Various Physical Stress Factors on Rat Sperm Motility, Integrity of Acrosome, and Plasma Membrane

Authors

  • Omer Varisli,

    1. Department of Animal Reproduction and Artificial Insemination, School of Veterinary Medicine, University of Ankara, Ankara, Turkey
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  • Cevdet Uguz,

    1. Department of Medical Biology and Genetics, School of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkey
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  • Cansu Agca,

    1. Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri.
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  • Yuksel Agca

    Corresponding author
    1. Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri.
      College of Veterinary Medicine, University of Missouri, 1600 East Rollins Road, Room W191, Columbia, MO 65211 (e-mail: agcay@missouri.edu).
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College of Veterinary Medicine, University of Missouri, 1600 East Rollins Road, Room W191, Columbia, MO 65211 (e-mail: agcay@missouri.edu).

Abstract

ABSTRACT: The objective of this study was to determine the effects of various physical interventions such as centrifugation regimes, Percoll gradient separation, and repeated pipetting on various viability parameters of epididymal sperm of Fischer 344 (F-344) and Sprague-Dawley (SD) rat strains. Three experiments were conducted. In experiment 1, sperm motility and acrosomal and membrane integrity were compared after exposing sperm samples to 200, 400, 600, and 800 × g centrifugal forces for 5, 10, or 15 minutes. In experiment 2, sperm motility and acrosomal and membrane integrity were compared after passing them through a Percoll separation using centrifugal forces of 600, 800, 1000, and 1200 × g for either 15 or 30 minutes. In experiment 3, the effect of repeated pipetting (2, 4, 6, 8, and 10 times) on motility and membrane integrity of rat sperm was compared with that on mouse, ram, bull, and boar sperm. The results revealed that both F-344 and SD rat sperm motility and membrane integrity were significantly affected by centrifugation (P < .05). The acrosomal integrity of SD rat sperm was affected after using 800 × g centrifugation force for 10 or 15 minutes (P < .05), whereas F-344 rat sperm acrosomal integrity was not affected by any centrifugation regimes (P > .05). Sperm from SD rats also had higher motility and membrane integrity loss than did sperm from F-344 rats after centrifugation and pipetting (P < .05). Percoll gradient separation did not cause significant motility loss or acrosomal damage to either F-344 or SD sperm (P > .05). Repeated pipetting had a dramatic adverse effect on both rat and mouse sperm motility (P < .05) as compared with sperm from bull, boar, and ram, which were not affected at all (P > .05). These data suggest that rat sperm have unique properties that need to be considered during centrifugation, Percoll gradient separation, and pipetting procedures.

Rats are one of the most widely used laboratory animal species in studies involving genomic research, reproductive biology and toxicology, drug testing, behavioral, neurological, cardiovascular, and transplantation studies (Gibbs et al, 2004; Agca and Critser, 2005; Lazar et al, 2005). Because of recent advancements in the development of novel gene modification techniques, use of genetically modified rats in biomedical research is expected to increase significantly in the near future (Lois et al, 2002; Zan et al, 2003; Zhou et al, 2003; Tesson et al, 2005). Availability of optimal conditions for sperm, oocyte, and embryo recovery and their in vitro culture is required for successful reproductive, cryobiologic, cellular, and molecular studies. There are several drawbacks with the manipulation of rat germplasm, such as: 1) sensitivity of rat sperm to physical interventions, which is the topic of the current study; 2) spontaneous activation of metaphase II oocytes during their retrieval from oviduct and during their in vitro culture (Zernicka-Goetz, 1991; Ben-Yosef et al, 1995); and 3) poor developmental competence of rat zygotes to blastocyst-stage embryos under entirely in vitro conditions (Matsumoto and Sugawara, 1998; Nishikimi et al, 2000). Poor viability caused by mishandling of gametes and embryos not only causes the need to repeat the procedures multiple times, but also contributes to potential confounding effects on the experimental procedures, which must be avoided.

In many cases, sperm-handling procedures are performed consecutively, and thus adverse effects are expected to be cumulative at the end of the entire procedure. During the course of sperm handling upon recovery, sperm samples are usually first introduced in a physiologic media (ie, Tyrode lactate or Dulbecco phosphate buffered saline) at appropriate osmolality, temperature, and pH. They then undergo multiple pipettings and centrifugation in order to remove seminal/epididymal fluid. In addition, semen extenders and cryoprotectants are added before routine or experimental analysis or sperm cryopreservation. In some cases, centrifugation may be combined with various gradient separation methods such as Percoll in order to remove concomitant somatic and blood cells and nonviable sperm fraction. There is a potential for substantial motility loss because of mishandling of the sperm samples even before the intended reproductive procedure is performed. Thus, determination of optimal conditions for sperm washing, pipetting, centrifugation, and Percoll gradient separation is required to obtain high-quantity and high-quality sperm samples. In the context of sperm cryopreservation, rats appear to be one of the most challenging mammalian species (Nakatsukasa et al, 2001). Thus, minimizing motility loss prior to rat sperm cryopreservation is necessary to increase overall efficiency. In addition, postthaw removal of sperm extenders (eg, egg yolk and skim milk) and cryoprotectants (eg, glycerol and raffinose) from sperm also requires centrifugation and pipetting, which would further affect sperm viability (Agca et al, 2002).

Survival of sperm from several mammalian species (ie, bull, boar, human, and mouse) after various handling procedures has been well investigated (Hammerstedt et al, 1990; Holt, 2000). However, there are only a few methodological reports that describe the mechanical sensitivity of mouse sperm. These reports are limited to only different centrifugation regimes (Katkov and Mazur, 1998, 1999). One of the earliest reports suggested the vulnerability of rat sperm to mechanical distortions (Cardullo and Cone, 1986). However, to date, effects of these stress factors on the viability of epididymal rat sperm have not been systematically studied; thus, our current knowledge of appropriate rat sperm manipulation is very limited. Therefore, determination of optimal rat sperm handling methods would have great importance for many areas of biomedical fields where gene modification and common assisted reproductive techniques such as genome cryobanking, in vitro fertilization, and artificial insemination are routinely performed (Toyoda and Chang, 1974; Oh et al, 1998; Nakatsukasa et al, 2001, 2003). Here, we performed a series of studies to determine the extent of sperm sensitivity to various mechanical effects that are created during commonly used sperm manipulation procedures such as centrifugation, Percoll gradient separation, and pipetting.

Materials and Methods

Animals

Sexually mature (12–20 weeks old) male rats (inbred Fischer 344 [F-344] and outbred Sprague-Dawley [SD] strains) were used as sperm donors. Mouse sperm was collected from 12- to 14-week-old outbred ICR male mice (Harlan Sprague Dawley Inc. Indianapolis, Indiana). The rats and mice were housed in accordance with the policies of the University of Missouri Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals. Boars, bulls, and rams were maintained at the University of Missouri research farm.

Sperm Collection

All chemicals were purchased from Sigma Chemical (St Louis, Missouri) unless otherwise stated. Male rats and mice were euthanized by CO2 inhalation. The cauda epididymides were excised and then placed in 35-mm petri dishes containing HEPES-buffered Tyrode lactate (TL-HEPES) solution containing 3 mg/mL bovine serum albumin. Each cauda epididymis was then cut at several places using a fine scissors to allow sperm to swim out for 10–15 minutes at 37°C. The sperm suspension was then gently drawn into a plastic Samco (San Fernando, California) transfer pipette with 2-mm inner diameter and placed in 5-mL tubes for further experimentations. The initial sperm concentrations for SD and F-344 rats and ICR mice were approximately 30 × 106, 15 × 106, and 9 × 106 sperm/mL, respectively. The final sperm concentration during motility analysis was approximately 1 × 105 sperm/mL. Ram and bull sperm were collected by using an electroejaculator, and boar semen were collected by artificial vagina in a prewarmed 50-mL plastic test tube. The ejaculates from ram, bull, and boar were kept in an insulated Styrofoam box containing warm heat pads and transported to the laboratory within 30 minutes of semen collection.

Experiment 1: Effect of Centrifugation

One hundred microliters of sperm suspension was loaded in 1.5-mL Eppendorf centrifuge tubes and then 900 μL TL-HEPES was added and gently mixed with the sperm suspension before centrifugation using 200, 400, 600, and 800 × g average forces for 5, 10, and 15 minutes with an Eppendorf centrifuge under controlled temperature (30°C). At the end of the centrifugation procedure, the supernatant was gently removed and 500 μL TL-HEPES solution was gently added to the tube containing the sperm pellet to resuspend the sperm pellet by gentle rotation of the tubes. Sperm motility characteristics were subsequently evaluated using a computer-assisted sperm analysis (CASA) system 10 minutes after centrifugation at 37°C. The experiments were replicated at least 6 times.

Experiment 2. Effect of Percoll Gradient Separation

Preparation of isosmotic Percoll solution is required for cell separation (Vincent and Nadeau, 1984). Sperm separation procedure via Percoll gradient was done as previously described by Parrish et al (1995). To prepare working Percoll solution, aniso-osmotic Percoll solution was mixed 9:1 with a concentrated solution containing 31 mM KC1, 800 mM NaCl, 3 mM NaH2PO4, and 100 mM HEPES. The pH of the concentrated solution was previously adjusted to pH 7.3 with 1 N NaOH. The following chemicals were then added (mM final concentrations): CaCl2 (2.0), MgCl2 (0.4), lactic acid (21.6), and NaHCO3 (25.0). The 90% Percoll solution had a final osmolality of about 290 milliosmolal/kg as measured by freezing point depression (VAPRO 5520;Wescor Inc, Logan, Utah). To prepare the 45% Percoll solution, the 90% Percoll solution was mixed 1:1 with TL-HEPES. Rat sperm was layered on a discontinuous gradient of 45% and 90% (vol/vol) Percoll. The gradient consisted of 150 μL rat sperm layered over 0.5 mL of 45% Percoll and 0.5 mL of 90% Percoll in a 1.5-mL Eppendorf centrifuge tube. The tubes were then centrifuged using 600, 800, 1000, and 1200 × g average force for either 15 or 30 minutes in each centrifugation force at 30°C. At the end of the centrifugation procedure, the supernatant was gently removed and 500 μL TL-HEPES solution was added in the tube containing the sperm pellet to resuspend them by gentle rotation. Sperm motility characteristics were subsequently evaluated using a CASA system 10 minutes after centrifugation.

Experiment 3. Effect of Pipetting

One milliliter of either rat, mouse, bull, boar, or ram sperm suspension in TL-HEPES was transferred into a 1.5-mL Eppendorf centrifuge tube and subjected to either 2, 4, 6, 8, or 10 times successive pipetting using 1-mL-capacity pipette tips (Pipetman P-1000; Gilson Inc, Middleton, Wisconsin) attached to a blue tip (Gilson) at 30°C. Ten-microliter sperm samples were taken from each treatment to evaluate motility characteristics using a CASA system in 10 minutes.

CASA

Computer-assisted rat sperm analysis has previously been published (Chapin et al, 1992; Yeung et al, 1992; Slott et al, 1993; Moore and Akhondi 1996; Perreault, 2002). CASA (M2030; Hamilton Thorne Biosciences Inc, Beverly, Massachusetts) was used to analyze rat, mouse, bull, boar, and ram sperm motility. The CASA system we utilized has been widely accepted and used for evaluating motility of human as well as ram, boar, and bull sperm. However, some considerations needed to be taken when evaluating motility of rodent sperm such as rat and mouse (Slott et al, 1993). Because of larger head size and tail length and unusual morphology of mouse and rat sperm compared with bull and ram sperm, an 80-μm-deep dual-sided chamber (2× CELL; Hamilton Thorne Biosciences) was used to evaluate mouse and rat sperm, whereas a 10-μm-deep Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) was used to evaluate bull, boar, and ram sperm at 37°C. Motility estimates were always validated manually by the video playback option of the instrument. The setting parameters and the definition of measured sperm motion parameters for the CASA were: frames per second, 20; duration of tracking time, 0.7 second; medium average path velocity (VAP) cutoff, 25.0 μm/sec; low VAP cutoff, 5.0 μm/sec; count slow as motile, yes.

Fluorescent Microscopic Evaluation of Plasma Membrane and Acrosome Integrity

Acrosome integrity and plasma membrane integrity were assessed by using the procedure previously described (Si et al, 2006). Propidium iodide (PI)/SYBR-14 live and dead stain and Alexa Fluor-488-PNA (peanut agglutinin) conjugate (Molecular Probes, Eugene, Oregon) were used to determine rat sperm plasma membrane and acrosomal integrity, respectively. For acrosomal integrity the treated and control sperm samples were smeared onto microscopic slides and air-dried. The specimens were then fixed with 99% methanol and kept at room temperature until fluorescence staining. For staining, slides were incubated with 20 μg/mL Alexa Fluor-488-PNA at 37°C for 30 minutes, washed with PBS, and then analyzed under epifluorescence microscope (Zeiss Axiophot, Oberkochem, Germany) by using an appropriate filter set. The observed images of rat spermatozoa stained with Alexa Fluor-488-PNA were classified into 2 groups. Spermatozoa displaying intensively and moderately bright fluorescence in the acrosomal region were considered as intact acrosome, and spermatozoa displaying weak, patchy, or no fluorescence in the acrosomal region were considered as damaged acrosome. For plasma membrane integrity, treated and control sperm samples were incubated with 5 μM PI and 1 nM SYBR-14 at 37°C for 30 minutes. After staining, 10 μL of sperm sample was placed on a microscope slide, covered with a coverslip, and observed under an epifluorescence microscope using an appropriate filter set. In each treatment, 80–100 sperm per sample were counted and a total of 5–6 replicates were examined.

Statistical Analysis

Statistical analysis was performed by using analysis of variance general linear models of SAS version 9.1 (SAS Institute Inc, 1985) to determine the effects of strain, centrifugation force and time, and pipetting on motility, velocity, and acrosomal and plasma membrane integrity. The means were separated using Duncan's multiple range test. Values were given as the mean ± standard error of the mean. For all statistical tests the level of statistical significance was chosen as P < .05.

Results

Experiment 1: Effect of Centrifugation on Rat Sperm Motility, Acrosome Integrity, and Membrane Integrity

Figure 1 shows the effects of centrifugation forces (200, 400, 600, and 800 × g) and time (5, 10, or 15 minutes) on SD and F-344 rat epididymal sperm motility. Table 1 provides percentage progressive motility and corresponding VAP values after using the same centrifugation forces and time. Overall, there was a significant motility difference between SD and F-344 sperm in their response to centrifugation (P < .05). Motility loss of SD rat sperm was significantly greater than that of F-344 sperm (P < .05). Motility of neither F-344 nor SD rat sperm was further affected because of centrifugation when the sperm was centrifuged above 200 × g (400, 600, and 800 × g; P > .05). There was no interaction between centrifugation force and time with regard to motility (P > .05). Although progressive motility was recorded lower than previously reported (Slott et al, 1993), progressive motility of sperm from both strains was similarly and significantly affected by centrifugation (P < .05). However, the corresponding sperm VAP was not affected by centrifugation for either strain (P > .05). Despite significant difference in motility loss between F-344 and SD sperm, the ideal centrifugation regimes for both strains were found to be around 400 × g for 5 minutes. For SD sperm, 50%–65% motility loss occurred at 200 × g, but no additional motility loss was observed after centrifuging using 800 × g force for up to 15 minutes. The same was true for F-344 sperm in that centrifuging F-344 sperm at 200 × g for 15 minutes or at 800 × g for 15 minutes showed comparable motility loss (40% and 50%, respectively). Whereas F-344 rat sperm motility was affected (P < .05) by the duration of centrifugation, centrifugation force had no effect on the motility (P > .05) of SD sperm.

Figure 1.

. Percentage motility (mean ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to different centrifugation forces (200, 400, 600, and 800 × g) and times (5, 10, and 15 minutes).

Table 1. . Percentage progressive motility and average path velocity values (mean ± SEM) of epididymal Fischer 344 and Sprague-Dawley rat sperm that were subjected to different centrifugation forces (200, 400, 600, and 800 × g) and times (5, 10, and 15 min)
  
  
ForceTime, minProgressive Motility, %Average Path Velocity, μm/sProgressive Motility, %Average Path Velocity, μm/s
  1. aP < .05 vs control.

Control25.20 ± 2.45128.40 ± 4.9220.18 ± 1.82110.18 ± 5.67
200518.40 ± 2.42a131.70 ± 6.6711.21 ± 2.01a113.29 ± 7.49
2001019.10 ± 2.03a122.60 ± 4.4710.33 ± 2.93a91.41 ± 11.22
2001514.40 ± 1.57a116.80 ± 11.148.58 ± 2.35a97.70 ± 10.03
400515.30 ± 1.95a134.00 ± 6.2610.92 ± 2.71a104.83 ± 14.26
4001017.30 ± 2.31a132.40 ± 3.368.36 ± 2.04a90.14 ± 11.03
4001515.67 ± 2.16a139.00 ± 7.0512.75 ± 2.59a126.33 ± 12.53
600518.40 ± 1.73a119.00 ± 3.6711.91 ± 2.70a121.20 ± 10.82
6001014.10 ± 1.56a125.70 ± 4.1012.00 ± 3.47a116.64 ± 13.03
6001514.50 ± 1.85a124.50 ± 4.309.14 ± 2.08a127.57 ± 13.63
800513.20 ± 2.30a115.60 ± 4.5111.09 ± 2.96a108.91 ± 10.84
8001013.56 ± 1.90a122.33 ± 8.109.70 ± 2.33a99.60 ± 13.39
8001512.80 ± 1.90a122.20 ± 7.3510.50 ± 2.76a106.90 ± 10.98

Figure 2 shows the effects of centrifugation forces (200, 400, 600, and 800 × g) for 5, 10, or 15 minutes on SD and F-344 rat epididymal sperm acrosomal integrity. The centrifugation forces and duration of centrifugation had no effect on the acrosomal integrity of F-344 rat sperm (P > .05). Sperm from SD rats had 20%–25% less intact acrosome than control after they were subjected to 800 × g force for 10 or 15 minutes (P < .05). Figure 3 depicts the effects of various centrifugation forces and duration on rat sperm membrane integrity. Similar to motility, membrane integrity was also significantly affected by centrifugation time, and this effect was much higher for SD sperm than for F-344 sperm (P < .05). Whereas the optimal membrane integrity recovery for SD sperm was achieved using 200 × g for 5 minutes, the corresponding values for F-344 sperm were either 200 × g or 400 × g for 5 minutes. Figure 4 depicts representative images of rat sperm acrosome and plasma membrane integrity following staining with Alexa Fluor-488-PNA or PI /SYBR-14.

Figure 2.

. Percentage acrosomal integrity (mean ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to different centrifugation forces (200, 400, 600, and 800 × g) and times (5, 10, and 15 minutes).

Figure 3.

. Percentage membrane integrity (mean ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to different centrifugation forces (200, 400, 600, and 800 × g) and times (5, 10, and 15 minutes).

Figure 4.

. Representative fluorescent images of rat sperm after staining with Alexa Fluor-488-PNA, PI, or SYBR-14. (A) Solid line indicates intact acrosome, arrow heads indicates partially damaged and lost acrosome; (B) membrane-damaged; and (C) membrane-intact rat sperm. Color figure available online at www.andrologyjournal.org.

Experiment 2: Effect of Percoll Gradient Separation on Rat Sperm Motility and Acrosome and Membrane Integrity

Figures 5 and 6 show the effects of centrifugation force (600, 800, 1000, or 1200 × g) and duration (15 or 30 minutes) during Percoll gradient separation on epididymal SD and F-344 rat sperm motility characteristics and acrosomal integrity, respectively. Table 2 shows percentage progressive motility and corresponding VAP values of SD and F-344 sperm after Percoll separation. Percentage progressive motility and VAP were generally increased after Percoll separation of F-344 sperm (P < .05), whereas there was no change in VAP values for SD sperm (P > .05).

Figure 5.

. Percentage motility (means ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to Percoll gradient separation using different centrifugation forces (600, 800, 1000, and 1200 × g) and times (15 and 30 minutes).

Figure 6.

. Percentage acrosomal integrity (mean ± SEM) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to Percoll gradient separation using different centrifugation forces (600, 800, 1000, and 1200 × g) and times (15 and 30 minutes).

Table 2. . Percentage progressive motility and average path velocity values (mean ± SEM) of epididymal Fischer 344 and Sprague-Dawley rat sperm that were subjected to Percoll gradient separation using different centrifugation forces (600, 800, 1000, and 1200 × g) and times (15 and 30 min)
  
  
ForceTime, minProgressive Motility, %Average Path Velocity, μm/sProgressive Motility, %Average Path Velocity, μm/s
  1. aP < .05 vs control.

Control22.83 ± 3.53125.50 ± 4.5223.40 ± 2.16119.10 ± 3.80
6001528.17 ± 2.73124.00 ± 12.6527.00 ± 3.20125.00 ± 5.82
6003028.83 ± 1.85142.67 ± 4.66a25.70 ± 3.04112.30 ± 14.02
8001529.67 ± 6.25137.33 ± 9.40a33.50 ± 3.76a129.90 ± 17.13
8003028.00 ± 3.06138.50 ± 4.26a30.20 ± 1.60a124.90 ± 6.23
10001528.33 ± 3.32141.50 ± 6.16a31.80 ± 2.04a134.00 ± 4.46
10003034.50 ± 4.40a131.67 ± 7.0629.80 ± 1.42a127.60 ± 4.71
12001533.83 ± 2.55a146.17 ± 7.72a32.75 ± 5.95a130.13 ± 5.67
12003030.50 ± 4.66a139.00 ± 3.31a25.70 ± 2.47120.90 ± 5.66

Percoll gradient separation had no adverse effect on the acrosomal integrity of either F-344 or SD rat sperm (P > .05). Figure 7 shows the percentage of membrane-intact rat sperm following Percoll separation using various centrifugation forces and durations. As expected, the percentage of membrane-intact sperm significantly increased following Percoll separation for all centrifugation forces and durations for both SD and F-344 rat strains (P < .05). The optimal Percoll separation regime with regard to membrane integrity for both strains was 600 × g for 15 minutes without the need to further increase the centrifugation force and duration.

Figure 7.

. Percentage membrane integrity (mean ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm that were subjected to Percoll gradient separation using different centrifugation forces (600, 800, 1000, and 1200 × g) and times (15 and 30 minutes).

Experiment 3: Effect of Pipetting on Rat, Mouse, Ram, Bull, and Boar Sperm Motility and Membrane Integrity

Figure 8 shows the effects of repeated pipetting (2, 4, 6, 8, and 10 times) on epididymal and/or ejaculated rat, mouse, ram, bull, and boar sperm motility characteristics. Table 3 shows percentage progressive motility and corresponding VAP values of SD and F-344 sperm after repeated pipetting. Compared with the control, there was about 40% and 25% reduction in sperm motility even after 2 times pipetting of SD and F-344 rat sperm, respectively (P < .05). Overall, SD rat sperm showed more motility loss than did F-344 rat sperm (P < .05). Mouse sperm was also significantly affected by pipetting, more similarly to F-344 sperm than to SD sperm (P < .05). There was only a slight decrease in sperm motility for ejaculated bull and boar sperm as the number of pipettings increased (P > .05). There was no difference with regard to sperm motility between epididymal and ejaculated ram sperm that were subjected to the pipetting procedure 10 times (P > .05). For both strains, progressive motility and velocity were dramatically reduced after pipetting, and the pipetting effect was more adverse for SD than for F-344 sperm (P < .05). Figure 9 shows percentage membrane integrity of SD and F-344 sperm following repeated pipetting procedure. Pipetting SD or F-344 sperm as few as 2 times caused significant loss (approximately 50%) of membrane integrity, although this effect was much higher for SD than F344 sperm (P < .05).

Figure 8.

. Percentage motility (mean ± SEM; *P < .05 vs control) of epididymal rat (Sprague-Dawley [SD] and Fischer 344 [F-344]), mouse, and ram sperm and ejaculated ram, bull, and boar sperm after being subjected to pipetting procedure 2, 4, 6, 8, and 10 times.

Table 3. . Percentage progressive motility and average path velocity values (mean ± SEM) of epididymal Fischer 344 and Sprague-Dawley rat sperm after being subjected to 2, 4, 6, 8, or 10 times pipetting
 
 
Times PipettingProgressive Motility, %Average Path Velocity, μm/sProgressive Motility, %Average Path Velocity, μm/s
  1. aP < .05 vs control.

Control26.19 ± 2.12129.69 ± 8.8127.29 ± 1.73118.14 ± 4.52
215.50 ± 1.20a115.88 ± 4.8711.25 ± 3.35a95.13 ± 9.69
410.13 ± 1.64a86.69 ± 6.84a5.50 ± 1.70a73.00 ± 10.62a
65.44 ± 1.83a89.00 ± 13.04a4.50 ± 1.41a66.75 ± 12.24a
85.80 ± 1.20a83.13 ± 9.12a0.63 ± 0.42a39.63 ± 13.59a
104.27 ± 1.13a51.20 ± 12.95a0.29 ± 0.29a12.43 ± 8.08a
Figure 9.

. Percentage membrane integrity (mean ± SEM; *P < .05 vs control) of epididymal Sprague-Dawley (SD) and Fischer 344 (F-344) rat sperm after being subjected to pipetting procedure 2, 4, 6, 8, and 10 times.

Discussion

Collection of epididymal rat sperm and its subsequent dilution, pipetting, and centrifugation are the most commonly used laboratory practices by basic reproductive biology and toxicology laboratories as well as genome resource centers. Centrifugation procedure is one of the necessary steps during sperm washing in order to eliminate seminal fluid and semen extenders (ie, skim milk, egg yolk) and to increase sperm concentration. However, it may cause major physical stress on sperm. It was evident from this study that significant motility loss took place (30%–35% and 50%–65% for F-344 and SD, respectively) after using a minimal (200 × g) centrifugation force, which clearly demonstrated sensitivity of rat sperm to the centrifugation process. It was also very interesting to see no further loss when centrifugation force was increased from 200 × g to 400, 600, or 800 × g. Overall, for each centrifugation force there was a time-dependent decrease in motility and plasma membrane integrity as duration of centrifugation was extended from 5 minutes to 10 or 15 minutes, suggesting time-dependent rather than centrifugation force—dependent detrimental effects on motility.

Although there is no exact explanation with regard to how centrifugation affects rat sperm cells, mechanical effects and pellet formation during the centrifugation procedure have been proposed to be the major culprits in cell death and subsequent motility loss (Abidor et al, 1994; Katkov and Mazur, 1998). It has been reported that mouse and boar sperm are more affected if they are highly packed during the pellet state and distortion of the sperm pellet following centrifugation (Carvajal et al, 2004; Katkov and Mazur, 1998). If this were true for rat sperm, because higher centrifugation force and period would result in a tighter sperm pellet, we should have obtained higher motility loss as the centrifugation force was increased. However, no further motility loss was observed when centrifugation force was increased from 200 × g to 600 or 800 × g. These findings suggest that centrifugation time is a more important determinant of rat sperm motility than centrifugation force, and thus one should not centrifuge rat sperm for more time than necessary. Moreover, particular consideration should be given to strain differences during centrifugation because of different levels of sensitivity between strains.

Sensitivity of rat sperm to physical/mechanical stress such as osmotically driven volume excursion has also been previously reported. Si et al (2006) reported that rat sperm is also sensitive to aniso-osmotic stress, which was created by nonionic compounds such as sucrose. Sensitivity of human sperm to mechanical stress has been reported by several groups (Makler and Jakobi, 1981; Ng et al, 1990; Agarwal et al, 1994). Makler and Jakobi (1981) reported that human sperm is adversely affected by shaking for 30 seconds or more and that centrifugation force of 580 × g is detrimental. One of the early studies by Mack and Zaneveld (1987) suggested that centrifugation causes acrosomal damage in human sperm. Similarly to human sperm, SD sperm also showed more than a 20% decline in acrosomal integrity after being subjected to 800 × g centrifugation force for 15 or 30 minutes. However, we did not find significant change in acrosomal integrity for F-344 rat sperm after any centrifugation regimes tested.

Katkov and Mazur (1998) suggested that centrifugation of mouse sperm using 400 × g force for 15 minutes resulted in optimal sperm recovery for outbred ICR mouse sperm. In their study, increasing centrifugation force to 800 × g caused a 43% decrease in sperm motility compared with control even after 5 minutes. In 1 related study, Nakatsukasa et al (2001) centrifuged SD rat sperm using only 700 × g force for 5 minutes. They observed about 35% motility loss after 1 time centrifugation. The motility declined further after 2 and 3 times centrifugation of the same samples. These current studies are overall in agreement with their results using similar centrifugation forces, although our results showed about 50% motility loss after using 800 × g force for 5 minutes. As for some other species, Rijsselaere et al (2002) found that centrifugation at 720 × g for 5 minutes was optimal for dog sperm. Interestingly, Brinsko et al (2000) found that centrifugation and partial removal of stallion seminal plasma increases progressive motility.

In addition to centrifugation, Percoll gradient separation is one of the most commonly used techniques for semen enrichment as well as elimination of undesired contaminants, fraction of immotile sperm, blood, epithelial cells, and microbial agents from the semen. In frozen-thawed bovine spermatozoa, about 90% of the spermatozoa loaded on the Percoll gradient are recovered from centrifugation (Parrish et al, 1995). Moreover, using this protocol, bull sperm viability and acrosomal integrity are maintained after Percoll separation (Somfai et al, 2002). Boar sperm have also been successfully separated with Percoll gradient separation, using 900 × g for 15 minutes, and effectively used for in vitro fertilization studies (Grant et al, 1994; Suzuki and Nagai, 2003). In this study, depending on the centrifugation force and time, Percoll-separated sperm had a higher rate of progressive motility than controls. This may suggest that although centrifugation alone had detrimental effect on rat sperm motility, centrifugation during the course of Percoll gradient separation is not as harmful even after using 1000 or 1200 × g force for up to 30 minutes. Whereas the optimal Percoll separation conditions to obtain improved motility for SD sperm were determined as 800 × g for 15 minutes; 1200 × g for 15 minutes was optimal for F-344 rat sperm. Furimsky et al (2005) reported that Percoll-separated epididymal mouse sperm had significantly higher fertilizing ability than their nonseparated counterparts, and concluded that Percoll separation may be useful during mouse in vitro fertilization (IVF). Based on our current results for rat sperm. Percoll separation may be recommended to select the most competent sperm fraction for optimal IVF outcome. In addition, Percoll separation may be a useful procedure for frozen-thawed rat sperm in order to eliminate dead sperm and freezing extender components such as egg yolk (Nakatsukasa et al, 2001). On the other hand, Percoll separation would not be appropriate for toxicology studies, in which one needs to consider all sperm when comparing control vs treated rats or samples.

It is also important to note that, compared with the control, Percoll treatment made significant improvement on the sperm samples that had a higher rate of intact plasma membrane than motility. This was particularly apparent when we used 600 × g force for 15 minutes for SD (approximately 50%) and F-344 (approximately 30%). These data overall suggest that although membrane damage caused by centrifugation can be somewhat compensated for by Percoll separation, a relatively lesser extent of motility enrichment can be achieved. In this study, none of the Percoll-separation regimes had any detrimental effect on acrosomal integrity of rat sperm, suggesting that rat sperm acrosome is not sensitive to the centrifugation procedure of Percoll separation.

The current results dramatically showed that both rat and mouse sperm are exceptionally sensitive to repeated pipetting compared with sperm from other species tested. For both rat strains, pipetting 4 times caused a more than 50% decrease in both motility and membrane integrity, although SD rat sperm were more affected than F-344 rat sperm. We also compared epididymal and ejaculated ram sperm for their sensitivity to repeated pipetting in this study. Interestingly, motility of neither ejaculated nor epididymal ram sperm declined even after 10 times pipetting, indicating its strong resistance to such pipetting force. There may be some reasonable explanations with regard to the nature of the susceptibility of epididymal rat and mouse sperm to pipetting. During the pipetting procedure using standard pipette tips, there exists strong shear force, by which rat sperm is detrimentally affected. However, we cannot explain why the sperm of other species studied was not similarly affected. We speculate that the mechanism underlying the extreme sensitivity of epididymal sperm cannot be explained only by the source of the sperm being epididymal or ejaculate, because there was no difference in motility loss between epididymal and ejaculated ram sperm regardless of number of pipettings. However, it should be pointed out that sperm flagella are mainly responsible for motility, and both mouse (approximately 120 μm) and particularly rat (approximately 190 μm) spermatozoa have relatively longer flagella than do other mammalian species, including bull, boar, ram, and also human, which range from 38 to 60 μm long (Gao et al, 1997). Thus, to some extent the significant motility loss of rat sperm after being pipetted may be attributed to flagellar length, because mouse sperm also showed high sensitivity to such physical effects in the present study. In conclusion, physical interventions alone are lethal to epididymal rat spermatozoa, and thus for optimal rat sperm recovery one should consider the present information prior to planned reproductive studies.

Acknowledgement

The authors thank the Institute of Health Sciences of Ankara University and the Scientific and Technical Research Council of Turkey (TUBITAK) for their partial support.

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