner were frozen from 5°C to –5°C at a rate of 6°C/min, frozen from –5°C to –80°C at a rate of 40°C/min, held for 30 seconds at –80°C, and then cooled from 70°C/min to –150°C using a programmable cell freezer (IceCube 1810, Minitüb, Germany). After reaching –150°C, the straws were plunged into liquid nitrogen and stored in a liquid nitrogen container.
Frozen sperm samples were thawed in a circulating water bath at 37°C for 20 seconds. After thawing, sperm suspensions were diluted at 37°C in BTS (1:1 [vol/vol]) and held in the water bath for 150 minutes.
Sperm Quality Parameters
Objective Sperm Motility—
A 4-µL sperm sample was placed on a prewarmed (37°C) Makler chamber
(Sefi Medical Instruments, Haifa, Israel) and analyzed for kinematics using a
computer-assisted semen motility analysis system (Sperm Class Analyzer;
Microptic, Barcelona, Spain). Observations were made at a magnification of
100x on a positive phase-contrast microscope (Labophot; Nikon, Tokyo,
Japan) with a warmed (39°C) stage. Three fields per sample were analyzed,
and the total number of spermatozoa analyzed in each sperm sample was a
minimum of 100 spermatozoa. The computer-assisted sperm analysis
(CASA)-derived motility characteristics studied were curvilinear velocity
(VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity
(VAP, µm/s), linearity of the curvilinear trajectory (LIN, ratio of
VSL/VCL, %), straightness (STR, ratio of VSL/VAP, %), amplitude of lateral
head displacement (ALH, µm), mean dance (DNM, mean value of VCL x
ALH, µm2/s), and beat cross-frequency (BCF, Hz). The definition of these
descriptors can be found in previous publications (eg,
Davis and Siemers, 1995).
Total sperm motility (TSM, %) was defined as the percentage of spermatozoa
that showed an average path velocity greater than 10 µm/s, and rapid motile
spermatozoa (RMS, %) was defined as the percentage of spermatozoa that showed
an average path velocity greater than 50 µm/s.
Plasma Membrane Integrity, Mitochondrial Function, and Acrosomal Integrity— 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 the 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 assay (FITC-PNA). Three hundred spermatozoa were counted at 1000x magnification (Eclipse E800, Nikon) using a BV-2A filter (a 400- to 440-nm excitation filter, an emission wavelength of 455 nm, and a 470-nm barrier filter). Four staining patterns could be discerned: 1) viable sperm with an intact acrosome (VS, with no staining of the head and the midpiece of the sperm tail stained green), 2) viable sperm with an acrosome reaction (VS-AR, with both the midpiece of sperm tail and the acrosome region stained green), 3) nonviable sperm with an intact acrosome (DS, with the sperm head stained red), and 4) nonviable sperm with an acrosome reaction (DS-AR, with the sperm head stained red and the acrosome region stained green. Values were expressed as percentages.
Capacitation-Like Changes— Capacitation-like changes were evaluated by fluorescent microscopy with chlortetracycline (CTC) staining (Maxwell and Johnson, 1997) that included the antibiotic CTC to visualize the course of capacitation and the acrosome reaction. Two hundred spermatozoa were counted at 1000x magnification (Eclipse E800, Nikon), using a BV-2A filter (a 400- to 440-nm excitation filter, an emission wavelength of 455 nm, and a 470-nm barrier filter). Sperm were classified according to CTC staining patterns using the nomenclature described by Fraser et al (1995): F (uniform fluorescent head— uncapacitated and acrosome intact), B (fluorescence-free band on the postacrosomal region—capacitated), and AR (nonfluorescent head or a thin fluorescent band on the equatorial segment— acrosome reacted). Values were expressed as percentages.
In Vitro Penetrating Ability— In vitro penetrating ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Martínez et al, 1996). Two hundred forty oocytes per replicate (80 oocytes per centrifugation regime) were processed. The fixed oocytes were stained with 1% lacmoid and examined for evidence of sperm penetration under phase-contrast microscopy at 400x magnification. Immature oocytes with a broken oolemma or abnormal-looking cytoplasm were classified as degenerated (unhealthy oocytes) and were not evaluated. Healthy immature oocytes at the germinal vesicle stage were considered penetrated when spermatozoa with swollen or un-swollen heads and their corresponding tails were found in the vitellus. The oocyte penetrating ability parameters evaluated were as follows: oocyte penetration rate (percentage of the number of oocytes penetrated/the total number of viable oocytes, %) and the number of spermatozoa per penetrated oocyte (the mean number of spermatozoa in penetrated oocytes).
Lipid Peroxidation Determination
Lipid peroxidation levels were quantified using the endpoint generation of
malondialdehyde (MDA) with the thiobarbituric acid reactive substance (TBARS)
test (Comaschi et al, 1989) and
were expressed as the simple concentration of MDA (pmol
MDA/108cells). The MDA concentration was measured by a
spectrophotometer (UNICAM PU 8610 Kinetics Spectrophotometer; PHILIPS,
Cambridge, United Kingdom) at 534 nm.
Experimental Design
Experiment 1: Effect of the Centrifugation Regimes on Boar Sperm Recovery and Yield—
The factors incorporated in this experiment (4 x 2 factorial) were as
follows: g-force (400 x g, 800 x g,
1600 x g, and 2400 x g) and centrifugation time
(3 and 5 minutes). The standard centrifugation regime, 800 x g
for 10 minutes (Westendorf et al,
1975) was used as a reference. The diluted and pooled sperm-rich
fractions were divided into 9–50 mL aliquots and transferred to 50
mL-Corning centrifugation tubes. After centrifugation at 17°C, the
supernatants were discarded, and pelleted spermatozoa were gently rediluted
with BTS to the initial volume (50 mL). Before (BC) and after centrifugation
(AC), the sperm concentration (SC; Bürker Chamber) and the percentage of
viable sperm with intact acrosome (VS; using the triple fluorescent stain)
were assessed. This experiment was performed 3 times. Sperm recovery (SR) and
sperm yield (SY) were expressed in percentages and were calculated as follows:
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Experiment 2: Effect of Centrifugation Regimes Before Freezing on Boar Sperm Cryosurvival and Postthaw Lipid Peroxidation— To perform this experiment, 2 centrifugation regimes of experiment 1 were chosen: 2400 x g for 3 minutes (C1) and 1600 x g for 5 minutes (C2). The standard centrifugation regime, 800 x g for 10 minutes (Westendorf et al, 1975), was used as a reference (C3). The diluted and pooled sperm-rich fractions were divided into 3 aliquots and transferred to 50-mL Corning centrifugation tubes. After centrifugation at 17°C, the samples were processed following the cryopreservation protocol previously described. The postthaw evaluation was performed as follows: motility, viability, and capacitation-like change parameters were assessed at 30 and 150 minutes. Oocyte penetrating ability parameters were evaluated just after thawing. MDA production was measured after 0, 30, and 150 minutes of postthawing. This experiment was performed 5 times.
Statistical Analysis
Data were analyzed using the SPSS 10.0 PC statistics package (SPSS Inc,
Chicago, Ill). The sperm parameters evaluated both in experiment 1
(g-forces and centrifugation time) and in experiment 2
(centrifugation regime and postthawing evaluation time) were analyzed using a
2-way analysis of variance. In the second experiment, when no significant
effect of postthawing evaluation time was observed, the results were combined
and analyzed as a complete data set. When the analysis of variance showed a
significant effect, the values were compared by the Tukey test. In the second
experiment, the percentage of oocytes penetrated and the number of spermatozoa
per penetrated oocyte were analyzed using the chi-square test and an analysis
of variance, respectively. Values are presented as the mean ± the
standard error of the mean, and the level of statistical significance was
considered P < .05.
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Results |
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Three regimes (2400 x g for 5 minutes, 1600 x g for 5 minutes, and 2400 x g for 3 minutes) provided the highest (P < .05) postcentrifugation sperm recovery. Two of them, 2400 x g for 5 minutes (97.26% ± 0.94%) and 1600 x g for 5 minutes (93.40% ± 0.93%) reached significantly (P < .05) higher sperm recoveries than the reference, 800 x g for 10 minutes (80.58% ± 4.6%).
The highest (P < .05) value for postcentrifugation sperm yield
was achieved (P < .05) both when using these 3 centrifugation
regimes, 2400 x g for 5 minutes (75.03% ± 0.50%), 1600
x g for 5 minutes (74.70% ± 0.79%), and 2400 x
g for 3 minutes (69.57% ± 0.77%), and when using the reference
regime (72.28% ±
0.29%).
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The low speed/short time of centrifugation (400 x g for 5 minutes, 800 x g for 3 minutes, and 400 x g for 3 minutes) pelleted about half of the sperm; consequently, the sperm recovery and yield achieved with them were significantly (P < .05) lower than with the reference.
Experiment 2: Effect of Centrifugation Regimes Before Freezing on Boar Sperm Cryosurvival and Postthaw Lipid Peroxidation
The centrifugation regime used had a significant influence on all
postthawing sperm quality parameters. Sperm motility indexes (TSM and RMS)
were significantly (P < .05) higher for frozen-thawed spermatozoa
centrifuged with C1 before freezing than for the rest of the regimes (Table).
In relation to kinetics parameters, the only difference noted among
centrifugation regimes was for ALH, which was higher (P < .05)
when using C1 and C2 than when using C3.
The centrifugation regimes C1 (55.10% ± 1.17%) and C2 (57.50% ± 1.48%) showed significantly (P < .05) higher percentages (Figure 2) of frozen-thawed viable spermatozoa with an intact acrosome (VS) than did C3 (49.80% ± 1.94%). Likewise, lower percentages (P < .05) of nonviable spermatozoa with an intact acrosome (DS) were observed for C1 (17.10% ± 0.38%) and C2 (17.80% ± 0.48%) than for C3 (23.60% ± 1.22%).
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Capacitation-like change results are shown in Figure 3. The centrifugation regimes C1 (47.15% ± 2.09%) and C2 (48.15% ± 1.45%) showed significantly (P < .05) higher percentages of frozen-thawed uncapacitated spermatozoa (F) than did C3 (40.70% ± 2.14%). Besides, C3 (33.65% ± 2.19%) had a significantly (P < .05) higher percentage of frozen-thawed capacitated spermatozoa than did C1 (27.05% ± 2.16%) and C2 (26.75% ± 1.86%).
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The oocyte penetrating ability parameters, in terms of the oocyte penetration rate and the number of spermatozoa per penetrated oocyte (Figure 4), were significantly (P < .05) higher for frozen-thawed spermatozoa centrifuged with C1 before freezing than for those centrifuged using the C2 and C3 regimes.
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The different centrifugation regimes had no effect on MDA production (P > .05), a measure of lipid peroxidation. All of the centrifugation regimes showed the same behavior: relatively low levels of MDA 0 and 30 minutes after postthawing incubation time and a significant increase (P < .05) after 150 minutes (Figure 5).
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Discussion |
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In the first experiment, centrifugation with the standard centrifugation regime used in cryopreservation protocols, 800 x g for 10 minutes (Westendorf et al, 1975), brought about a 19% loss of sperm caused by their being discarded with the supernatant, which was similar to the losses reported by Almlid et al (1987), 24%, and Eriksson et al (2002), 20%–25%. Our study shows that the centrifugation regime influences this sperm loss and that it therefore influences sperm recovery. Using the lowest g-forces and the shortest times of centrifugation (800 and 400 x g for 3 minutes) led to the lowest sperm recovery, because of a lack of complete pelleting and, consequently, the loss of unpelleted cells upon supernatant removal. By contrast, using the highest g-forces and the longest times of centrifugation (2400 and 1600 x g for 5 minutes) led to greater spermatozoa sedimentation. Moreover, the percentage of sperm recovered achieved with these regimes remained almost unchanged when using the highest g-force and reduced centrifugation time (2400 x g for 3 minutes). This suggests that it is possible to achieve high sperm recovery using a relatively high g-force and a short time of centrifugation.
On the other hand, the primary reason for centrifuging cell suspensions is to recover the maximum number of sperm from the initial population with minimum adverse effects on their viability. Centrifugation has been shown to be a potentially sperm-damaging step during human semen processing, inducing sublethal damage (Mortimer, 1991; Alvarez et al, 1993). In some mammals, such as the rat (Cardullo and Cone, 1986), human (Makler and Jakobi, 1981; Ng et al, 1990; Agarwal et al, 1994), and mouse (Schreuders et al, 1996), spermatozoa have been shown to be very sensitive to mechanical and centrifugal forces and must manipulated under carefully defined conditions to reduce sperm damage. Spermatozoa from other species, such as equine and bovine (Picket et al, 1975; Katkov and Ostashko, 1996; Crockett et al, 2001), are somewhat less sensitive to centrifugation, which indicates that species specificity is very important with respect to spermatozoa injury caused by centrifugation.
With regard to boar spermatozoa, no studies have been made regarding the effect of centrifugation on spermatozoa viability, to our knowledge. In the present study, there were no differences between the percentages of viability of the population recovered by each one of the centrifugation regimes, including the reference treatment (data not shown), immediately after centrifugation. From the point of view of using frozen-thawed spermatozoa for AI, the most important objective is to achieve the maximum total number of viable sperm with intact acrosomes after the freezing-thawing process and, therefore, to provide an optimal number of doses per cryopreserved ejaculate. Consequently, a high percentage of viability may mean little if the population measured is a small subset of the original, as happened with those samples centrifuged using low g-forces and short times of centrifugation. Therefore, we focused on the effect of centrifugation on the sperm yield and not on the percentage of viability achieved. The centrifugation regime used influenced both sperm recovery and yield in a similar fashion. The poor sperm yield that occurred with the lowest g-forces combined with the shortest time was obviously due to the low sperm recovery values that were obtained using those regimes. Using the highest g-forces with the longest times of centrifugation (2400 and 1600 x g for 5 minutes) or the highest g-force combined with the shortest centrifugation time (2400 x g for 3 minutes) provided the highest (P < .05) fraction of viable sperm from the initial sperm suspension. This suggests that we can use a relatively high g-force combined with a short centrifugation time without any negative effect on sperm yield, at least when viability is assessed immediately after centrifugation.
The influence of different centrifugation regimes on spermatozoa viability has been the objective of some research. The effect of different g-forces at a given centrifugation time on sperm motility and viability has been evaluated in humans (Fredricsson and Kinnari, 1979; Mack and Zaneveld, 1987) immediately after centrifugation and also in dogs (Rijsselaere et al, 2002), both immediately after centrifugation and daily, during 3 days of posterior storage at 4°C. These studies observed that high centrifugal forces depressed both sperm motility and viability; they therefore recommended the use of a medium g-force (in humans, 300–600 x g for 10–20 minutes, and in dogs, 720 x g for 5 minutes). Katkov and Mazur (1998), in a study of the effect of different centrifugation g-force/time combinations on mouse sperm motility (assessed after centrifugation and 2.5 hours later, stored at room temperature), recommend the use of 400 x g for 10–12 minutes to achieve the maximum number of motile sperm or the use of 200 x g for 5 minutes to maximize the percentage of motility. In any case, all of these studies recommend the use of g-forces below 800 x g. Data obtained in our study indicate that the highest g-force (2400 x g) for any of the centrifugation times tested (3 or 5 minutes) achieved high sperm recovery and yield and did not reduce boar sperm viability, at least immediately after centrifugation. However, we must note that the potentially harmful effect of centrifugation on spermatozoa is not observed immediately after centrifugation but that it develops with the passage of time (Alvarez et al, 1993). Therefore, it could be that the potentially harmful effect of centrifugation becomes evident with the passage of time, after the manipulation or processing (incubation, cool storage, or cryopreservation) of centrifuged boar sperm. For this reason, we designed the second experiment to evaluate the possible damage that the centrifugation step of the cryopreservation protocol had on frozen-thawed boar spermatozoa.
There is not much literature about the impact that centrifugation before freezing has on the quality of frozen-thawed spermatozoa. With regard to boar sperm, Salamon (1973) showed that centrifugation before freezing had an adverse effect on the motility of frozen-thawed spermatozoa. On the other hand, currently, centrifugation is the only technique used for separating boar spermatozoa from seminal plasma in cryopreservation protocols. In spite of this, to our knowledge, no study has been published concerning the effect of centrifugation regimes on the cryosurvival of boar sperm.
In this paper, we evaluated the effect of 3 centrifugation regimes: 2 were experimental (2400 x g for 3 minutes and 1600 x g for 5 minutes), and 1 was normally used in cryopreservation protocols (800 x g for 10 minutes) (Westendorf et al, 1975) as a reference. Both of the tested centrifugation regimes showed better postthaw sperm quality than the reference treatment. Because the procedure followed for cryopreservation was the same for all of the centrifugation regimes, the reason for these differences must lie in the centrifugation step itself. Therefore, our results suggest that the cryopreservation process facilitated the observation of presumed sublethal damage caused by centrifugation prior to freezing. The less harmful treatment appeared to be 2400 x g for 3 minutes, because it yielded the highest oocyte penetrating ability. By contrast, the reference treatment (800 x g for 10 minutes) appeared to be more damaging than 2400 x g for 3 minutes and 1600 x g for 5 minutes. These findings seem to prove that the time of centrifugation is more critical than the g-force for inducing boar sperm damage. Shekarriz et al (1995) reached a similar conclusion for human sperm and recommended the use of short-term centrifugation.
There is still no complete or accepted explanation of how centrifugation induces sperm damage. However, it has been hypothesized that the damage to the spermatozoa is due to a direct mechanical effect on the sperm membranes (Alvarez et al, 1993) as well as to an indirect adverse effect caused by excessive ROS formation (Aitken and Clarkson, 1988; Mortimer, 1991). High levels of ROS are associated with sperm membrane damage through lipid peroxidation (Shekarriz et al, 1995), which may alter sperm function, leading to loss in motility and viability and, in the end, reduced fertility of spermatozoa. Since no differences in MDA production (an indicator of lipid peroxidation) were observed between the centrifugation regimes tested in the second experiment, it could be assumed that the sperm sublethal damage was caused by the mechanical effect of centrifugation. Abidor et al (1994) postulated that the cell damage is not a consequence of higher accelerations or higher sedimentation rates per se, but is a result of the cells being packed in a pellet at the tube bottom. The closer and the longer the cells stand packed, the more damaged they become (Katkov and Mazur, 1998). These affirmations would explain our results: higher postthaw sperm quality was achieved when using 3 and 5 minutes than when using 10 minutes, because cells centrifuged for 3 and 5 minutes spent less time in the pelleted state. Moreover, the centrifugation for 3 minutes achieved better postthaw oocyte penetrating ability than did the other centrifugations, because the time that the sperm remained packed was the lowest.
In conclusion, using relatively high g-forces combined with short centrifugation times has no negative effect on sperm recovery and yield, at least when viability is assessed immediately after centrifugation. Moreover, the centrifugation regime used in the centrifugation step of the cryopreservation process influences boar sperm cryosurvival; hence, we recommend the use of short-term centrifugation with a relatively high g-force (2400 x g for 3 minutes) to minimize the sublethal damage of boar spermatozoa, which will thus enhance postthawing sperm quality.
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Acknowledgments |
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Footnotes |
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) and yield (
) of diluted (1:1 [vol/vol] with
Beltsville Thawing Solution [BTS]) pooled sperm-rich fractions of boar semen
cooled to 17°C; (a–g) indicate significant differences among
groups (P < .05).
; viable sperm
with acrosome reaction), DS (
-), and C3 (800 x g for 10 minutes; reference;
—
—), on malondialdehyde (MDA; indicator of lipid
peroxidation) concentration (pmol MDA/108cells) produced by
frozen-thawed boar spermatozoa 0, 30, and 150 minutes after postthawing
incubation.