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,
From the * Department of Reproductive Sciences,
Center for Species Survival, Smithsonian's National Zoological Park, Front
Royal, Virginia; and the Cheetah Conservation
Fund, Otjiwarongo, Namibia.
Present address: Namibian Ministry of
Environment and Tourism, Private Bag 13306, Windhoek, Namibia.
| Correspondence to: Dr Adrienne E. Crosier, Smithsonian's National Zoological Park, Conservation and Research Center, Department of Reproductive Sciences, 1500 Remount Rd, Front Royal, VA 22630 (e-mail: crosiera{at}si.edu). |
| Received for publication July 8, 2008; accepted for publication September 23, 2008. |
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Abstract |
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Key words: Felid, acrosome, cryopreservation, cryoprotectant, genome resource banking
The preservation of germplasm, especially spermatozoa, can assist with conserving genetic diversity in captive breeding programs for select wildlife species (Holt and Pickard, 1999; Wildt et al, 2001; Agca and Critser, 2002). For the cheetah, the organized collection and storage of spermatozoa in a genome resource bank would allow genetic management of this species on a global scale potentially using a variety of assisted reproductive techniques (Wildt et al, 1997). Sperm already have been collected and frozen from a wild cheetah, transferred intercontinentally, and used via artificial insemination (AI) to produce offspring (Wildt et al, 1997; Howard and Wildt, 2009). Additionally, cryopreserved cheetah sperm have been used successfully for in vitro production of embryos in North America and Namibia (Crosier et al, unpublished data). If more effective, this global approach has the potential for large-scale infusions of new genetic material into the captive population without removing more cheetahs from the wild to maintain genetic diversity among zoological collections. The use of cryopreserved sperm for assisted breeding also overcomes the common problem of sexual incompatibility between male and female cheetahs (Caro, 1993; Wildt et al, 1993) and enables managers to move genetic material between institutions while eliminating the stresses associated with transporting live, wild animals (Wildt et al, 1997, 2001).
Cryopreserved cheetah sperm experience loss of membrane and acrosomal integrity in as many as 40% of cells following freeze-thawing using current laboratory protocols (Crosier et al, 2006). Spermatozoa typically are cryopreserved using the permeating cryoprotectant glycerol, which in turn, contributes to fluid flux and osmotic stress that can disrupt cell membrane integrity (Gao et al, 1995; Pukazhenthi et al, 2002). Most damage during cryopreservation is caused by cryoprotectant and water movement across sperm membranes during initial equilibration before freezing and again during thawing as the cryoprotectant is removed and the cell rehydrated. Spermatozoa from teratospermic animals, such as the cheetah, are especially vulnerable to osmotic (Pukazhenthi et al, 2001) and freezing-induced damage (Crosier et al, 2006). We have determined that the sperm acrosome in teratospermic felids is especially vulnerable to damage during cryopreservation and thawing (Crosier et al, 2006; Pukazhenthi et al, 2006).
Assisted reproductive techniques with cryopreserved sperm routinely use postthaw processing methods to concentrate cellular suspensions, remove egg yolk prior to intrauterine insemination, and select spermatozoa with good motility (Henkel and Schill, 2003). For example, sperm samples collected for AI in felids generally are washed (resuspended in medium and centrifuged) with the resulting pellet directly mixed with insemination medium to maximize sperm numbers (Howard et al, 1992, 1997). In comparison, cryopreserved felid sperm used for in vitro fertilization (IVF) usually are subjected to swim-up processing to enhance sperm motility and to improve the number of structurally normal cells available (Comizzoli et al, 2003). However, the swim-up technique reduces sperm concentration (Howard et al, 1990), which is of special concern in a species producing approximately 75% pleiomorphic cells in an average ejaculate. Maintaining cellular integrity also is critical, and we have evidence that multistep cryoprotectant addition and removal using a buffered solution is beneficial in felids (Pukazhenthi et al, 2002). Furthermore, we recently demonstrated that cheetah spermatozoa withstand incubation in glycerol for up to 60 minutes at room temperature before cryopreservation with little detrimental impact on postthaw acrosomal integrity (Crosier et al, 2006). This observation suggests that the majority of damage to cheetah sperm integrity occurs during cryoprotectant removal from cells after thawing.
It was logical that the next step toward enhancing the viability of thawed cheetah spermatozoa was to explore novel glycerol removal approaches for enhancing both motility and acrosomal integrity. Sperm centrifugation through a density gradient (eg, Percoll) is widely used in domestic livestock and humans for separating sperm from seminal fluid (McLean et al, 1998), separating motile from nonmotile cells (Sbracia et al, 1996), and removing cryoprotectant from thawed sperm samples (Long and Kulkarni, 2004). Recently, Accudenz (formerly known as Nycodenz) has gained popularity as an inert and chemically dense substance that prevents endotoxin precipitation (Sbracia et al, 1996). Accudenz also is noted for yielding sperm fractions with better motility longevity compared with those filtered through a Percoll gradient (Froman and McLean, 1996; King et al, 2000). Centrifugation through Accudenz gradients of increasing density has been shown to mitigate the damage arising from removing cryoprotectant from spermatozoa and facilitates a slower transfer of permeating cryoprotectant and water across cell membranes (Long and Kulkarni, 2004). For poultry spermatozoa, the use of Accudenz improved fertility after AI compared with a standard Percoll gradient (Long and Kulkarni, 2004).
All evidence to date strongly suggests that the most significant damage induced by contemporary cryopreservation protocols for cheetah spermatozoa is related to cryoprotectant removal after thawing. Further, we suspect that mitigating the osmotic stress associated with glycerol elimination after thawing cheetah spermatozoa would coincidentally enhance acrosomal integrity while maintaining sperm motility. Thus, the objective of the present study was to determine if postthaw sperm quality was improved significantly by processing through a gradient filtration compared with the current protocols of traditional sperm washing, multistep resuspension, and swim-up processing. We hypothesized that use of an Accudenz gradient would effectively remove glycerol from thawed cheetah spermatozoa, which in turn, would enhance sperm motility and acrosomal integrity.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Anesthesia was induced in all animals using a combination of tiletamine hydrochloride and zolazepam (4–6 mg/kg body weight; Telazol; Fort Dodge Laboratories, Fort Dodge, Iowa) (Wildt et al, 1987; Crosier et al, 2007) or a combination of medetomidine hydrochloride (1.5–2 µg/kg body weight; Domitor; Pfizer Inc, La Jolla, California) and tiletamine hydrochloride/zolazepam (2 mg/kg body weight; Telazol; Fort Dodge Laboratories) (Crosier et al, 2007) administered intramuscularly. Testicular length and width were measured using laboratory calipers and then converted to total testes volume per male (Howard et al, 1990). A rectal probe of 1.6 or 1.9 cm in diameter with 3 longitudinal electrodes and an electrostimulator (P.T. Electronics, Boring, Oregon) were used to provide a total of 80 stimuli (at a low voltage of 2–5 volts) over a 30-minute interval (Howard, 1993). Ejaculates (n = 21) were collected in sterile prewarmed collection vials as previously described (Wildt et al, 1983; Howard, 1993; Crosier et al, 2006, 2007).
Each raw ejaculate was examined for volume, and an aliquot (3 µL) was immediately assessed for sperm percent motility (% M) and forward progressive status (FPS; scale, 0–5 with a 5 rating equivalent to rapid, straightforward progression [Howard, 1993]) using a prewarmed microscope slide and coverslip. A sperm motility index (SMI) was calculated using the formula (% M + [FPS x 20]) ÷ 2 (Howard et al, 1990). A 15-µL aliquot of raw semen was fixed in 100 µL of 0.3% glutaraldehyde in phosphate-buffered saline (pH 7.4, 340 mOsm) (Pukazhenthi et al, 2000) for assessing sperm morphology. A 15-µL aliquot of raw semen also was fixed in 500 µL of 4% paraformaldehyde for evaluating acrosomal integrity. The remainder of the raw ejaculate was diluted 1:1 (vol/vol) with sterile Ham F10 culture medium (HF10; Irvine Scientific, Santa Ana, California) supplemented with 20 mM HEPES, 5% (vol/vol) fetal calf serum (Irvine Scientific), pyruvate (1 mM), L-glutamine (2 mM), 10 000 IU/mL penicillin, 10 mg/mL streptomycin, and 20 mg/mL neomycin (Sigma Chemical Co, St Louis, Missouri). The final handling medium had an osmolarity of approximately 285 mOsm. Sperm concentration was determined using a hemocytometer (Wildt et al, 1983; Howard, 1993; Crosier et al, 2006).
Sperm Cryopreservation
Each ejaculate diluted in HF10 was centrifuged (Mini-Spin; Eppendorf,
Hamburg, Germany) for 8 minutes at 100 x g and the supernatant
aspirated from the sperm pellet. The resulting sperm pellet was resuspended in
TEST-yolk buffer (TYB refrigeration medium; Irvine Scientific) containing 0%
glycerol at ambient temperature. A modified TYB containing 8% glycerol
(vol/vol) was prepared as a 2:1 mixture of the "freezing medium –
TYB with 12% glycerol" (Irvine Scientific) and "refrigeration
medium without glycerol." Each sample was placed in a water bath (350 mL
at ambient temperature) and slow cooled (approximately 3.5 hours) to 5°C.
After reaching this temperature, TYB plus 8% glycerol was added in 3 aliquots
over 30 minutes (adding one-fourth volume and waiting 15 minutes, adding
one-fourth volume and waiting 15 minutes, and then adding the remaining half
volume). Samples were loaded into 0.25-mL straws containing 80 to 120 µL of
sperm suspension with an average final sperm concentration of 67.8 ±
5.3 x 106 motile cells/mL (range, 30–120 x
106 motile cells/mL) and frozen over liquid nitrogen (LN) using a
2-step cryomethod (Crosier et al,
2006). Briefly, straws were placed 7.62 cm above LN for 1 minute,
then 2.54 cm above liquid for an additional 1 minute, and then plunged into
LN.
Study 1: Efficacy of an Accudenz Gradient for Glycerol Removal
Cryopreserved straws (n = 9 ejaculates total from 5 males) were thawed
individually for 10 seconds in air followed by immersion in a 37°C water
bath for 30 seconds. Each straw was dried, and its contents emptied into a
sterile Eppendorf tube (1–2 straws/tube; approximately 150 µL of
suspension total). The thawed aliquot immediately was assessed for % M and
FPS, and 10 µL was fixed to subsequently determine percent intact acrosomal
membranes (% IA). Remaining sperm suspension in the Eppendorf tube then was
divided equally (40–50 µL of sperm suspension/aliquot) among 3
processing treatments: 1) sperm washing (control), 2) multistep resuspension,
or 3) Accudenz (Accurate Chemical and Scientific Corp, Westbury, New York).
These treatments were selected to represent different rates of glycerol
removal from cryopreserved sperm
(Pukazhenthi et al, 2002). The
control treatment (traditional method for handling cryopreserved cheetah sperm
[Howard, 1990]) represented "rapid" glycerol removal in that a
single aliquot of culture medium (HF10) was pipetted into the sample followed
by centrifugation to pellet the sperm. The multistep treatment represented
"moderate" glycerol removal through its slow addition (in multiple
steps) of a greater volume of medium (1400 µL) compared with the control
(300 µL). The third treatment (Accudenz gradient, which separates cells in
suspension based on density [Sbracia et
al, 1996]) represented the first examination of the influence of a
gradient approach on a carnivore ejaculate and specifically from a species
well known for its low sperm density, prevalence of malformed cells, and
acrosomal sensitivity to cooling and thawing
(Pukazhenthi et al, 2001;
Crosier et al, 2006).
For the control treatment, sperm aliquots in TYB were diluted with 300 µL of HF10 added dropwise over 1 minute. Samples in HF10 were centrifuged (8 minutes at 100 x g), the supernatant removed, and the sperm pellet resuspended in 200 µL of HF10 added dropwise over 1 minute. For the multistep treatment, sperm aliquots in TYB were slowly diluted (added in 7 equal 200-µL aliquots over 3 minutes) with 1400 µL of HF10. The sample in HF10 then was centrifuged (8 minutes at 100 x g), the supernatant removed, and resulting pellet resuspended in 200 µL of HF10 added dropwise over 1 minute. For the Accudenz treatment, a modification of the technique described by Long and Kalkarni (2004) was used. The gradient was created by layering 100 µL of 10% (wt/vol) Accudenz in HF10 underneath 500 µL of 4% (wt/vol) Accudenz in HF10 in an Eppendorf tube. The thawed sperm sample in TYB was layered on top of the Accudenz gradient and centrifuged (8 minutes at 100 x g). After centrifugation, the entire suspension formed 3 distinctive layers: the top layer, predominantly composed of TYB; intermediate containing a high concentration of motile sperm; and bottom pellet of less motile cells. The intermediate layer was removed, diluted in 1 mL of HF10, and centrifuged a second time (8 minutes at 100 x g) to remove residual Accudenz. The resulting pellet was resuspended in 200 µL of HF10 added dropwise over 1 minute. Each sample from all treatments was assessed for % M, FPS, and % IA postdilution (precentrifugation), postcentrifugation, and at hourly intervals for 4 hours (while being maintained in Eppendorf tubes at a constant ambient temperature and protected from light).
Study 2: Efficacy of a Single Centrifugation With the Accudenz Gradient for Improving Sperm Motility
Cryopreserved straws (n = 6 ejaculates total from 5 males) were thawed (as
in study 1; 3 of 5 donors were the same males as in study 1). In this study,
the contents of 2 straws per ejaculate were emptied into a sterile Eppendorf
tube (approximately 150 µL of suspension total) and immediately evaluated
for % M, FPS, and % IA. The remaining sperm suspension then was divided
equally (approximately 75 µL of suspension per aliquot) between 2
treatments, the control procedure and the Accudenz gradient. The control
treatment was modified slightly to include an initially larger volume of HF10
(1 mL [compared with 300 µL for study 1]) to allow more thorough glycerol
removal. Specifically, a sperm aliquot in TYB (approximately 75 µL) was
diluted slowly with 1 mL of HF10 added dropwise over 1 minute followed by
centrifugation (8 minutes at 100 x g) and supernatant removal.
The resulting sperm pellet was resuspended in 200 µL of HF10 added dropwise
over 1 minute and then evaluated.
The Accudenz protocol was modified to include only a single centrifugation step (to eliminate the detrimental effects of a second centrifugation on sperm motility; see study 1 results). The Accudenz gradient was prepared as in study 1, and then each thawed sample in TYB (approximately 80 µL) was layered on top of the gradient and the tube centrifuged (8 minutes at 100 x g). The intermediate layer (approximately 80 µL) was removed and resuspended with fresh HF10 to a total volume of 200 µL. All samples were assessed for % M, FPS, and % IA immediately postthaw, postdilution before centrifugation, postcentrifugation, and hourly for 4 hours.
Study 3: Efficacy of the Accudenz Gradient Compared With Swim-up Processing for Enhancing Sperm Morphology
Cryopreserved straws (n = 6 ejaculates total from 6 males) were thawed (as
in studies 1 and 2; 3 of 6 donors were the same males as in studies 1 and 2).
To allow more comparative examinations, the contents of 2 or 3 straws were
emptied into a single Eppendorf tube (approximately 240 µL of suspension
total) and then assessed immediately for % M, FPS, and % IA. The collective
content of each tube was divided equally (approximately 80 µL of suspension
per aliquot) among 3 processing treatments: the control (washing) procedure,
swim-up, and the Accudenz gradient. The control and Accudenz treatments were
identical to those described for study 2. For swim-up, a thawed sample was
resuspended in 1 mL of HF10 and centrifuged (8 minutes at 100 x
g), the supernatant removed, and the sperm pellet overlaid gently
with 60 µL of HF10. Sperm were allowed to swim-up into the medium for 30
minutes after which 50 µL of the supernatant was removed and resuspended in
150 µL of HF10 for a final volume of 200 µL. An aliquot (20 µL) was
removed to determine sperm structural morphology and concentration immediately
after swim-up processing. Samples from all treatments were assessed for % M,
FPS, and % IA immediately postthaw, following the allocated swim-up time (30
minutes), and at hourly intervals for 4 hours.
Assessment of Sperm Morphology and Acrosomal Integrity
Sperm morphology was evaluated using phase-contrast microscopy at
x1000 (Wildt et al,
1987; Howard,
1993; Crosier et al,
2007). Sperm were classified as normal or as having 1 of the
following abnormalities: 1) head anomalies, including microcephalic,
macrocephalic, and bicephalic or tricephalic; 2) acrosomal aberrations,
including missing, loose, or damaged acrosomal membrane; 3) midpiece
deviations, including abnormal or missing midpiece, bent midpiece with
retained cytoplasmic droplet, or bent midpiece with no droplet; 4) flagellar
irregularities, including tightly coiled flagellum, bent flagellum with
retained cytoplasmic droplet, bent flagellum with no droplet, biflagellate or
triflagellate, retained proximal cytoplasmic droplet, or retained distal
droplet; and 5) other abnormalities, including spermatid, bent neck and
detached head, or detached flagellum
(Crosier et al, 2007). If an
individual spermatozoon had an abnormality of either the head or acrosome in
addition to an anomaly of either the midpiece or flagellum, the cell was
classified as having a head or acrosomal defect. Similarly, if an individual
sperm had both a midpiece as well as a flagellar abnormality, the cell was
classified as having a midpiece defect.
Sperm samples were evaluated for acrosomal integrity using a modified Coomassie staining technique (Larson and Miller, 1999; Crosier et al, 2006). Briefly, aliquots fixed in 4% paraformaldehyde were centrifuged for 8 minutes at 2000 x g and the supernatant discarded. Resulting sperm pellets were washed twice with 500 µL of 0.1 M ammonium acetate (pH 9.0) and the final pellet resuspended in approximately 50 µL of the ammonium acetate solution. This sperm suspension was smeared onto a single microscope slide and allowed to dry at ambient temperature. Each slide was flooded with Coomassie stain (0.22% of Coomassie Blue G-250; Fisher Biotech, Springfield, New Jersey) for 90 seconds, rinsed with deionized water, dried at ambient temperature, and permanently preserved by placing a coverslip over a drop of mounting medium (Krystalon; EM Science, Gibbstown, New Jersey). For each sample, either 100 or 200 spermatozoa (dependent on sperm concentration) were assessed individually for acrosomal integrity using bright-field microscopy at x1000. Each spermatozoon was categorized as having an intact or nonintact acrosome, and the number of sperm per sample with intact membranes was converted to a percentage (Crosier et al, 2006). Sperm considered to have intact acrosomes displayed uniform staining overlying the entire acrosomal region (Figure 1A) compared with patchy or no staining for cells with damaged or missing acrosomes, respectively (Figure 1B).
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Results |
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Study 1: Efficacy of an Accudenz Gradient for Glycerol Removal
Cryopreserved cheetah sperm samples processed for glycerol removal using
the control, multistep, or Accudenz approaches maintained similar (P
> .05) sperm motility (SMI, approximately 60) through the washing
(postcentrifugation) step after thawing
(Figure 2A). Compared with
controls, sperm subjected to multistep resuspension experienced a reduced
(P < .05) SMI both immediately after resuspension
(precentrifugation) as well as after centrifugation
(Figure 2B). The SMI was
similar (P > .05) between controls and Accudenz for all time
points except an approximately 10% reduction for the latter treatment at 4
hours (Figure 2C).
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Study 3: Efficacy of the Accudenz Gradient Compared With Swim-up Processing for Enhancing Sperm Morphology
Compared with the control, both swim-up and Accudenz processing increased
(P < .05) the proportion of structurally normal sperm recovered
(Table 3). Additionally, the
proportion of head anomalies was reduced (P < .05) for Accudenz
samples compared with both the control and swim-up treatments. However, there
was no difference (P > .05) among treatments in the proportion of
midpiece, flagellar, or "other" deformities observed
(Table 3).
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Because the control treatment involved pelleting the entire sperm population after washing, the concentration of spermatozoa was highest (P < .05) for this treatment compared with either swim-up or Accudenz (Table 3). The total number of motile sperm was higher (P < .05) for the control compared with swim-up samples but was comparable (P > .05) to Accudenz (Table 3). Because of the increase in percent normal sperm from both the swim-up and Accudenz treatments, there was no difference (P > .05) among groups in number of total motile normal sperm recovered (Table 3).
For sperm motility, the SMI was comparable (P > .05) for all treatments and time points through 1.5 hours of postthawing (Figure 4). However, centrifugation through Accudenz resulted in superior (P < .05) sperm motility compared with control and swim-up treatments at 2.5 and 3.5 hours postthaw. There was 1 time point (2.5 hours after Accudenz) at which there was an improvement (P < .05) in sperm acrosomal morphology (8%–10%), a difference that was not sustained for the 2 later evaluation periods (Table 4).
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Discussion |
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Standard protocols for cryopreserving felid sperm rely on adding glycerol slowly to cells before freezing, either at room temperature or after cooling (Pukazhenthi et al, 2001, 2006; Crosier et al, 2006). Intracellular water is replaced by glycerol, and upon thawing, the reverse occurs. During glycerol removal, spermatozoa swell in response to a rapid influx of extracellular water, resulting in extreme volume excursions, which can cause cell membrane disruptions (Noiles et al, 1993). Cheetah spermatozoa are not particularly sensitive to osmotic changes during equilibrium prefreeze but are highly vulnerable to injury during the thawing and resuspension processes. The density gradient, Accudenz, appeared to slow the movement of water and/or cryoprotectant across the membranes, thereby maintaining higher levels of acrosomal integrity compared with conventional 1-step or multistep removal protocol. In this context, our findings were similar to an earlier study of poultry spermatozoa (Sbracia et al, 1996), demonstrating for the first time the applicability of Accudenz to carnivore sperm, even in a species that usually produces extraordinary numbers of pleiomorphic spermatozoa.
Other commonly used separation gradients (ie, Percoll, PureSperm, IxaPrep) function in much the same way as Accudenz by creating layers of increasing osmolarity. Since the withdrawal of Percoll from use in human assisted reproduction in 1996, alternative nontoxic density gradients, including Accudenz, have been investigated. This gradient has been popularized for use in human and domestic poultry sperm samples, both to remove seminal plasma from fresh ejaculates as well as to remove cryoprotectants from thawed suspensions (Sbracia et al, 1996; King et al, 2000; Long and Kulkarni, 2004). This is important for use in human intrauterine AI procedures because Accudenz does not have to be removed completely from a sperm sample before AI or other assisted reproductive techniques. In the present study, we were initially interested in determining if using Accudenz on thawed cheetah sperm influenced membrane integrity as well as motility over time. Therefore, the objective of study 1 was to thoroughly remove Accudenz from each sperm sample by 2 centrifugation steps and resuspending the pellet in fresh, gradient-free medium. This approach obviously exerted additional mechanical stress that did not accelerate motility decline nor did it improve overall sperm motility. Because Accudenz is nontoxic to cells (Sbracia et al, 1996), we then investigated a less intensive centrifugation protocol that involved direct removal of the interphase gradient layer containing the highest proportion of motile and acrosome-intact sperm. The result was improved sperm motility as well as acrosomal integrity, which agreed with earlier reports in human sperm samples (Sbracia et al, 1996).
Although securing significant populations of acrosome-intact sperm postthawing is important for AI, we also envision a role for embryo technologies in the genetic management of the cheetah. We recently made significant progress in producing cheetah embryos in vitro (Crosier et al, unpublished), a process that also requires membrane-intact, motile spermatozoa. IVF in felids has traditionally relied on the use of the swim-up technique, which is based on the concept that the most motile spermatozoa will actively swim from a centrifuged seminal pellet into an overlying medium that, in turn, serves as a resource for the inseminate (Howard et al, 1990; Henkel and Schill, 2003). This approach works well for felid IVF, producing sperm samples with improved motility and normal morphology and decreased debris compared with controls (Howard et al, 1990). The disadvantage is that swim-up processing results in recovery of low numbers of spermatozoa. The present study revealed that, although swim-up processing and exposure to the Accudenz gradient produced reduced sperm concentrations compared with the traditional washing protocol, both of these treatments significantly improved the proportions of recoverable normal spermatozoa and in similar proportions. We envision that the Accudenz gradient might be the most practical application for assisted breeding in cheetahs that could benefit from IVF. Our most recent studies determined that as many as 40% of cheetah oocytes matured in vitro formed cleaved embryos after insemination with as few as approximately 1.0 x 105 motile sperm/mL (Crosier et al, unpublished results). Extrapolating to findings from study 3 here, we recovered an average of 4.0 x 105 total motile sperm with intact acrosomal membranes from each 80-µL aliquot of TYB exposed to Accudenz (Table 4). Therefore, given an average of approximately 80 x 106 total motile sperm in a raw sample (Table 1) and sperm recovery postthaw, it is potentially possible to produce up to 60 doses of high-quality IVF inseminates from a single cheetah ejaculate.
The Accudenz gradient approach also has been touted as a means for restoring sperm cells to a chemically defined environment, including allowing the removal of 98% of seminal plasma proteins (McLean et al, 1998). Novel approaches to cheetah conservation have involved the development of a genome resource bank (ie, an organized frozen repository including germplasm) for the explicit purpose of improved genetic management by moving sperm and embryos rather than whole, living animals (Wildt et al, 1997; Holt and Pickard, 1999). It is well known that the cheetah appears vulnerable to infectious diseases related no doubt to the species' lack of genetic diversity and homozygosity at the level of the major histocompatibility complex (O'Brien et al, 1983, 1985). For other species, the transmission of pathogens generally occurs via seminal plasma rather than the sperm cell itself, which resists incorporating viral genetic material (Couto et al, 2004). Therefore, it is worth noting that passing cheetah sperm through a concentration gradient to remove virtually all seminal plasma has the added benefit of substantially lessening (or totally eliminating) the potential of disease transmission as sperm samples begin to be transported intercontinentally to benefit genetic management. Further studies designed to investigate the efficacy of Accudenz for removing infectious agents are warranted.
In conclusion, this investigation confirmed that the cheetah spermatozoon is highly sensitive to the processes of thawing and cryoprotectant removal, which cause a more than 35% reduction in cells with intact acrosomes and more than 15% decline in cellular motility. Traditional postthaw processing, including the use of swim-up separation, fails to reduce this damage that appears related to the removal of glycerol from the spermatozoon. However, it is clear that most of the postthaw damage can be mitigated by Accudenz gradient processing, which alleviates a significant portion of the injurious effects of glycerol permeating the cell membrane. The benefit is largely expressed by the retention of acrosomal integrity in a higher proportion of spermatozoa. The biological impact of this improved quality factor now can be assessed in our contemporary IVF studies geared to eventually applying cryopreserved sperm to enhanced reproduction and genetic management.
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Acknowledgments |
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Footnotes |
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