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From the * California National Primate Research
Center, University of California, Davis, California; the
School of Environmental Science and Public
Health, Wenzhou Medical College, Wenzhou, China; and the
Department of Obstetrics and Gynecology,
School of Medicine, University of California, Davis, California.
| Correspondence to: Dr Catherine A. VandeVoort, PhD, California National Primate Research Center, University of California, Davis, CA 95616 (e-mail: cavandevoort{at}ucdavis.edu). |
| Received for publication July 24, 2008; accepted for publication October 9, 2008. |
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Abstract |
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Key words: Epididymides, assisted reproduction, semen, Macaca mulatta, nonhuman primates
Earlier studies hypothesized that LDL, especially its phospholipids, could bind or adhere to sperm membranes (direct association) and provide protection by forming a protective film (Quinn et al, 1980) or replacing lost phospholipids (Foulkes et al, 1980; Graham and Foote, 1987; Trimeche et al, 1996) at the surface of sperm membranes. Alternatively, recent studies suggest that LDL may offer protection to sperm by reducing the deleterious effect of seminal plasma proteins on the sperm membrane (indirect association; Bergeron and Manjunath, 2006). In this latter hypothesis, a family of lipid-binding proteins present in bull seminal plasma (BSP proteins) was found to interact with LDL (Manjunath and Thérien, 2002; Bergeron et al, 2004). These BSP proteins are secreted by the seminal vesicles upon ejaculation, and their binding to the sperm membrane induces cholesterol and phospholipid efflux (Thérien et al, 1998, 1999), which in turn results in decreased resistance to cold shock and freezing (see Witte and Schäfer-Somi, 2007, for review). When semen is diluted with extenders containing egg yolk, LDL sequesters most of the BSP proteins present in semen (hereafter referred to as the "LDL-BSP hypothesis"), thus preventing lipid efflux from the sperm membrane, and consequently sperm are more resistant to cryodamage (Bergeron and Manjunath, 2006). Because homologs of BSP proteins from other species were also found to bind LDL, this mechanism seems to be responsible for the ubiquitous protective role of egg yolk in sperm cryopreservation of various mammals (Bergeron and Manjunath, 2006).
Based on the LDL-BSP hypothesis, Bergeron and Manjunath (2006) predicted that increasing the proportion of egg yolk in the extender, doubling the extender volume generally used to dilute each ejaculate, or earlier addition of egg yolk after sperm collection could prevent the detrimental effect of BSP proteins on sperm, and thus the sperm could survive the cryopreservation process better. Similarly, if the LDL-BSP hypothesis is the main mechanism responsible for egg yolk's protective role in the cryopreservation process, epididymal sperm, which is devoid of seminal plasma proteins, should tolerate the freezing-thawing equally well in media with or without egg yolk.
In rhesus monkeys, the use of cryopreserved sperm in standard, intravaginal artificial insemination (AI) has not been successful and live births have been reported only through intrauterine insemination (Sánchez-Partida et al, 2000) or intracytoplasmic sperm injection (ICSI; Yeoman et al, 2005). These latter techniques are not readily available at most primate colonies around the world; therefore, propagation by using frozen-thawed sperm with standard AI would be very valuable for practical use. Previous success with intrauterine insemination or ICSI suggests that frozen-thawed sperm is capable of fertilizing eggs in rhesus monkeys, and the failure of standard AI may well indicate that sperm are incapable of swimming up through the cervical canal to meet the egg. This failure may be caused by barriers created by the mucus or the unique tortuous cervical canal of rhesus monkeys (Hafez and Jaszczak, 1972). Therefore, substantial improvement of motility may allow frozen-thawed sperm to have a better chance at success in standard AI procedures.
As we discussed in an earlier paper, systematic procedure optimization of the series of sequential steps involved in sperm cryopreservation could reduce the cumulative loss and may consequently help retain better postthaw motility (Dong et al, 2008b). As in studies in most mammalian species, egg yolk has been one of the essential components in extenders for sperm cryopreservation in nonhuman primates. However, it is unknown what percentage of egg yolk should be added and when and how the egg yolk should be added to effect the most protection for ejaculated sperm in nonhuman primates. It is also unknown whether the egg yolk is equally essential to epididymal sperm as it is to ejaculated sperm in monkeys. Considering the recently proposed LDL-BSP hypothesis, it is possible that the same mechanism observed in bulls may also explain the protective role of egg yolk in nonhuman primates. Consequently, modification of the amount of egg yolk, the dilution method, and the addition time may help improve postthaw motility for rhesus monkey sperm. Therefore, as part of our continuous efforts in systematic procedure optimization for sperm cryopreservation in nonhuman primates, the present study evaluated the effect of egg yolk on cryopreservation of ejaculated and epididymal sperm of rhesus macaques.
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Materials and Methods |
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A dilution of 1:20 (vol/vol) of semen to TL-BSA was used for sperm motility estimation of fresh semen, and a second dilution of 1:20 (vol/vol) of sperm to distilled water was used for hemacytometer counts (Hausser Scientific, Horsham, Pennsylvania). The mean sperm density ranged from 3.1 x 108 mL–1 to 62.0 x 108 mL–1 (Table 1). TES-Tris (TEST)–yolk solution was prepared with 43.25 g TES, 10.265 g Tris, and 10 g glucose in 1 L distilled water (modified from Tollner et al, 1990) and with the addition of 20% egg yolk (vol/vol) unless specified otherwise. Sperm were frozen without washing at a final concentration of 5 x 107 cells mL–1 with 3% glycerol. Glycerol was added to the sperm in a single step. All chemicals used for preparation of solutions were of reagent grade (Sigma Chemical Corporation, St Louis, Missouri).
Freezing and Thawing Procedure
Aliquots (50 µL) of sperm suspensions were drawn into 0.25-mL French
straws (IMV International, Minneapolis, Minnesota) manually with a 1-mL
syringe and were heat-sealed (MP-4 Impulse Sealer; J. J. Elemer Corp, St
Louis, Missouri). Straws were placed into a 600-mL glass beaker containing 500
mL of room temperature distilled water, and equilibrated at 4°C in a
refrigerator for 2 hours before initiation of the freezing process. Freezing
followed the methods described previously
(Dong et al, 2008b). In brief,
a Styrofoam box (inside dimensions: 33x24x23 cm) was filled with a
depth of 4 cm liquid nitrogen and a 1-cm-thick or 0.4-cm-thick Styrofoam
"boat" was floated on top of it for 10 minutes; then, straws were
placed on top of the "boat" for 10 minutes before being plunged
into liquid nitrogen. The cooling rate was measured using a data logger
thermometer (Type T thermocouple; Omega, Stamford, Connecticut) with the wire
inserted into a 0.25-mL straw filled with TEST-20% yolk–3% glycerol, and
a minimum of 5 measurements were recorded. The average cooling rate from
–10°C to –70°C was about 220°C min–1
for the 1-cm boat and about 400°C min–1 for the 0.4-cm
boat. The high cooling rates were used because our preliminary trials
indicated consistently higher postthaw motility in fast-cooled (220°C
min–1 or above) vs slow-cooled (e.g., 5°C to 29°C
min–1) conditions for rhesus monkey ejaculated sperm. For
postthaw motility estimation, 4 straws per treatment were thawed in a 37°C
water bath for 30 seconds (ISOTEMP 102; Fisher Scientific, Pittsburgh,
Pennsylvania). Samples were evaluated after a minimum of 2 days' storage in
liquid nitrogen.
Motility Estimation
A 10-µL drop of prefreeze or postthaw semen, covered with a 22-mm square
cover glass, was visualized with a x20 positive-phase objective and a
condenser setting of 100 (pseudodark field) on an Olympus BH-series
phase-contrast microscope (Scientific Instrument Co, Sunnyvale, California).
An air curtain incubator (Sage Instruments, Model 279; Orion Research Inc,
Cambridge, Massachusetts) maintained the microscope stage at 37°C. The
initial motility was in the range of 70%–95%
(Table 1). Postthaw motility
was estimated without any dilution or washing immediately after thawing or
following incubation at 37°C in 5% CO2 in air for 1 or 4 hours.
Forward progression was estimated with an adjusted motility index (AMI) as
described previously (Dong et al,
2008b). In brief, the percentage forward progression was
subjectively estimated with a 5-point scale, and this was integrated with the
percentage motility into 1 number with the formula as follows: AMI = (Scale
value/4) x percentage motility. Samples were presented in random order
each time so that the operator did not know their identity.
Effect of Egg Yolk Concentration in TEST
There were 2 trials in this experiment. For the first trial, ejaculated
semen from 5 males was used, and each ejaculate was divided into 5 samples for
the following treatments: freeze with 3% glycerol in TEST combined with 10%,
20%, 30%, 40%, and 50% egg yolk. Samples were frozen at 220°C
min–1 and 400°C min–1 in liquid nitrogen
vapor as described above. Postthaw motility and forward progression were
estimated immediately after thawing and after 4 hours incubation at 37°C.
For the second trial, ejaculated semen from 6 males were used to test the egg
yolk concentrations at a lower range of 2%, 4%, 6%, 8%, 10%, and 20% in TEST
combined with 3% glycerol. Samples were frozen at 220°C
min–1 and postthaw motility and forward progression were
estimated immediately after thawing and after 1 hour incubation at
37°C.
Effect of the Dilution Method of TEST-Yolk
Ejaculated semen from 8 males was used in this experiment. Each ejaculate
was divided into 2 equal portions, with 1 suspended in a single volume of
TEST-egg yolk with a resulting sperm density of 2 x 108
mL–1 and a final concentration of 20% (vol/vol) egg yolk, and
the other in a double volume of TEST-egg yolk with a resulting sperm density
of approximately 1 x 108 mL–1 and a final
concentration of 20% egg yolk. Sperm were preincubated with single vs double
volumes of TEST-egg yolk for 1 hour at room temperature before exposure to
glycerol. Samples were adjusted to a final sperm concentration of 5 x
107 mL–1 in 3% glycerol and cooled at 220°C
min–1. Postthaw motility and forward progression were
estimated as described in "Motility Estimation."
Effect of Delay in the Addition of TEST-Yolk
A total of 12 ejaculates from 8 males were used in this experiment. Each
ejaculate was divided into 5 samples, and samples were left undiluted at room
temperature for 0, 1, 2, 3, and 4 hours before the addition of TEST-20% egg
yolk, which corresponds to 1, 2, 3, 4, and 5 hours, respectively, after
ejaculation when including the 1-hour processing time for sample collection
and liquefaction. Approximately 0.5 hours after the addition of TEST-yolk at
the 4-hour treatment, samples were suspended in 3% glycerol and cooled at
220°C min–1. Postthaw motility and forward progression
were estimated as described in "Motility Estimation."
Effect of Egg Yolk on Ejaculated and Epididymal Sperm
There were 2 trials in this experiment; for the first trial, ejaculates
collected from 4 males were used to compare the effect of freezing with 3%
glycerol suspended in TEST-20% egg yolk and TEST alone without egg yolk. For
the second trial, sperm collected from epididymides of 5 males were used for
the same treatments. Samples were frozen at 220°C min–1,
and postthaw motility and forward progression were estimated as described in
"Motility Estimation."
Data Analysis
Data were analyzed using repeated-measures analysis of variance (SAS 9.1;
SAS Institute, Cary, North Carolina). When a significant difference
(P = .05) was observed among treatments, Tukey's studentized range
test was used for posttest comparisons. Percentage motility was arcsine-square
root transformed and means of 4 straws per treatment were used for analysis.
Values presented are means ± SD.
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Results |
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Effect of the Dilution Method of TEST-Yolk
The 2 different dilution methods of TEST-20% egg yolk yielded no
differences in motility (P = .544) or AMI (P = .729) for
samples at 0 or 4 hours after thawing
(Table 4). Samples incubated at
37°C for 4 hours after thawing showed a significant loss of motility and
AMI.
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Effect of Delay in the Addition of TEST-Yolk
There were no differences in motility (P = .676) or AMI
(P = .982) among samples treated with waiting periods of 0, 1, 2, 3,
and 4 hours between sperm collection and the addition of TEST-yolk extender
(Table 5). Prolonged incubation
of 4 hours after thawing resulted in a significant loss of motility and
AMI.
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Effect of Egg Yolk on Ejaculated and Epididymal Sperm
Samples frozen in TEST-glycerol with the presence of egg yolk had
significantly higher motility and AMI at 0 and 4 hours after thawing than
those frozen in TEST-glycerol alone (Table
6). This was true for both ejaculated and epididymal sperm. An
incubation time of 4 hours after thawing yielded significantly lower motility
and AMI.
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Discussion |
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The present study also demonstrated that neither the dilution method nor the time delay after ejaculation before adding egg yolk-TEST extender has a significant effect on postthaw motility for ejaculated rhesus monkey sperm. In fact, sperm can be stored undiluted at room temperature for up to 5 hours without significant effect on their postthaw motility. Thus, continuous exposure to seminal plasma proteins in rhesus monkeys had minimal effect on sperm quality. For practical use, our findings suggest a wide time window for sample processing after sperm collection. It appears that our data on rhesus monkey sperm did not support the LDL-BSP hypothesis in that exposure to seminal plasma proteins for a time span of 5 hours was not detrimental to sperm survival after cryopreservation. We suspect that mechanisms other than LDL-BSP binding may be responsible for the protective role of egg yolk in sperm cryopreservation of rhesus monkeys. Sperm washing prior to freezing is a common practice for most mammals, and one would expect less seminal plasma protein to be present in washed sperm suspension. However, egg yolk remains the major constituent of extenders for cryopreservation of washed sperm. In our previous studies with rhesus monkeys, egg yolk has been found to be necessary in cryopreservation of washed sperm (Dong et al, 2009).
In the present study, undiluted semen was collected into empty 50-mL tubes upon ejaculation. Generally, semen was collected from 4 males at the same time and it took approximately 30 minutes to complete the collection process, and semen was then delivered to the laboratory immediately. Samples were further allowed to liquefy for 30 minutes before processing; thus, the earliest time point for TEST-yolk extender addition (for the 0-hour treatment) was 1 hour after ejaculation. Because the cholesterol efflux stimulated by BSP proteins is time- and concentration-dependent (Manjunath and Thérien, 2002), the continuous exposure of sperm to BSP proteins would result in continuous lipid removal. Therefore, it is possible that damage associated with seminal plasma proteins, if it exists, might already have occurred prior to processing in our case with rhesus monkey sperm, which could lead to no effect for subsequent treatments of egg yolk content, the dilution method, or delay in addition time, as found in this study. However, if this is true, our findings would further suggest that mechanisms other than the LDL-BSP binding of egg yolk prevented sperm from cryodamage, because ejaculated sperm processed in the same way yielded significantly higher postthaw motility and AMI in TEST-yolk extender than in TEST alone.
If the main protective mechanism of egg yolk in sperm cryopreservation of rhesus monkeys is sequestration of detrimental seminal plasma proteins, as suggested by the LDL-BSP hypothesis, one would expect that epididymal sperm could tolerate the freezing-thawing process in the absence of egg yolk, because BSPs are rare on the plasma membrane of epididymal spermatozoa (Miller et al, 1990). However, the addition of egg yolk has been found to have a significant beneficial effect on protecting epididymal sperm from cold shock or freezing and thawing in domestic cat Felis catus (Harris et al, 2001; Hermansson and Axner, 2007), red deer Cervus elaphus hispanicus (Fernández-Santos et al, 2006), Spanish ibex Capra pyrenaica (Santiago-Moreno et al, 2007), and rhesus monkey (the present study). These findings suggest that mechanisms other than LDL-BSP are responsible for the protective role of egg yolk in the cryopreservation of epididymal sperm.
Because LDL consists of 86%–89% lipids and 12.5% proteins (Martin et al, 1963), lipids have been the main focus for the study of the protective role of egg yolk (eg, Purdy and Graham, 2004; Ricker et al, 2006). However, besides egg yolk, other additives such as skimmed milk, soybean proteins, and bovine serum albumin (Stoss and Holtz, 1983; Foote et al, 2002) have also been used successfully in sperm cryopreservation as membrane stabilizers. For example, skim milk was found to offer an equal amount of protection to sperm as compared with whole milk, even though skim milk contains no lipids (Foote et al, 2002). In skim milk, the major protein component, casein micelles, has been suspected to be the main protective constituent because it can protect sperm during cold storage and freezing (Choong and Wales, 1963; Batellier et al, 1997; Leboeuf et al, 2003). Moussa et al (2002) suggest that adsorption and gelation of LDL apoproteins around the spermatozoa membrane could form a protective film against ice crystals generated during freezing. The beneficial effect of soybean proteins and BSA as mentioned above also suggests an important role of proteins in membrane stabilization during sperm cryopreservation. Therefore, it is possible that the apoproteins of LDL may play a significant role in the egg yolk protective mechanism; or similarly to the function of egg yolk in the food-emulsifying process (Mine, 1998), that the combination of the lipid-protein complex in LDL interacts with sperm membrane.
In summary, our findings reveal that rhesus monkey ejaculated sperm can be frozen in TEST-yolk media with a low percentage of egg yolk; the dilution methods and addition time of egg yolk did not result in significant improvement of postthaw motility. Although all appear to be negative, our results suggested that the presence of egg yolk, rather than the amount of egg yolk, the dilution method, or when to add egg yolk, affects the freezing outcome. Future understanding of the mechanism for the protective role of egg yolk in rhesus monkey sperm may ultimately lead to the rapid improvement of postthaw motility and in turn to possible success in standard AI trials. The finding that egg yolk is equally as necessary for protecting epididymal sperm as it is for ejaculated sperm indicates that protocols optimized on the basis of ejaculated sperm may applicable to epididymal sperm from the same species. This is important in a practical sense for nonhuman primates, because epididymal sperm are rarely available, but their preserving could be essential in cases in which valuable animals die unexpectedly and to preserve the genetic diversity of colonies (Beehler et al, 1982).
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Acknowledgments |
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Footnotes |
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