| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the * Department of Animal Reproduction and
Artificial Insemination, School of Veterinary Medicine, University of Ankara,
Ankara, Turkey; the Department of Medical
Biology and Genetics, School of Veterinary Medicine, Afyon Kocatepe
University, Afyon, Turkey; and the Department
of Veterinary Pathobiology, College of Veterinary Medicine, University of
Missouri, Columbia, Missouri.
| Correspondence to: Dr Yuksel Agca, College of Veterinary Medicine, University of Missouri, 1600 East Rollins Road, Room W191, Columbia, MO 65211 (e-mail: agcay{at}missouri.edu). |
| Received for publication October 4, 2007; accepted for publication July 29, 2008. |
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Key words: Centrifugation, Percoll gradient, pipetting
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 |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
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
x 106, 15 x 106, and 9 x
106 sperm/mL, respectively. The final sperm concentration during
motility analysis was approximately 1 x 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
x 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 x
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
(2x 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 |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Figure 2 shows the effects of centrifugation forces (200, 400, 600, and 800 x 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 x 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 x g for 5 minutes, the corresponding values for F-344 sperm were either 200 x g or 400 x 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.
|
|
|
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 x 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).
|
|
|
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 x g for 15 minutes without the need to further increase the centrifugation force and duration.
|
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).
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
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 x g to 600 or 800 x 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 x 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 x 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 x g force for 15 minutes resulted in optimal sperm recovery for outbred ICR mouse sperm. In their study, increasing centrifugation force to 800 x 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 x 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 x g force for 5 minutes. As for some other species, Rijsselaere et al (2002) found that centrifugation at 720 x 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 x 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 x g force for up to 30 minutes. Whereas the optimal Percoll separation conditions to obtain improved motility for SD sperm were determined as 800 x g for 15 minutes; 1200 x 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 x 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.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Agarwal A, Ikemoto I, Loughlin KR. Effect of sperm washing on levels of reactive oxygen species in semen. Arch Androl. 1994;33: 157 –162.[Medline]
Agca Y, Critser JK. Assisted reproductive technologies and genetic engineering in rats. In: Suckow MA, Weisbroth SH, Franklin CL, eds. The Laboratory Rats. 2nd ed. San Diego, CA: Academic Press; 2005: 165–190.
Agca Y, Gilmore J, Byers M, Woods EJ, Liu J, Critser JK. Osmotic
characteristics of mouse spermatozoa in the presence of extenders and sugars.
Biol Reprod. 2002; 67: 1493
–1501.
Ben-Yosef D, Oron Y, Shalgi R. Low temperature and fertilization-induced Ca+2 changes in rat eggs. Mol Reprod Dev. 1995;42: 122 –129.[CrossRef][Medline]
Brinsko SP, Crockett EC, Squires EL. Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. Theriogenology. 2000; 54: 129 –136.[CrossRef][Medline]
Cardullo RA, Cone RA. Mechanical immobilization of rat sperm does not change their oxygen consumption rate. Biol Reprod. 1986; 34: 820 –830.[Abstract]
Carvajal G, Cuello C, Ruiz M, Vazquez JM, Martinez EA, Roca J.
Effects of centrifugation before freezing on boar sperm cryosurvival.
J Androl. 2004;25: 389
–396.
Chapin RE, Filler RS, Gulati D, Heindel JJ, Katz DF, Mebus CA, Obasaju F, Perreault SD, Russell SR, Schrader S, Slott V, Sokol RZ, Toth G. Methods for assessing rat sperm motility. Reprod Toxicol. 1992;6: 267 –273.[CrossRef][Medline]
Furimsky A, Vuong N, Xu H, Kumarathasan P, Xu M, Weerachatyanukul
W, Khalil MB, Kates M, Tanphaichitr N. Percoll gradient-centrifuged
capacitated mouse sperm have increased fertilizing ability and higher contents
of sulfogalactosylglycerolipid and docosahexaenoic acid-containing
phosphatidylcholine compared to washed capacitated mouse sperm.
Biol Reprod. 2005; 72: 574
–583.
Gao D, Mazur P, Critser JK. Fundamental cryobiology of mammalian spermatozoa. In: Karow A, Critser JK, eds. Reproductive Tissue Banking: Scientific Principles. San Diego, CA: Academic Press; 1997 : 263–328.
Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, et al. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature. 2004; 428: 493 –521.[CrossRef][Medline]
Grant SA, Long SE, Parkinson TJ. Fertilizability and structural
properties of boar spermatozoa prepared by Percoll gradient centrifugation.
J Reprod Fertil. 1994; 100: 477
–483.
Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian
sperm: what we ask them to survive. J Androl. 1990; 11: 73
–88.
Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology. 2000; 3: 47 –58.
Katkov II, Mazur P. Influence of centrifugation regimes on
motility, yield, and cell associations of mouse spermatozoa. J
Androl. 1998;19: 232
–241.
Katkov II, Mazur P. Factors affecting yield and survival of cells when suspensions are subjected to centrifugation. Influence of centrifugal acceleration, time of centrifugation, and length of the suspension column in quasi-homogeneous centrifugal fields. Cell Biochem Biophys. 1999;31: 231 –245.[Medline]
Lazar J, Moreno C, Jacob HJ, Kwitek AE. Impact of genomics on
research in the rat. Genome Res. 2005; 15: 1717
–1728.
Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline
transmission and tissue-specific expression of transgenes delivered by
lentiviral vectors. Science. 2002; 295: 868
–872.
Mack SR, Zaneveld LJ. Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res. 1987;18: 375 –383.[CrossRef][Medline]
Makler A, Jakobi P. Effects of shaking and centrifugation on human sperm motility. Arch Androl. 1981; 7: 21 –26.[Medline]
Matsumoto H, Sugawara S. Effect of phosphate on the second cleavage
division of the rat embryo. Hum Reprod. 1998; 13: 398
–402.
Moore HD, Akhondi MA. Fertilizing capacity of rat spermatozoa is
correlated with decline in straight-line velocity measured by continuous
computer-aided sperm analysis: epididymal rat spermatozoa from the proximal
cauda have a greater fertilizing capacity in vitro than those from the distal
cauda or vas deferens. J Androl. 1996; 17: 50
–60.
Nakatsukasa E, Inomata T, Ikeda T, Shino M, Kashiwazaki N. Generation of live rat offspring by intrauterine insemination with epididymal spermatozoa cryopreserved at –196 degrees C. Reproduction. 2001; 122: 463 –467.[Abstract]
Nakatsukasa E, Kashiwazaki N, Takizawa A, Shino M, Kitada K, Serikawa T, Hakamata Y, Kobayashi E, Takahashi R, Ueda M, Nakashima T, Nakagata N. Cryopreservation of spermatozoa from closed colonies, and inbred, spontaneous mutant, and transgenic strains of rats. Comp Med. 2003;53: 639 –641.[Medline]
Ng SC, Bongso TA, Sathananthan H, Tok VC, Ratnam SS.
Micro-centrifugation of human spermatozoa: its effect on fertilization of
hamster oocytes after micro-insemination spermatozoal transfer. Hum
Reprod. 1990;5: 209
–211.
Nishikimi A, Uekawa N, Yamada M. Involvement of glycolytic metabolism in developmental inhibition of rat two-cell embryos by phosphate. J Exp Zool. 2000; 287: 503 –509.[CrossRef][Medline]
Oh SH, Miyoshi K, Funahashi H. Rat oocytes fertilized in modified
rat 1-cell embryo culture medium containing a high sodium chloride
concentration and bovine serum albumin maintain developmental ability to the
blastocyst stage. Biol Reprod. 1998; 59: 884
–889.
Parrish JJ, Krogenaes A, Susko-Parrish JL. Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology. 1995; 44: 859 –869.[CrossRef][Medline]
Perreault SD. Smart use of computer aided sperm analysis (CASA) to characterize sperm motion. In: Robaire B, Hinton BH, eds. The Epididymis. New York, NY: Kluwer Academic/Plenum Publishers; 2002 : 459–471.
Rijsselaere T, Van Soom A, Maes D, de Kruif A. Effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. Theriogenology. 2002; 57: 1669 –1681.[CrossRef][Medline]
SAS Institute Inc. SAS User's Guide: Statistics. Cary, NC: SAS Institute Inc; 1985 .
Si W, Benson JD, Men H, Critser JK. Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm. Cryobiology. 2006; 53: 336 –348.[Medline]
Slott VL, Suarez JD, Poss PM, Linder RE, Strader LF, Perreault SD. Optimization of the Hamilton-Thorn computerized sperm motility analysis system for use with rat spermatozoa in toxicological studies. Fundam Appl Toxicol. 1993;21: 298 –307.[CrossRef][Medline]
Somfai T, Bodo S, Nagy S, Papp AB, Ivancsics J, Baranyai B, Gocza E, Kovacs A. Effect of swim up and Percoll treatment on viability and acrosome integrity of frozen-thawed bull spermatozoa. Reprod Domest Anim. 2002;37: 285 –290.[CrossRef][Medline]
Suzuki K, Nagai T. In vitro fertility and motility characteristics of frozen-thawed boar epididymal spermatozoa separated by Percoll. Theriogenology. 2003; 60: 1481 –1494.[CrossRef][Medline]
Tesson L, Cozzi J, Menoret S, Remy S, Usal C, Fraichard A, Anegon I. Transgenic modifications of the rat genome. Transgenic Res. 2005;14: 531 –546.[CrossRef][Medline]
Toyoda Y, Chang MC. Fertilization of rat eggs in vitro by
epididymal spermatozoa and the development of eggs following transfer.
J Reprod Fertil. 1974; 36: 9
–22.
Vincent R, Nadeau D. Adjustment of the osmolality of Percoll for the isotonic separation of cells and cell organelles. Anal Biochem. 1984;141: 322 –328.[CrossRef][Medline]
Yeung CH, Oberländer G, Cooper TG. Characterization of the
motility of maturing rat spermatozoa by computer-aided objective measurement.
J Reprod Fertil. 1992; 96: 427
–441.
Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN. Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol. 2003; 21: 645 –651.[CrossRef][Medline]
Zernicka-Goetz M. Spontaneous and induced activation of rat oocytes. Mol Reprod Dev. 1991; 28: 169 –176.[CrossRef][Medline]
Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y,
Fraichard A, Cozzi J. Generation of fertile cloned rats by regulating oocyte
activation. Science. 2003; 302: 1179
.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
