Info

d a Values are means ± SEM. Semen was diluted (1:3; semen:diluent) in TEST or SFS diluent, cooled slowly and maintained at 4°C for 48 hours; b,c,d Within a time period, means with different superscripts are different (p < 0.05).

d a Values are means ± SEM. Semen was diluted (1:3; semen:diluent) in TEST or SFS diluent, cooled slowly and maintained at 4°C for 48 hours; b,c,d Within a time period, means with different superscripts are different (p < 0.05).

highest sperm motility (>65%) over 48 hours (see Table 7.5). These data confirm that short-term, cold storage at 4°C is effective for maintaining excellent sperm motility and intact acrosomes for at least 48 hours in the giant panda.

Benefits of semen cryopreservation and a genome resource bank

Having access to an organised Giant Panda Genome Resource Bank (GRB; a repository of sperm as well as tissue, blood and DNA) would be a valuable resource for helping to maintain genetic diversity in the ex-situ giant panda population, both inside and outside China (Wildt et al., 1997; Howard, 1999; see Chapter 21). Frozen semen could be used to move genes among geographically disparate breeding centres to avoid inbreeding. Although most AI procedures in China rely on fresh semen, AI with frozen-thawed sperm has been used with modest success, the first birth occurring in 1980 at the Chengdu Zoo (Ye et al., 1991; Zhang et al., 1991). A more recent evaluation of available data is presented in this book (see Chapter 20).

To survive 'cryo-stress', sperm require an appropriate:

1. seminal cryodiluent and cryoprotectant;

2. cooling rate (to a temperature just above freezing);

3. storage package;

4. freezing method/rate;

5. thawing method/rate;

6. post-thaw dilution to remove cryoprotectant.

The Biomedical Survey allowed the systematic examination of the following:

Impact of freezing method and cryodiluent

The traditional method for freezing giant panda sperm is pelleting, a technique originally developed for bull, boar, dog and cat semen (Platz et al., 1983; Howard et al., 1986; Chen et al., 1992; Howard, 1993). This cryotechnique was first used in 1979 for the giant panda Hsing Hsing (SB 121) at the Smithsonian's National Zoological Park and involved a cryodiluent of 20% egg yolk, 11% lactose and 4% glycerol (Platz et al, 1983). The method itself consists of pipetting cooled, liquid seminal drops into indentations on a block of dry ice (i.e. solid carbon dioxide at —96°C) and then leaving them in place for three minutes before plunging into liquid nitrogen. In China, this pelleting method had been modified to avoid the need for dry ice. Rather a plastic tray or wire-mesh screen was suspended over liquid nitrogen vapour (see Fig. 7.4).

Initially during the survey, the Chinese pelleting technique and SFS cryodiluent were tested, but resulted in wide-ranging, unreliable post-thaw sperm motility and acrosomal ratings. We speculated that discrepancies were related to inconsistent:

1. heights above the nitrogen vapour causing varied temperatures on the tray or mesh during pelleting;

2. pellet sizes; and/or

3. length of time pellets remained on the mesh or tray before plunging into liquid nitrogen.

Each of these factors are well known to influence freezing rate and, thus, the incidence of intracellular ice formation that can cause membrane damage and cell death (Mazur, 1974; Howard et al, 1986; Hammerstedt et al., 1990).

To promote consistency in technique, we developed a means to stabilize the plastic tray or wire-mesh screen above the liquid nitrogen to achieve a constant temperature of —96°C during pelleting (to mimic dry ice temperature). A high-quality pipetting device was used to ensure a standardised pellet size of 40 ml. We also decided to test another cryomethod, specifically, a manual straw container technique. This simple two-step straw method of placing the straws on a test-tube rack at two different levels in liquid nitrogen vapour proved to be an excellent and highly portable method for cryopreservation of panda spermatozoa (see Fig. 7.5; Table 7.6). Two tissue culture solutions (Ham's F10 and Tyrode's 199) also were evaluated as thawing media for pellets and dilution media for straws. For the former method, one pellet was thawed in 0.5-ml of each medium at 37°C. For the straw method, one 0.25-ml straw was thawed in a 37°C water-bath for 45 seconds, then the thawed semen split into two aliquots and diluted in Ham's F10 or Tyrodes 199 culture medium at 37°C.

Neither cryodiluent nor freezing method influenced (p > 0.05) sperm viability (see Table 7.6). There was no difference (p > 0.05) in pre-freeze sperm motility or forward progression after dilution in the TEST egg-yolk diluent (commercially available) or SFS egg-yolk diluent (made fresh daily), each containing 5% glycerol (see Table 7.6). Although sperm quality was not affected, motility traits were visualised more easily microscopically in the TEST compared to the SFS diluent. This was no doubt due to the sophisticated processing of TEST during preparation by the manufacturer. In contrast, SFS was not filtered, thus making it difficult to view sperm easily because of particulate matter in the egg yolk. Overall, diluting giant panda semen in TEST 5% and SFS 5% cryodi-luents at 37°C provided similar (p > 0.05) sperm protection during freezing and thawing (see Table 7.6). Likewise, post-thaw sperm motility, forward progression, longevity of sperm motility in vitro (data not shown) and acrosomal integrity (Table 7.6) were similar after cryopreservation in TEST or SFS using the pellet-versus-straw method. Tissue culture medium used for thawing and dilution also had no impact (p > 0.05) on sperm viability (including acrosomal integrity) after thawing (data not shown).

Impact of freezing rate and cryomethod

The challenge during sperm freezing is not the cell's endurance to an ultra-low temperature (—80 to —196°C) (Mazur, 1974). Rather, the hazard is the intermediate temperature zone (—15 to —60°C) that the

Table 7.6. Influence of cryodiluents (TEST vs. SFS) and freezing methods (straws vs. pellets) on cryopreservation of giant panda semen (n = 14 males)11

Acrosomal integrity (%)

Sperm Forward Normal apical Damaged apical Missing apical Loose acrosomal motility (%) progression6 ridge ridge ridge cap

Pre-freeze

SFS 5% glycerol 67.9 ± 4.2 2.9 ± 0.1 95.7 Post-thaw

TEST 5% glycerol/straws 59.3 ± 3.9 3.5 ± 0.2 75.5

TEST 5% glycerol/pellets 56.7 ± 5.6 3.5 ± 0.2 67.0

SFS 5% glycerol/pellets 55.0 ± 7.5 3.4 ± 0.2 62.6

a Values are means ± SEM. Fresh semen was diluted (1:3; semen:cryodiluent) in TEST 5% glycerol or SFS 5% glycerol at 37°C, cooled to 4°C over 4 hours, then frozen in 0.25-ml straws or 40-/il pellets. Straws were thawed at 37°C, then diluted in Ham's F10/HEPES medium at 37°C. Pellets were thawed in Ham's F10/HEPES medium at 37°C. Sperm acrosomes were evaluated immediately post thaw. Data presented for percentage sperm motility and forward progression were at 60 minutes post-thaw; h Sperm forward progression was based on a scale of 0 to 5; 5 = best.

cell must traverse twice, once during freezing and once during thawing. Different approaches for packaging semen affect the freezing rate through this critical zone by providing different surface-to-volume ratios (Mazur, 1974; Hammerstedt et al., 1990). The 40-ml pellet and straw freezing techniques have very rapid freezing rates (~100oC per minute and ~70oC per minute, respectively; see Fig. 7.7), largely because of the significant surface area exposed to liquid nitrogen. And, as we saw above, when these rates were applied to giant panda sperm, the result was high post-thaw sperm viability and acrosomal integrity. Because larger volumes freeze more slowly, the impact of a slow freezing rate can be assessed by cryopreserving semen in large volumes such as in a bulk vial. Therefore, we took the opportunity to evaluate a cryovial method for giant panda sperm by placing 0.5-ml of semen in a 1.8-ml cryovial, cooling slowly to 4oC, then transferring the container onto a Styrofoam platform floating on 7.5 cm of liquid nitrogen for 15 minutes before plunging into liquid nitrogen. As anticipated, the content of the cryovial froze slowly (at about —20oC per minute) due to the larger volume of semen and low surface area of the vial (see Fig. 7.7). Therefore, to further assess cryomethods, this study examined the efficacy of the cryovial versus the 0.25-ml straw versus two sizes of pellets. The first was the standard 40-ml pellet that freezes at about 100oC per minute versus an 80-ml drop that freezes at about 50oC per minute. All semen samples were diluted in TEST, cooled slowly to 4oC over 4 hours and then frozen using one of these four methods.

Freezing rate had a profound influence on post-thaw sperm survival in the giant panda (Table 7.7). The faster freezing rates associated with either pelleting method or with the 0.25-ml straw consistently produced excellent post-thaw sperm motility (>70%) and acrosomal integrity (>85%). Slower freezing via a cryovial constantly resulted in almost a 20% decrease in sperm motility and a more than 30% reduction in the number of sperm with normal acrosomes (p < 0.05). One result was many more sperm (p < 0.05) with a damaged (35%) or missing (11%) apical ridge. Overall, results confirm that giant panda sperm prefer a rapid cryopreservation rate, whereas slower freezing damages both motility and the acrosomal apparatus.

Impact of glycerol temperature and duration of exposure

The temperature of glycerol when added to the semen and the duration of glycerol exposure can influence sperm viability. In livestock species,

Table 7.7. Impact of freezing rate using pellets, straws and cryovials on cryopreseiyation of giant panda sperm (n = 6 malesf

Acrosomal integrity (%)

Sperm Forward Normal apical Damaged apical Missing apical Loose acrosomal motility (%) progression6 ridge ridge ridge cap

Pre-freeze

TEST cryodiluent 76.7 ± 6.0C 3.1 ± 0.2 96.2 ± 1.3C 2.3 ± 0.6C 1.5 ± 0.9C 0 ± 0 Post-thaw

40 |il pellets (~100°C/min) 70.8 ± 5.2C 4.0 ± 0.1 86.3 ± 1.7d 5.8 ± 1.2d 6.7 ± 0.8d 1.2 ± 0.3

80 |il pellets (~50°C/min) 73.3 ± 6.0C 4.0 ± 0.1 87.2 ± 1.6d 5.8 ± l.ld 5.5 ± 0.8d 1.5 ± 0.6

0.25 ml straws (~70°C/min) 70.8 ± 4.4C 4.1 ± 0.2 87.5 ± 1.9d 5.5 ± 0.8d 5.7 ± 1.8d 1.3 ± 0.4

0.5 ml cryovial (~20°C/min) 52.8 ± 4.2d 3.4 ± 0.2 54.0 ± 2.9e 34.8 ± 3.2e 10.7 ± 4.5d 0.5 ± 0.3

a Values are means ± SEM. Semen was diluted (1:2; semen:cryodiluent) with TEST 0% or 5% cryodiluent at 37°C, cooled to 4°C over 4 hours and frozen in pellets, straws or ciyovials at a 4% final glycerol concentration. Sperm acrosomes were evaluated immediately post-thaw. Data presented for percentage sperm motility and forward progression were at 60 minutes post-thaw; h Sperm forward progression was based on a scale of 0 to 5; 5 = best; cAe Within columns, values with different superscripts differ (p < 0.05).

glycerol must often be added to the semen at 4°C to minimise osmotic injury to sperm (Hammerstedt et al., 1990). As discussed above, we initially discovered that exposing giant panda sperm to TEST or SFS with glycerol at 37°C, followed by slow cooling to 4°C before freezing, produced a high incidence of intact acrosomes (see Table 7.6). Here, we further assessed the sensitivity of giant panda sperm to glycerol by examining addition at 37°C versus 4°C and a duration of glycerol exposure of 4 hours versus 1 hour. A treatment involving short-term glycerol exposure at a lower temperature (4°C for only 1 hour following a 3-hour cooling interval) allowed evaluating the protective or toxic effects of glycerol. Thus, for this study, semen was diluted in TEST or SFS with 0% or 5% glycerol at 37°C, cooled slowly to 4°C over 3 hours and then diluted in TEST or SFS with 8% or 5% glycerol for an additional 1 hour of cooling before freezing in 0.25-ml straws. Adding glycerol at 4°C for only 1 hour failed to enhance or compromise (p > 0.05) sperm quality post-thawing (Table 7.8). Sperm motility traits and the incidence of normal acrosomes were similar (p > 0.05) after cryopreservation regardless of the temperature or duration of glycerol exposure.

Sperm capacitation, the acrosome reaction and decondensation

For optimal post-thaw functionality, a spermatozoon must have progressive motility, an intact acrosome and the ability to undergo capaci-tation, the acrosome reaction, zona pellucida penetration and decondensation in the oocyte's cytoplasm (Yanagimachi, 1994). Although giant panda sperm capacitation (Sun et al., 1996), the acrosome reaction (Chen et al, 1989a) and oocyte penetration in vitro (Moore et al., 1984; Chen et al., 1989a,b) have been studied, these functional events have not been evaluated with frozen-thawed sperm.

During the Biomedical Survey, giant panda sperm capacitation and the acrosome reaction were evaluated by exposing fresh and thawed sperm to heterologous (cat) solubilised zonae pellucidae (Spindler et al, 2004). Results revealed that giant panda sperm were capable of capacitating in vitro over six hours (Table 7.9). Because the acrosome reaction was elicited using heterologous (felid) zonae emulsions, our findings supported earlier data indicating that the triggers to this phenomenon are not species specific (Yanagimachi, 1994). Most importantly, freeze-thawing had no detrimental influence on these functional

Table 7.8. Influence of cryodiluent (TEST vs. SFS) and temperature of glycerol addition (37°C vs. 4°C) using the straw method on sperm cryopreservation in the giant panda (n = 6 males)11

TEST/glycerol at 37°C TEST/glycerol at 4°C SFS/glycerol at 37°C SFS/glycerol at 4°C

Pre-freeze

Sperm Forward Normal apical motility (%) progression6 ridge

Post-thaw

Sperm Forward Normal apical motility (%) progression6 ridge

a Values are means ± SEM. Fresh semen was diluted (1:2) in TEST 0% or 5% glycerol or SFS 0% or 5% glycerol at 37°C and cooled to 4°C over 3 hours. Aliquots were then diluted in TEST 8% or 5% (1:1) or SFS 8% or 5% (1:1) for a final 4% glycerol concentration. Samples were cooled for an additional hour, then frozen in 0.25 ml straws. For thawing, straws were held at 37°C for 45 seconds, then diluted in Ham's F10/HEPES medium at 37°C. Sperm acrosomes were evaluated immediately post-thaw. Data presented for percentage sperm motility and forward progression were at 60 minutes post-thaw; h Sperm forward progression was based on a scale of 0 to 5; 5 = best.

Table 7.9. Percentage (mean ± SEM) of fresh and frozen-thawed giant panda spermatozoa that demonstrated capacitation after exposure to heterologous (cat) solubilised zonae pellucidae (n = 9 males )a

Time 0 hour 3 hours 6 hours 9 hours

Fresh sperm - capacitated 3.0 ± 0.2 28.0 ± 0.7 43.6 ± 1.1 41.3 ± 1.1

Frozen-thawed sperm - capacitated 5.3 ± 0.5 27.7 ± 1.2 43.3 ± 1.1 49.6 ± 1.1b a Capacitation was defined as the proportion of sperm with intact acrosomes after exposure to solubilised cat zonae pellucidae subtracted from the proportion of sperm with intact acrosomes after exposure to only Ham's F10 medium with no zonae (control). For cryopreservation, semen was diluted in TEST egg-yolk diluent with 5% glycerol, cooled slowly and frozen in 0.25-ml straws; b Different from fresh counterparts (p < 0.05).

events and did not compromise the ability of giant panda sperm to undergo capacitation and the acrosome reaction.

Once zona penetration has been achieved, decondensation is essential to fertilisation, whereby disulphide bonds in the spermatozoon are released so that the chromosomes become accessible to the oocyte (Mahi & Yanagimachi, 1975). Since cryopreservation exposes the cell to expansion, shrinkage, dehydration and significant temperature fluctuations which can cause lethal damage to chromatin structure and bond stability (Gao et al., 1997), we also examined the impact of cryo-preservation on the sperm nucleus and chromatin decondensation in the giant panda. Following pre-incubation and exposure to a solubilized cat zonae solution (to induce capacitation and the acrosome reaction), fresh and frozen (TEST/straw)-thawed giant panda sperm were exposed to heterologous (cat) oocyte cytoplasmic emulsion for assessment of decondensation. There was no effect of cryopreservation on the subsequent ability of giant panda sperm to decondense (Table 7.10). More than half of the fresh sperm underwent decondensation after two hours of incubation in the cytoplasmic emulsion with nearly three-quarters after four hours. The total number of thawed sperm undergoing decondensation did not differ (p > 0.05) from fresh counterparts at any time point (see Table 7.10). Thus, we conclude that cryopreserved as well as fresh giant panda sperm have equivalent capacity for maintaining chromosomal and nuclear stability.

Table 7.10. Percentage (mean ± SEM) of fresh and frozen-thawed giant panda spermatozoa that demonstrated decondensation during incubation in cat oocyte cytoplasmic emulsion (n = 8 males)a

Time

0 hour

2 hours

4 hours

Fresh sperm - decondensed

2.8 ± 0.4

51.4 ± 3.7

69.8 ± 5.9

Frozen-thawed sperm - decondensed

3.8 ± 0.4

58.1 ± 6.2

71.5 ± 4.9

a Giant panda spermatozoa were pre-incubated in Ham's F10 medium for 6 hours to induce sperm capacitation, then exposed to a cat solubilised zonae pellucidae solution for 30 minutes to induce the acrosome reaction prior to the assessment of decondensation during incubation in cat oocyte cytoplasmic emulsion. For cryopreservation, semen was diluted in TEST egg-yolk diluent with 5% glycerol, cooled slowly and frozen in 0.25-ml straws.

a Giant panda spermatozoa were pre-incubated in Ham's F10 medium for 6 hours to induce sperm capacitation, then exposed to a cat solubilised zonae pellucidae solution for 30 minutes to induce the acrosome reaction prior to the assessment of decondensation during incubation in cat oocyte cytoplasmic emulsion. For cryopreservation, semen was diluted in TEST egg-yolk diluent with 5% glycerol, cooled slowly and frozen in 0.25-ml straws.

Sperm-ovum interaction and zona penetration

The ultimate assay for assessing sperm function after freezing is zona penetration and fertilisation of oocytes in vivo or in vitro (Drobnis et al, 1988). But, of course, testing functionality of giant panda sperm on giant panda oocytes is limited because of a lack of eggs from the latter. One alternative is to use the oocytes of another species. A salt-stored zona penetration assay, developed in the hamster (Yanagimachi et al, 1979; Boatman et al., 1988) and adapted for cat oocytes (Andrews et al, 1992), may be one future approach for testing the ability of panda sperm to bind and penetrate the zona pellucida, the oocyte's primary barrier to fertilisation. This could, for example, be valuable in testing new sperm cryopreservation protocols. Salt-stored zonae also retain the ability to distinguish between capacitated and non-capacitated sperm (Boatman et al., 1988; Andrews et al., 1992).

Our preliminary studies have demonstrated that zona-intact, salt-stored cat oocytes can be penetrated by fresh and thawed giant panda spermatozoa (see Fig. 7.8). Giant panda sperm binding and penetration of cat oocytes appeared similar to what has been measured with salt-stored giant panda oocytes recovered from ovaries post-mortem (see Fig. 7.8). This interesting finding implies that the zona receptor on the giant panda spermatozoon and the ligand on the cat zona pellucida may be the same, that is, conserved across these quite different species. Due to this non-specificity, we predict that the ready availability of cat eggs, and thus their zonae, could be a useful tool for studying sperm function and gamete interaction in the giant panda.

Was this article helpful?

0 0

Post a comment