- Top of page
- Materials and Methods
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.
- Top of page
- Materials and Methods
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.