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Figure 7.3. Acrosomal morphology of the giant panda spermatozoon with a (a) normal apical ridge, (b) damaged apical ridge, (c) missing apical ridge or (d) loose acrosomal cap. (See also Plate VIII.)

(i.e. macrocephaly, microcephaly or bicephaly); abnormal acrosome; coiled flagellum; bent midpiece with cytoplasmic droplet; bent mid-piece without cytoplasmic droplet; bent flagellum with cytoplasmic droplet; bent flagellum without cytoplasmic droplet; proximal cytoplas-mic droplet; and distal cytoplasmic droplet (Howard, 1993) (see Fig. 7.2). Sperm with an abaxial attachment of the neck were not considered abnormal. This unusual offset of the head with the neck region (most easily seen in Fig. 7.2a, f, h) is a common feature of the giant panda spermatozoon (Moore et al, 1984).

The acrosome is the outer membrane of the spermatozoon, which is critical to successful fertilisation. Damage to this structure as a result of inherent physiological anomalies, physical mishandling or suboptimal cryopreservation techniques of semen is a common cause of infertility. Acrosomal integrity was evaluated in the giant panda using the rose bengal/fast green stain (Pope et al., 1991) (Fig. 7.3; Plate VIII). Raw semen was diluted in Ham's F10 tissue culture medium (Irvine Scientific, Santa Ana, CA) supplemented with 25-mM HEPES buffer and 5% heat-treated fetal calf serum (one part semen to 10 parts Ham's). For

Figure 7.3. Acrosomal morphology of the giant panda spermatozoon with a (a) normal apical ridge, (b) damaged apical ridge, (c) missing apical ridge or (d) loose acrosomal cap. (See also Plate VIII.)

staining, 1 ml of diluted semen was added to 9 ml of rose bengal/fast green stain, incubated for 90 seconds and then smeared on a glass slide. Using bright-field microscopy (x1000), a minimum of 100 sperm acrosomes per sample was assessed as having a:

1. normal intact apical ridge (uniform staining of the acrosome over the anterior half of sperm head);

2. damaged apical ridge (non-uniform staining with ruffled or folded acrosome);

3. missing apical ridge (lack of staining due to acrosome absence);

4. loose acrosomal cap (loose membrane protruding above the level of the sperm head) (Howard, 1993) (see Fig. 7.3; Plate VIII).

Semen processing and sperm dilution

Successful sperm cryopreservation requires using a cryoprotectant -an antifreeze to protect the sperm from lethal ice damage during freezing and thawing. For the giant panda, the cryoprotectant of choice has been glycerol, which in this case was added to two cryodi-luents used historically by the USA and Chinese colleagues. A cryodi-luent called TEST was prepared in the USA using a commercially available 'Freezing Medium-TEST Yolk Buffer (TYB) with Glycerol', which is marketed by Irvine Scientific for freezing human sperm. This is a 20% egg-yolk solution containing 12% glycerol combined with the commercially available 'Refrigeration Medium-TEST Yolk Buffer (TYB) without Glycerol' to yield a 5% glycerol concentration. The medium was prepared in the USA, then re-frozen for shipment to China. The second cryodiluent, called SFS (for Chinese 'Sperm Freezing Solution'), was prepared fresh each day in China and consisted of 20% egg yolk, 12% sucrose and 5% glycerol. This SFS 5% glycerol cryodiluent was maintained at 4°C and not frozen. For comparative studies, both diluents were also prepared with no glycerol (i.e. TEST 0% and SFS 0% glycerol) for initial dilution of specific treatments.

Following collection, giant panda semen was diluted (1:3; semen: diluent) immediately with TEST or SFS diluent containing 0% or 5% glycerol at 37°C. Diluted aliquots were evaluated for sperm motility and acrosomal traits at 37°C, then placed in a 400-ml water-jacket for slow cooling over 4 hours to 4°C. Aliquots were either maintained at 4°C to assess duration of motility for 48 hours or further diluted in glycero-lated diluents and prepared for cryopreservation.

Sperm cryopreservation and thawing

Three freezing methods (pellets vs. straws vs. cryovials) were evaluated, with cooling rates monitored by a thermocouple device (Omega Engineering, Inc., Stanford, CT). After cooling, additional aliquots of glycero-lated TEST and SFS were added to the sperm suspensions to achieve a final 4% glycerol concentration before freezing. Two pellet-freezing techniques were tested that involved a plastic tray within a stainless-steel box or a metal wire-mesh screen in a Polystyrene box (Fig. 7.4). To compare the impact of pellet size, semen was also frozen in 40-p.l (~100°C per minute) or 80-p.l (~50°C per minute) droplets, then packaged in plastic cryovials (Vangard International, Inc.), placed on a metal cane and maintained immersed in liquid nitrogen. For straws, cooled semen was pipetted into 0.25-ml sterile plastic straws (Veterinary Concepts, Spring Valley, WI) and sealed at the open end (Fig. 7.5). A two-step

Figure 7.4. Semen cryopreservation using the pellet-freezing method on a plastic tray within (a) a stainless-steel box or (b) a wire mesh-screen in a Styrofoam box positioned at a consistent level above liquid nitrogen to achieve a constant temperature of —96°C. Pellets remained on the tray or wire mesh for three minutes before plunging into liquid nitrogen.

Figure 7.4. Semen cryopreservation using the pellet-freezing method on a plastic tray within (a) a stainless-steel box or (b) a wire mesh-screen in a Styrofoam box positioned at a consistent level above liquid nitrogen to achieve a constant temperature of —96°C. Pellets remained on the tray or wire mesh for three minutes before plunging into liquid nitrogen.

Figure 7.5. Semen cryopreservation using a two-step straw-freezing method in liquid nitrogen (LN) vapour. Straws (0.25-ml) containing cooled semen are placed at 7.5 cm (Level 1; —30°C; for one minute) and 2.5 cm (Level 2; —130°C; for one minute) above liquid nitrogen in a Styrofoam box, then plunged into liquid nitrogen.

Figure 7.5. Semen cryopreservation using a two-step straw-freezing method in liquid nitrogen (LN) vapour. Straws (0.25-ml) containing cooled semen are placed at 7.5 cm (Level 1; —30°C; for one minute) and 2.5 cm (Level 2; —130°C; for one minute) above liquid nitrogen in a Styrofoam box, then plunged into liquid nitrogen.

method was used whereby each straw was placed 7.5 cm over liquid nitrogen for 1 minute (Level 1; —30°C) and then 2.5 cm above liquid nitrogen for 1 minute (Level 2; —130°C) to achieve rapid cryopreservation (about — 70°C per minute) before plunging into liquid nitrogen (see Fig. 7.5). Finally, 0.5-ml aliquots of semen cooled in 1.8-ml cryovials (Vangard International, Inc.) were frozen on a thin Polystyrene platform floating on 7.5 cm of liquid nitrogen for 15 minutes before storing immersed in liquid nitrogen (Fig. 7.6). The cryovial method resulted in the slowest freezing rate (~20°C per minute; Fig. 7.7).

Two tissue culture media were evaluated for thawing and post-thaw sperm dilution, Ham's F10 medium and Tyrode's 199, each containing 25-mM HEPES buffer to maintain pH. The vial containing pellets was maintained in liquid nitrogen, while one pellet was removed for thawing. The pellet was held in air for 10 seconds, then placed into a sterile 12 x 75-mm glass tube containing 0.5 ml of either thaw medium (for 45 seconds in a 37°C water bath). The glass tube was agitated and the thawed suspension transferred into a plastic Eppendorf tube (Brink-man Instruments, Inc., Westbury, NY) and maintained for 90 minutes (at 37°C). For the straw method, each straw was held in air for 10

Figure 7.6. Semen cryopreservation using a cryovial method of freezing. Cooled semen (0.5-ml) in a 1.8-ml cryovial is placed and taped onto a thin Styrofoam platform floating on 7.5 cm of liquid nitrogen for 15 minutes before plunging into liquid nitrogen.
Figure 7.7. Comparison of freezing rates using the pellet, straw and cryovial method of sperm cryopreservation.

seconds, then plunged into the 37°C water bath for 45 seconds. The sealed end of the straw was cut off, then thawed semen was expelled and diluted in either thaw medium at 37°C (half in 1.25-ml Ham's; half in 1.25-ml Tyrode's 199). For the cryovial method, each vial was simply thawed in the 37°C water bath. All samples were maintained at 37°C while assessed for sperm motility, progression and acrosomal integrity at 0, 30, 60 and 90 minutes after thawing.

Sperm capacitation, acrosome reaction, zona penetration and decondensation

Most traditional sperm assessments have relied largely on simple evaluations of motility (cell movement). Modern fertility evaluations also test the functionality of spermatozoa by examining how effectively they undergo the biochemical and cellular processes associated with preparation for, or the actual, fertilisation event (Yanagimachi, 1994). In our studies, we chose several such phenomena, including the ability of sperm to undergo:

1. capacitation, or a series of physiological changes (alterations or removal of substances) on the sperm plasma membrane that renders the spermatozoon capable of undergoing the acrosome reaction;

2. the acrosome reaction, or multiple fusions between the outer acrosomal membrane and overlaying plasma membrane, that enable the contents of the acrosome to escape through fenestrated membranes.

For these two assessments, fresh or thawed semen was diluted in Ham's F10 (37°C) containing 5% fetal calf serum and 25-mM HEPES, centrifuged (200g, 8 minutes) and the pellet resuspended in fresh Ham's F10 (37°C). At 0, 3, 6 and 9 hours after resuspension, aliquots were removed and assessed for sperm motility and acrosomal integrity. Additional aliquots were removed and diluted in Ham's F10 only (control) or Ham's F10 containing heterologous (domestic cat) solubilized zonae pellucidae for 30 minutes. Following incubation, acrosomes were evaluated as above. Percentage of capacitated sperm was defined as the proportion of sperm with intact acrosomes after exposure to solu-bilized cat zonae subtracted from the proportion of sperm with intact acrosomes after exposure only to Ham's F10 medium with no zonae (control).

The impact of cryopreservation on the functional ability of the sperm nucleus to undergo decondensation, or the release of disul-phide bonds within the spermatozoon, also was evaluated. Nuclear

Figure 7.8. Sperm penetration of the zona pellucida of a (a) homologous giant panda and (b) heterologous cat salt-stored oocyte used to assess sperm function before and after cryopreservation. (See also Plate IX.)

decondensation allows the sperm chromosomes to become accessible to the oocyte (Mahi & Yanagimachi, 1975). Fresh and frozen-thawed giant panda sperm were pre-incubated in Ham's F10 for 6 hours to induce capacitation, followed by a cat solubilized zonae pellucidae solution for 30 minutes to induce the acrosome reaction (as described above). To assess decondensation, aliquots were then removed at 0, 2 and 4 hours and incubated in Ham's F10 only (control) or cat oocyte cytoplasmic emulsion at 37°C. Sperm were categorised as normal (condensed cytoplasm) or having partial or complete decondensation (based on sperm head transparency and enlargement).

The sperm penetration assay using zona-intact, salt-stored oocytes (Fig. 7.8; Plate IX) has been used to assess sperm-oocyte interaction in numerous species (Drobnis et al., 1988). Spermatozoa must undergo capacitation and the acrosome reaction for subsequent zona penetration; thus this assay evaluates several stages of fertilisation. Also, heter-ologous oocytes are easily obtained and provide an attractive approach for evaluating the ability of sperm to bind and penetrate the zona pellucida, an often limiting factor for fertilisation. In a preliminary study, the ability of giant panda sperm to bind and penetrate the zonae pellucidae of salt-stored, domestic cat oocytes (which were readily available) was investigated.

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