Cryopreservation of Rhesus Monkey (Macaca mulatta) Epididymal Spermatozoa Before and After Refrigerated Storage

Authors

  • Qiaoxiang Dong,

    1. California National Primate Research Center, University of California, Davis, California
    2. School of Environmental Science and Public Health, Wenzhou Medical College, Wenzhou, China
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  • Sarah E. Rodenburg,

    1. California National Primate Research Center, University of California, Davis, California
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  • Changjiang Huang,

    1. School of Environmental Science and Public Health, Wenzhou Medical College, Wenzhou, China
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  • Catherine A. Vandevoort

    Corresponding author
    1. California National Primate Research Center, University of California, Davis, California
    2. Department of Obstetrics and Gynecology, School of Medicine, University of California, Davis, California.
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California National Primate Research Center, University of California, Davis, CA 95616 (e-mail: cavandevoort@ucdavis.edu).

Abstract

ABSTRACT: Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of material for genetic resource banking; however, cryopreservation of epididymal sperm from the rhesus monkey has not been explored. This study evaluated the effect of prolonged refrigerated storage of the intact cauda epididymides at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether altering cryoprotectants and cooling methods could improve post-thaw motility for epididymal sperm after refrigerated storage. Motility before freezing decreased significantly after refrigerated storage (0°C) for a period of 24 or 48 hours. Although postthaw motility was not significantly different after 24 hours of refrigerated storage, epididymides stored at a higher temperature (4°C–10°C) yielded better results, but postthaw motility still decreased significantly after 48 hours of refrigerated storage at 4°C. Comparisons of glycerol and ethylene glycol at 3% and 6% revealed similar postthaw motility. However, consistently high postthaw motility was obtained with 3% glycerol throughout all freezing trials regardless of whether samples were collected fresh or after refrigerated storage for 24 or 48 hours. Cooling at a higher rate of 220°C/min was found to yield better postthaw motility than the slower rate of 29°C/min. Thawing time duration was evaluated, and a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-μL semen samples. An overall average of 42% postthaw motility was obtained for rhesus monkey epididymal sperm packed in 3% glycerol and cooled after 24 or 48 hours refrigerated storage. These postthaw motility results for epididymal sperm indicate that this method should be practical for use in preserving epididymal sperm, even if tissue must be shipped from sites remote from the cryopreservation laboratory.

Nonhuman primates serve as important research models for human health. With the increased demand for the development of treatments for human diseases, there is more pressure on captive-breeding programs to provide animals for research. In previous years there has been a significant loss of rhesus matrilines from captive populations (Duggleby, 1976; Smith, 1985). Although infant cross-fostering (Smith, 1986) can randomize breeding within a captive population, it cannot reverse the trend toward genetic subdivision of captive populations at different breeding facilities (Kanthaswamy and Smith, 2002). The National Primate Research Centers lose genetics and rare alleles when animals are lost to disease, trauma, or old age, and these cannot be replaced because India has long banned further exportation of rhesus monkeys. In addition, many nonhuman primates are endangered species, and captive breeding is the only hope for their future survival. Nevertheless, it is important to maintain the genetic diversity within a given population to avoid problems of inbreeding and loss of heterozygosity (Moore, 1992).

Establishing a cryobank to preserve the germplasm of nonhuman primates would help maintain biodiversity because it will allow those genes to be recovered as live, breeding animals. In particular, when combined with assisted reproductive techniques, such as artificial insemination (AI), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo transfer, sperm cryopreservation provides an effective tool for preserving the genetic diversity of the captive primate populations for biomedical research as well as for conservation programs. Semen banking also provides a way to easily transfer genetic materials among colonies of animals, because only a few straws of frozen semen must be transported.

Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of valuable genes for genetic resource banking (eg, Foote, 2000; Stilley, 2001; James, 2004; Guerrero, 2006). The ability to bank epididymal sperm from nonreproducing animals or males that die suddenly (Kusunoki et al, 2001) or from animals that are subjects of terminal experiments would prevent the loss of valuable genetic information from the future gene pool. In addition, protocols developed for domestic species or captive populations may apply to their exotic counterparts for which semen samples are precious and limited (eg, domestic cats as a model for wild felids; Pope et al, 1998). These protocols could also be used to aid in solving difficulties with mating performance and to increase the population of individuals that are currently underrepresented or those with desirable genetic traits.

Unlike sperm cryopreservation in humans, there have been mostly sporadic attempts at freezing nonhuman primate sperm over the last 40 years (see Morrell and Hodges, 1998, for review). Published reports that directly address sperm cryopreservation in nonhuman primates number less than 100, and reliable information on the most appropriate methodologies is limited. Compared with studies of ejaculated sperm, there are fewer reports (9 studies) on the freezing of epididymal spermatozoa from nonhuman primates (Table 1), and cryopreservation of epididymal sperm from rhesus monkey remains unexplored. The present study evaluated the effect of prolonged refrigerated storage at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether changing of cryoprotectants and cooling methods could improve postthaw motility for epididymal sperm after refrigerated storage.

Table 1. . Literature review of cryopreservation of nonhuman primate epididymal sperm
SpeciesCryoprotectantCoolingThawingSummary of findingsReference
  1. Abbreviations: NA indicates not available; IVF, in vitro fertilization; DMSO, dimethyl sulfoxide; PG, propylene glycol; ICSI-TET, intracytoplasmic sperm injection—tubal embryo transfer; vapor freezing, cooling samples in liquid nitrogen vapor; pellet freezing, cooling drops of extended semen on the surface of a dry ice block; controlled-rate, cooling with an automated cell freezer.

Chimpanzee2.5% and 5% glycerolVapor freezing37°C water40% motility, 69.6% viability, 52.8% intact acrosome, 1 live birthKusunoki et al, 2001
 3% glycerolPellet freezing37°C82.5% motility, 79% intact acrosomeO'Brien et al, 2003
Gorilla7% glycerolVapor freezingNA1 insemination, no conceptionBeehler et al, 1982
 3% glycerolPellet freezing37°C air40.7% normal sperm, 39.5% head abnormality, 0% fertilization (IVF), 1 developed embryo (injection)Lanzendorf et al, 1992
Japanese monkey5% glycerolVapor freezing37°C water43% fertilization, 19% cleaved, 4 at 8-cell stage (IVF)Sankai et al, 1997
Cynomolgus monkey4%, 6%, and 8% glycerol, 6% DMSO, 6% PGVapor freezing37°C water36% motility, 45.2% viability, 47% intact membraneFeradis et al, 2001
 6% glycerolVapor freezing37°C2 live births, 2 abortions (ICSI-TET)Ng et al, 2002
Baboon3% glycerolPellet freezing37°C42.5% motility, 59.5% intact acrosomeO'Brien et al, 2003
Marmoset monkey5% glycerolControlled-rate37°C31% fertilization (IVF)Holt et al, 1994
 10% glycerolControlled-rateRT (22–25°C)6 inseminated, 1 conceived, 1 live birthMorrell et al, 1998
 3% glycerolPellet freezing37°C43.5% motility, 74.5% intact acrosomeO'Brien et al, 2003

Materials and Methods

Collection of Epididymal Spermatozoa

A total of 19 pairs of testes were donated from adult rhesus monkeys subjected to necropsy from other research projects at the California National Primate Research Center, and the cauda epididymis was dissected from each testis. For each pair of epididymides, 1 epididymis was processed immediately for sperm retrieval (hereafter referred to as “without storage”), and the other epididymis (“after refrigeration”) was stored in 8-mL Dulbecco phosphate-buffered saline (DPBS) in a 15-mL centrifuge tube for various refrigerated storage treatments as described below. To retrieve sperm, the epididymis was rinsed with DPBS, placed into a 35-mm Petri dish containing 1 mL of TEST-yolk solution (∼ 350 mOsm/kg, pH 7.4; for detailed recipe, see Tollner et al, 1990), and cut repeatedly with surgical scissors, and sperm were allowed to swim out into the solution for 5 minutes at room temperature. The resulting suspension of spermatozoa was transferred into a 15-mL plastic centrifuge tube prior to use.

Preparation, Freezing, Thawing, and Evaluation

A dilution of 1:20 (vol/vol) of sperm suspension to a modified Tyrode medium supplemented with bovine serum albumin (VandeVoort, 2004) was used for sperm motility estimation of freshly collected semen, and a second dilution of 1:20 (vol/vol) of sperm to distilled water was used for hemacytometer counts (Hausser Scientific, Horsham, Pennsylvania). Sperm suspensions were adjusted to 1 × 108 cells/mL of total motile sperm (count × motility) with TEST-yolk solution, and were mixed with double-strength cryoprotectant solutions to obtain a final concentration of 5 × 107 cells/mL. All cryoprotectant solutions were prepared within 1 week of use with TEST-yolk at 350 mOsm/kg as the diluent and were stored at 4°C. All chemicals used for preparation of solutions were of reagent grade (Sigma Chemical Corporation, St Louis, Missouri).

Aliquants of 50-μL sperm suspensions with cryoprotectant (detailed below) were drawn into 0.25-mL French straws (IMV International, Minneapolis, Minnesota) manually with a 1-mL syringe. Straws were heat-sealed, 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 by Leibo et al (2007). In brief, straws were placed on a Styrofoam “boat” with the thickness of either 5 cm or 1 cm on top of liquid nitrogen, which was filled to a depth of 4 cm in a 33×24×23-cm (inside dimensions) Styrofoam box, and equilibrated for 10 minutes before being plunged into liquid nitrogen. The average cooling rate measured between −10°C to −70°C was ca 29°C/min for the 5-cm boat and ca 220°C/min for the 1-cm boat. After a minimum of 12 hours, 4 straws per treatment were thawed in a 37°C water bath (ISOTEMP 102; Fisher Scientific, Pittsburgh, Pennsylvania) to estimate the postthaw motility.

For motility estimation, a 10-μL drop of prefreeze or postthaw semen, covered with a 22-mm-square cover glass, was visualized with a ×20 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. Motility was expressed as the percentage of cells actively moving in a forward direction. Sperm vibrating in place were not considered to be motile. Initial motility refers to the sperm motility after dilution, but before the addition of cryoprotectants to the samples. Postthaw motility was estimated immediately after thawing without any dilution or washing. Samples were also presented in random order each time so that the operator did not know their identity.

Experimental Layout

Four experiments were performed (Table 2). Experiment 1 evaluated the effect of thawing time on postthaw motility, and only the epididymis without storage was used. Experiment 2 evaluated the effect of storage temperature on postthaw motility, and there were 3 trials in this experiment. In the first trial, epididymal tissue was stored at 4°C (refrigerator) for 24 hours. In the second trial, epididymal tissue in a 15-mL centrifuge tube was sealed with parafilm, packed in a Styrofoam shipping box (same size as the freezing apparatus) at 0°C (ice/water slurry), and processed 24 hours later. In the third trial, epididymal tissue in a 15-mL centrifuge tube was sealed with parafilm and packed in a Styrofoam shipping box between 2 ice packs (temperatures varied from 4°C to 10°C) and processed 24 hours later. Experiment 3 evaluated the effect of cryoprotectant on postthaw motility before and after refrigerated storage (at 0°C ice/water slurry as described in experiment 2.2). Experiment 4 evaluated the effect of cooling and glycerol concentration on postthaw motility before and after refrigerated storage. There were 2 trials in this experiment, with the first trial evaluating cooling methods and glycerol concentrations, and samples subjected to the same refrigeration treatment as that of experiment 2.3 (4°Cto10°Cfor 24 hours). The second trial evaluated optimal glycerol concentration before and after refrigerated storage at 4°C (refrigerator) for an extended period of 48 hours.

Table 2. . Experimental layout
ExpParametersRefrigerated storage treatmentMale numberCryoprotectantCoolingThawing
  1. Abbreviations: Exp, experiment; Gly, glycerol; EG, ethylene glycol.

1ThawingNo storage4, 14, 16, 173% Gly29°C/min37°C water, 10 s, 20 s, 30 s, 60 s
2.1RefrigerationNo storage vs 4°C, 24 h1, 13, 15, 16, 173% Gly29°C/min37°C water, 30 s
2.2RefrigerationNo storage vs 0°C, 24 h4, 5, 7, 11, 14, 183% Gly29°C/min37°C water, 30 s
2.3RefrigerationNo storage vs 4°C to 10°C, 24 h2, 6, 9, 103% Gly29°C/min37°C water, 30 s
3CryoprotectantNo storage7, 11, 183%, 6% Gly; 3%, 6% EG29°C/min37°C water, 30 s
  0°C, 24 h7, 11, 18, 4, 5, 143%, 6% Gly; 3%, 6% EG29°C/min37°C water, 30 s
4.1Cooling × Gly4°C to 10°C, 24 h2, 6, 9, 103%, 6%, 9%, 12% Gly29°C vs 220°C/min37°C water, 30 s
4.2Gly concentrationNo storage vs 4°C, 48 h3, 8, 12, 193%, 6%, 9%, 12% Gly220°C/min37°C water, 30 s

Data Analysis

Data were analyzed using 2-sample independent t tests and 1-way and 2-way analysis of variance (ANOVA) (Origin 7.0, OriginLab, Northampton, Massachusetts). When a significant difference (P = .05) was observed among treatments, Tukey's honestly significant difference procedure was used for pairwise comparisons. All data were arcsine-square root transformed and means of 4 straws per treatment were used for analysis. Levene's test for equal variance showed nonsignificant difference for all datasets subjected to ANOVA analysis. Values presented are means ± SD.

Results

Basic Characteristics of Epididymal Spermatozoa

A total of 19 pairs of epididymides were isolated from males that went to necropsy for other research projects (Table 3). Males (n = 19) were between 4 and 14 years old, with an average age of 7 ± 3 years, and their weights ranged from 6.2 to 18.4 kg, with an average of 10.6 ± 3.2 kg. The initial motility of epididymal spermatozoa collected on the same day as necropsy (83% ± 16%) was significantly higher (P < .001) than that of spermatozoa collected after refrigeration for either 24 or 48 hours (59% ± 22%). However, sperm density of samples without storage (9.2 ± 6.5 × 108 cell/mL) was not significantly different (P = .351) from sperm density of samples after refrigeration (11.6 ± 8.9 × 108 cell/mL).

Table 3. . Basic characteristics of epididymal spermatozoa of the 19 males used in this study
   
Male numberAge, yWeight, kgWithout storageAfter refrigerationaWithout storageAfter refrigerationa
  1. a Prefreezing motility after 24 or 48 h refrigerated storage.

11413.873474.04.0
21317.770708.49.8
31018.4606015.622.2
4108.685509.812.4
599.750303.45.7
6711.995908.68.4
7710.7956011.820.8
8712.7955012.419.8
969.2906019.631.0
1069.895908.212.4
1168.250207.01.3
1269.8953021.820.0
13610.6959019.815.2
1458.8956012.610.2
1559.172750.90.5
1648.478506.420.8
1746.990901.41.0
181310.490602.23.2
1946.295301.52.0
Mean ± SD7 ± 310.6 ± 3.283 ± 1659 ± 229.2 ± 6.511.6 ± 8.9

Effect of Thawing Time on Postthaw Motility

Postthaw motility percentages of sperm samples thawed for different time periods (Figure 1) were significantly different from one another (P = .024), and the highest postthaw motility was obtained for time periods of 30 seconds (46.7% ± 2.0%) and 60 seconds (47.3% ± 5.8%); these percentages were not significantly different from each other, but were significantly higher than samples thawed for 10 seconds (34.9% ± 7.4%). Therefore, thawing for a period of 30 seconds was chosen for subsequent experiments, because that time gave equally good motility as, but required less time per sample than, the 60-second period.

Figure 1.

. Postthaw motility of rhesus monkey epididymal sperm samples cryopreserved with 3% glycerol and thawed in a 37°C water bath for 10, 20, 30, and 60 seconds. Values presented are means of 4 straws for each male.

Effect of Storage Temperature on Postthaw Motility

Refrigerated storage of epididymides for 24 hours had no significant effect on postthaw motility (Figure 2). Despite this, postthaw motility of sperm samples without storage was generally higher than that of sperm that were frozen after 24 hours refrigerated storage at 4°C in the refrigerator (42.0% ± 16.8% vs 35.4% ± 17.1%, P = .565, Figure 2a) or packed in a Styrofoam shipping box at 0°C ice/water slurry (44.4% ± 8.0% vs 36.8% ± 13.4%, P = .253, Figure 2b). Interestingly, the opposite was found with the refrigerated storage at a higher temperature of 4°C to 10°C with samples stored between 2 ice packs in a Styrofoam shipping box (44.0% ± 4.3% vs 48.6% ± 2.4%, P = .117, Figure 2c). Individual males were also found to respond to cryopreservation differently, with male 13 yielding the highest postthaw motility of 60% and male 15 yielding the lowest postthaw motility of 10%.

Figure 2.

. Postthaw motility of rhesus monkey epididymal sperm samples cryopreserved with 3% glycerol before (light gray bars) and after (dark gray bars) 24 hours of refrigerated storage at various conditions: (a) 4°C refrigerator; (b) 0°C ice/water slurry in a shipping box; and (c) 4°C to 10°C between 2 ice packs in a shipping box. Values presented are means of 4 straws for each male.

Effect of Cryoprotectant on Postthaw Motility Before and After Refrigerated Storage

No significant difference in postthaw motility among different cryoprotectants was observed for sperm samples collected from epididymides without storage (P = .292, Figure 3a) or those after 24 hours of refrigerated storage (P = .710, Figure 3b). However, samples cryopreserved with 3% glycerol consistently yielded the highest postthaw motility for no storage (45.6% ± 11.5%) or after refrigeration (36.8% ± 13.4%). Consequently, subsequent experiments focused on further optimization of glycerol concentration. Similar to previous experiments, individual males responded to cryopreservation differently, with post-thaw motility of ∼60% for some males (eg, male 18), and less than 10% for others (eg, male 11).

Figure 3.

. Postthaw motility of rhesus monkey epididymal sperm samples cryopreserved with 3% and 6% glycerol (Gly), as well as 3% and 6% ethylene glycol (EG), immediately after collection (a) and after 24 hours refrigerated storage at 0°C ice/water slurry in a shipping box (b). Each bar represents 1 male, and bars with same patterns identify same males. Values presented are means of 4 straws for each male.

Effect of Cooling and Glycerol Concentration on Postthaw Motility Before and After Refrigerated Storage

There were 2 trials in this experiment. For the first trial (Figure 4), glycerol at 4 concentrations (3%, 6%, 9%, and 12%) was cross-designed with 2 cooling rates (29°C/min vs 220°C/min). Interactions of these 2 factors were found to be nonsignificant (P = .647), but there were significant differences among glycerol concentrations, with samples cryopreserved with 3% and 6% glycerol yielding significantly higher postthaw motility than samples cryopreserved with 12% glycerol (P < .05). Although there was no significant difference between the 2 cooling rates (P = .054), higher postthaw motility was found with samples cooled at 220°C/min, and the highest postthaw motility was found with 3% glycerol (55.8% ± 7.3%).

Figure 4.

. Postthaw motility of rhesus monkey epididymal sperm samples cryopreserved with 3%, 6%, 9%, and 12% glycerol after 24 hours of refrigerated storage at 4°C to 10°C between 2 ice packs in a shipping box, and cooled at 29°C/min or 220°C/min in liquid nitrogen vapor. Values presented are means of 4 straws for each male.

Based on the above results, the second trial employed the cooling rate of 220°C/min. Glycerol concentrations were further evaluated with samples before and after extended refrigerated storage of 48 hours (Figure 5). The interaction of glycerol concentration and sample status was not significantly different (P = .746). However, postthaw motility of samples without storage was significantly higher than that of samples after 48 hours of refrigerated storage (P < .001). Similar to previous experiments, samples cryopreserved with 3% glycerol consistently yielded the highest postthaw motility, which was significantly higher than that of samples with 12% glycerol (P < .05). The highest postthaw motility (53.9% ± 4.8%) was obtained with 3% glycerol for samples without storage, and the lowest (26.8% ± 11.0%) with 12% glycerol for samples with refrigerated storage.

Figure 5.

. Postthaw motility of rhesus monkey epididymal sperm samples cryopreserved with 3%, 6%, 9%, and 12% glycerol before (without storage) and after 48 hours of refrigerated storage (48 hours) at 4°C, and cooled at 220°C/min in liquid nitrogen vapor. Values presented are means of 4 straws for each male.

Discussion

In nonhuman primates, epididymal sperm have been cryopreserved in the chimpanzee, gorilla, baboon, Japanese and cynomolgus macaque, and marmoset (Table 1). Although sperm within the cauda epididymis of a number of species are considered fully mature (Mahony et al, 1996; Yeung et al, 1996; Van der Horst et al, 1999), slight differences in cellular membrane structure and composition and lack of exposure to seminal plasma have been postulated to make epididymal sperm more susceptible to cryopreservation damage than ejaculated sperm (Nagy et al, 1995; Morrell, 1997; Perchec-Poupard et al, 1997; Kundu et al, 2000; Feradis et al, 2001; Hori et al, 2004). However, studies with gorilla (Lanzendorf et al, 1992) and porcine (Rath and Niemann, 1997) epididymal and ejaculated sperm showed a higher viability or fertilization rate with postthaw epididymal sperm, whereas studies with caprine suggested equal tolerance between epididymal and ejaculated sperm (Blash et al, 2000). It appears that whether epididymal sperm is more susceptible to cryopreservation damage than its ejaculated counterpart is species-specific. Despite this, reviewing the literature of nonhuman primates reveals that freezing protocols for epididymal sperm were essentially the same as those used for ejaculated sperm. For example, most studies used TEST-yolk-glycerol as the freezing medium, and cooled samples either in liquid nitrogen vapor or as pellets on a dry ice block. The use of frozen-thawed epididymal sperm has yielded live births in the chimpanzee, cynomolgus monkey, and marmoset monkey (Table 1).

One of the important aspects involving cryopreservation of epididymal sperm is refrigerated storage and shipping. When valuable males die unexpectedly and facilities for sperm cryopreservation are not immediately accessible, temporary storage of epididymides and shipment to freezing facilities are required. Shipment of fresh epididymides to a genetic resource—banking center would also facilitate standardized sample processing and thus guarantee uniform handling of sperm. As yet no investigation has been done on the possible effects of refrigerated storage on the postthaw survival of nonhuman primate sperm. However, it has been shown that epididymides that shipped at 4°C within 24 hours, and the sperm of which was subsequently frozen-thawed, did yield a live birth in the chimpanzee (Kusunoki et al, 2001). In this study, motility before freezing was found to decrease significantly after refrigerated storage for an extended period of 24 or 48 hours, whereas postthaw motility was not significantly different after 24 hours of refrigerated storage, but epididymides stored at a higher temperature (4°C to 10°C) yielded better results. However, postthaw motility decreased significantly after 48 hours of refrigerated storage at 4°C. This pattern was similar to that seen in studies with refrigerated storage of epididymal sperm from other domestic animals such as canine (Stilley, 2001), caprine, equine, and bovine (James, 2004).

Except for nonhuman primates, refrigerated storage of epididymal sperm has been studied extensively for many other mammalian species. For example, storage of epididymides from nondomestic animals at temperatures near 0°C permits recovery of viable sperm up to 48 hours later (Graham et al, 1978). A 41-day pregnancy following AI with cryopreserved epididymal sperm harvested 27 hours postmortem was reported for a gaur bull (Bos gaurus) (Hopkins et al, 1988). An extreme example is found in mice in which pups were produced from epididymal sperm that was subjected to cooled storage for 7 days after death (An et al, 1999; Kishikawa et al, 1999). More recently, it was reported that bovine epididymal sperm stored at 5°C were viable for at least 60 hours when used for AI (Foote, 2000). All these studies indicate that epididymal sperm can tolerate refrigerated storage for an extended time period and are suitable for long distance transportation. However, the present study did find male variations in response to refrigerated storage and subsequent sperm cryopreservation. Therefore, sperm from some males (eg, male 11 in this study) may not be suitable for freezing after shipping or extended refrigerated storage. To apply this knowledge for endangered species, it is recommended that sperm from epididymides be cryopreserved as soon as samples are collected.

Glycerol has been the most widely used cryoprotectant in various mammalian species. The choice of appropriate cryoprotectant may be more related to reproduction mode or sperm characteristics rather than to species, because fish that employ internal fertilization (livebearers), and therefore resemble mammals, also showed the best protection with glycerol, which is different from fish with external fertilization (Huang et al, 2004). Glycerol ranging from 2.5% to 10% has been used to freeze epididymal sperm in nonhuman primates (Table 1). Comparisons among 6% glycerol, dimethyl sulfoxide, and propylene glycol with cynomolgus epididymal sperm indicated that glycerol was superior to the others (Feradis et al, 2001). In addition, the survival of marmoset epididymal sperm is not enhanced by the inclusion of dodecyl sulfate in the cryopreservation medium, compared to glycerol alone (Holt et al, 1994). Recently, measurement of rhesus monkey sperm membrane permeability coefficients for various cryoprotectants has led to the suggestion that ethylene glycol may be the most appropriate cryoprotectant for this species (Agca et al, 2005). Studies with ejaculated rhesus (Si et al, 2004) and cynomolgus (Li et al, 2005) monkey sperm also indicated similar cryoprotection between ethylene glycol and glycerol when comparing immediate postthaw motility and membrane and acrosome integrity. Thus, the present study compared glycerol and ethylene glycol at 3% and 6%, and our findings revealed similar postthaw motility between these 2 cryoprotectants. However, consistently higher postthaw motility was obtained with 3% glycerol throughout all freezing trials for samples cryopreserved both immediately after collection and after refrigerated storage for 24 or 48 hours, which was also in agreement with the findings for ejaculated rhesus monkey sperm (Dong et al, unpublished data).

It is worth noting that there were variations between individual males in response to different cryoprotectants at different concentrations (Figure 3). This variation in susceptibility to cryopreservation damage is similar to the male-to-male variations previously reported for ejaculated rhesus sperm (Leibo et al, 2007), as well as in other species such as dogs, bulls, boars, stallions, and humans (Leibo and Bradley, 1999; Holt, 2000).

Cooling of nonhuman primate sperm samples was often conducted either in liquid nitrogen vapor or as pellets on a dry ice block. Direct comparisons of these 2 methods have not been made until recently, where rhesus monkey ejaculated sperm cooled as pellets on a dry ice block was found to yield a fertilization rate (73%) comparable to that of fresh controls (Yeoman et al, 2005). However, whether pellet freezing would consistently result in better breeding outcomes still merits future validation. In contrast, freezing in liquid nitrogen vapor using the Styrofoam box has been proven to be cheap, easy, reliable, and repeatable in studies with rhesus monkey sperm across 3 different laboratories (Leibo et al, 2007). The present study adopted the freezing method described by Leibo et al (2007), and modified the thickness of the floating boat to produce various cooling rates. Cooling at a higher rate of 220°C/min yielded better postthaw motility than did the slower rate of 29°C/min. This may be because of the extended equilibration time period (2 hours at 4°C) before freezing, as dehydration may have occurred in response to osmotic equilibrium. Optimal cooling rate may also vary with cryoprotectants and thawing methods/rates (eg, Fiser and Fairfull, 1984, 1990; Mazur, 1985; Yu et al, 2002). To further optimize cryopreservation protocols for rhesus monkey sperm, future experiments should examine these multiple factors simultaneously and systematically.

Temperatures ranging from 5°C to 75°C (in air or water bath) have been used for thawing nonhuman primate sperm, with the most satisfactory results obtained from samples thawed in a 37°C water bath (eg, Younis et al, 1998). However, thawing duration has often been neglected in published reports, and the few of those that did specify rate indicated a wide range from 10 seconds (Leibo et al, 2007), 30 seconds (Feradis et al, 2001), 45–60 seconds (Sanchez-Partida et al, 2000; Okada et al, 2001), and 2 minutes (Si et al, 2000) to 5 minutes (Tollner et al, 1990). To confirm the best results, a comparison of thawing time durations was made in this study, and our findings revealed that a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-μL samples. In general, fast rates are preferred, because this should minimize damage associated with recrystallization upon thawing and thus aid the recovery of motile sperm (Watson, 1979).

Motility has been proven to be a good indicator of sperm quality in frozen-thawed sperm of nonhuman primates. Postthaw motility of ejaculated (Tollner et al, 1990) and epididymal sperm (Feradis et al, 2001) from cynomolgus macaque has been found to positively correlate with membrane integrity. Examination of spermatozoa from the same species suggests that motility is the most convincing parameter for sperm function analysis, whereas sperm acrosome integrity (evaluated with fluorescein isothiocyanate-conjugated peanut agglutinin [FITC-PNA]) is the one most resistant to freezing (Li et al, 2005). This was further evidenced by studies of osmotic tolerance of rhesus monkey sperm, which indicated that sperm motility was more sensitive than membrane integrity to deviations from isotonicity (Rutllant et al, 2003). Positive correlations between motility and live sperm were also found in the howler monkey (Alouatta caraya) (Valle et al, 2004).

Successful live births with frozen-thawed sperm may rely not only on the efficiency of the cryopreservation protocol in preserving the fertilizing ability of sperm, but also on the insemination method. This is especially true with the rhesus macaque because it has a tortuous cervical canal, which may restrict the successful application of standard AI involving sperm deposition into the vagina or cervix. However, successful live births were reported through intrauterine insemination with frozen-thawed sperm for cynomolgus (Tollner et al, 1990) and rhesus macaques (Sanchez-Partida et al, 2000). Compared with standard IVF, AI via intrauterine insemination offers a simpler and more cost-effective approach for utilization of frozen-thawed sperm for macaque propagation. The use of transabdominal deposition of sperm into the uterus not only circumvents the tortuous female anatomy, but also places fewer demands on the requirement of highly motile sperm after thawing. In contrast, a standard IVF program is costly, requires expensive equipment, and is very complicated because it involves hormone stimulation, oocyte retrieval and in vitro culture, and embryo transfer through surgery, with the potential for failure at each of these steps.

Although the absolute dependence of successful live birth on postthaw motility may vary with the assisted reproduction technique employed, it would be expected that higher postthaw motility would result in improved assisted reproduction outcomes through the provision of more functional spermatozoa. In fact, a recent study with rhesus monkey sperm has shown that there were no significant differences in chromosome damage between fresh sperm and frozen-thawed sperm when motile sperm were selected for ICSI (Li et al, 2007). In addition to percentage motility, recording the forward progression scale (Yeoman et al, 2005) and the sperm longevity may provide a more accurate estimation of sperm quality after thawing. Future studies should evaluate the possibility of developing a composite index that incorporates the progressive scale into the motility estimation as well as the sperm longevity after thawing.

In summary, the present study demonstrated that sperm from rhesus monkey epididymides could be cryopreserved with 3% glycerol after extended refrigerated storage of 24 hours, but there were variations between individual males. Future AI trials are needed to confirm the viability of rhesus monkey epididymal sperm cryopreserved after refrigerated storage. Nevertheless, techniques developed in this study should help gene banking of nonhuman primates, including diseased males.

Acknowledgement

We thank D. Hill and N. Sealey for assistance with cryopreservation.

Footnotes

  1. Supported by NIH grants RR00169 and RR13439.

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