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BOR - Papers in Press, published online ahead of print January 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025304
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BIOLOGY OF REPRODUCTION 70, 1340–1348 (2004)
DOI: 10.1095/biolreprod.103.025304
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Effect of Cryopreservation Methods and Precryopreservation Storage on Bottlenose Dolphin (Tursiops truncatus) Spermatozoa1

T.R. Robeck2,3, and J.K. O'Brien4

SeaWorld Texas,3 San Antonio, Texas 78251 Centre for Advanced Technologies in Animal Genetics and Reproduction,4 Faculty of Veterinary Science,University of Sydney, New South Wales 2006, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Research was conducted to develop an effective method for cryopreserving bottlenose dolphin (Tursiops truncatus) semen processed immediately after collection or after 24-h liquid storage. In each of two experiments, four ejaculates were collected from three males. In experiment 1, three cryopreservation methods (CM1, CM2, and CM3), two straw sizes (0.25 and 0.5 ml), and three thawing rates (slow, medium, and fast) were evaluated. Evaluations were conducted at collection, prefreeze, and 0-, 3-, and 6-h postthaw. A sperm motility index (SMI; total motility [TM] x % progressive motility [PPM] x kinetic rating [KR, scale of 0–5]) was calculated and expressed as a percentage MI of the initial ejaculate. For all ejaculates, initial TM and PPM were greater than 85%, and KR was five. At 0-h postthaw, differences in SMI among cryopreservation methods and thaw rates were observed (P < 0.05), but no effect of straw size was observed. In experiment 2, ejaculates were divided into four aliquots for dilution (1:1) and storage at 4°C with a skim milk- glucose or a N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)-TRIS egg yolk solution and at 21°C with a Hepes-Tyrode balanced salt solution (containing bovine albumin and HEPES) (TALP) medium or no dilution. After 24 h, samples were frozen and thawed (CM3, 0.5-ml straws, fast thawing rate) at 20 x 106 spermatozoa ml–1 (low concentration) or at 100 x 106 spermatozoa ml–1 (standard concentration). The SMI at 0-h postthaw was higher for samples stored at 4°C than for samples stored at 21°C (P < 0.001), and at 6-h postthaw, the SMI was higher for samples frozen at the standard concentration than for samples frozen at the low concentration (P < 0.05). For both experiments, acrosome integrity was similar across treatments. In summary, a semen cryopreservation protocol applied to fresh or liquid-stored semen maintained high levels of initial ejaculate sperm characteristics.

assisted reproductive technology, gamete biology, male sexual function, sperm, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the last 14 years, survivorship, recruitment, and reproductive success of captive bottlenose dolphin (Tursiops truncatus) populations have paralleled those of wild populations [1, 2]. As a result, essentially no additional founder animals have been integrated into the North American captive population [3]. Traditionally, genetic management among the numerous facilities housing bottlenose dolphins has not been cohesive, and social groups are often without breeding-age males or females. Consequently, fewer than 58% and 51% of founder males and females, respectively, have contributed to the captive gene pool [4]. This implies a potential loss of almost half the original genetic variability of the founder population (D.A. Duffield, personal communication).

Wild bottlenose dolphin populations are usually comprised of three primary social groups of four to seven individuals (range, 1 to >30). These social units (pods) include 1) females and their most recent calves, 2) juveniles of mixed gender, and 3) adult males, either singly or in pairs [2]. Thus, adult males are usually associated with female-comprised pods only during periods of breeding activity. Maintaining this social organization in captivity can be difficult because of the need for multiple, adequate-sized enclosures. Strategies are required to address the risk of excessive intermale competition and conflict in captive dolphins as a result of the growing population and high male: female ratio (as compared to social groups in the wild).

The ability to predetermine the sex of offspring through sperm sorting [4] combined with artificial insemination (AI) is under development in domestic [5–7] and a number of exotic [8–10] species. In combination with genome resource banking, application of sperm-sorting technology to the bottlenose dolphin would aid in the management of sex ratios that facilitate normal social behavior and contribute to improved genetic management of dolphin populations. Flow cytometric analysis demonstrated a large difference in DNA content between X chromosome- and Y chromosome-bearing dolphin spermatozoa (3.9–4.0%) and high resolution of X and Y sperm populations (unpublished results). Thus, there appears to be strong potential for the development of sperm sorting in the bottlenose dolphin. Application of sperm-sorting technology to species management is limited in situations where the sperm sorter is located a long distance from the animal facility [9]. Before these and other assisted reproductive technologies can be applied to bottlenose dolphin population, however, semen cryopreservation and liquid storage methods need to be critically evaluated.

Seager et al. [11] were the first to describe and cryopreserve bottlenose dolphin semen using one ejaculate collected by electroejaculation. In that report, sperm concentration was high (189 x 107 ml–1), as was initial motility (85%) and the progressive motility rating (5 on a 0–5 scale). After cryopreservation as pellets on dry ice using a canine cryodiluent (20% egg yolk [v/v], 11% lactose [w/ v], 4% glycerol [v/v]), samples retained high levels of initial motility (80%; progressive motility rating, 5). Evidence for the in vitro functional capacity of frozen-thawed dolphin spermatozoa from the aforementioned study [12] was provided by some ability of spermatozoa to penetrate hamster oocytes after incubation in a canine capacitation medium for 22 h.

The early success of bottlenose dolphin cryopreservation was not repeated until methods for the voluntary collection of semen were developed [13]. A male bottlenose dolphin was trained for voluntary semen collection, and numerous ejaculates were characterized and cryopreserved using the canine pellet method [14]. Optimum postthaw motility (60%) and progressive motility rating (5) were obtained by freezing pellets in liquid nitrogen vapor compared with freezing pellets on dry ice (freezing rate and pellet size were not specified). Direct comparisons of freezing methods could not be made, however, because different ejaculates were used for each treatment. This research also resulted in, to our knowledge, the only published data regarding seasonal semen characteristics of a male bottlenose dolphin [15]. Using pellets, cryovials, and one ejaculate from two males, Durrant et al. [16] provided a brief description of effects of different freezing rates, incubation times, and addition of glycerol before or after cooling to 4°C. Results suggested that the most effective protocol comprised a moderate freezing rate (–12.8°C min–1), with glycerol (4%) added after cooling of diluted (1:1 [v/v]) semen in a N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)-TRIS yolk buffer (TYB) to 4°C. Despite these limited successes with a small number of males, controlled studies utilizing numerous ejaculates from several males are required to determine optimum methods for semen storage and cryopreservation.

The objectives of the present research were 1) to establish baseline characteristics of bottlenose dolphin ejaculates, 2) to determine an effective method for the cryopreservation and thawing of fresh semen, and 3) to establish a precryopreservation semen storage protocol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Three adult, proven breeding male bottlenose dolphins (males 1, 2, and 3) housed at SeaWorld California were used for semen collection. Males 1, 2, and 3 weighed 224, 324, and 283 kg, respectively, and were aged 14, 15, and 34 yr, respectively. The dolphins were housed in two outdoor pools holding 850 m3 of natural processed saltwater at an ambient water temperature of 18°C during the study. Two of the animals (males 1 and 2) were housed together; the third was housed with another male not used in the study. Animals were fed a diet of frozen-thawed whole fish (herring, Clupea harengus, and Columbia River smelt, Thaleichthys pacificus) at approximately 4% to 5% of their body weight per day.

Ethics of Experimentation

All samples were collected using routine husbandry training and on a voluntary basis. When possible, training of husbandry behavior to facilitate biological sample collection is currently viewed as the standard of care for marine mammals. All procedures described within were reviewed and approved by the SeaWorld Institutional Animal Care and Use Committee and were performed in accordance with the NRC Guiding Principles for the Care and Use of Animals.

Experimental Design

Two experiments were conducted to evaluate the effects of treatments on sperm characteristics (motility, viability, morphology, and acrosomal status) in vitro. In experiment 1, fresh semen was collected from three bottlenose dolphins (n = 4 ejaculates per male) and used to compare the effects of three cryopreservation methods, two straw sizes, and three thawing rates on sperm characteristics (3 x 2 x 3 factorial). In experiment 2, fresh semen was collected from the same three males (n = 4 ejaculates per male) and was used to compare characteristics of spermatozoa cryopreserved at a low and a standard sperm concentration after storage for 24 h in four storage medium/temperature combinations (2 x 4 factorial).

Semen Collection

The animals were trained for voluntary semen collection as previously described [13]. Briefly, using a positive-reinforcement schedule combined with operant conditioning, the animals received various tactile stimulations to elicit voluntary extrusion of the penis from the genital groove. After an erection was obtained, animals were conditioned to ejaculate by stimulation, often directed toward the perineal area. For the experiment, once the animals subjectively appeared in a pre-ejaculatory state, the penis was grasped with a gloved hand (Nitrisoft, Nitrile latex-free examination glove; Sintex, Houston, TX) and the ejaculate directed into a 24-ounce WHIRL- PAK (NASCO, Fort Atkinson, WI). Collections were made from each male one to four times in succession until an ejaculate or combined ejaculates contained adequate numbers of viable spermatozoa for the respective experiments. Collection sessions (n = 24) for both experiments were conducted a maximum of twice per day at a minimum of 6 h apart.

Semen Processing and Analysis

Sperm samples were held at room temperature (21°C) and processed within 30 min of collection by a standard semen processing and analysis (SSPA) method. Ejaculate concentration, volume, color, pH (pH indicator strips; Whatman, Inc., Ann Arbor, MI), as well as sperm motility, viability (plasma membrane integrity), acrosomal status, and morphology were determined.

The percentage of motile spermatozoa was subjectively determined to the nearest 5% by analyzing four to five fields of view of diluted sperm sample (1:25, sperm diluted with Androhep Enduraguard [AE; Minitube of America, Verona, WI], pH adjusted to 7.2, warmed to 35°C) placed on heated slides (35°C) using bright-field optics (400x; Olympus, Tokyo, Japan). Total motility (TM), percentage progressive motility (PPM), and kinetic rating (KR; scale, 0–5, where 0 = no movement and 5 = rapid forward progressive movement) were subjectively determined. For data analysis and sample comparisons, these values were then transformed into a sperm motility index (SMI; modified from that described by Howard et al. [17]) as follows:

For assessment of viability, 10 µl of sample were mixed with 40 µl of a live-dead exclusion stain (eosin-nigrosin; IMV International Corp., Maple Grove, MN) for 30 sec. A smear was made and allowed to air-dry for evaluation on the same day. For evaluation, 200 spermatozoa per sample were examined using bright-field optics (1000x). Spermatozoa were placed into one of two groups based on stain uptake by the sperm head: live (no stain uptake) or dead (partial or complete stain uptake).

For analysis of the acrosome and morphology, 2 µl of the sperm sample were diluted with 10 µl of AE at room temperature on a glass slide, smeared, and allowed to air-dry for 5 min. The slide was then fixed with formal saline (Spermac Stain; Minitube of America) and stained within 2 wk of fixation. Acrosomes were evaluated with bright-field optics (1000x) and were classified as normal or abnormal (100 per sample). A normal acrosome had a distinct outline and was stained blue-green. Acrosomes were classified as abnormal if they were partially or completely lost or if obvious membrane irregularities (pitting, vacuolation) were present [18]. For morphology, spermatozoa (200 per sample) were evaluated under bright-field optics (1000x) for gross structural abnormalities.

Experiment 1: Effect of Cryopreservation Method, Straw Size, and Thawing Rate on In Vitro Sperm Characteristics

Concentrations of all samples were standardized to approximately 400 x 106 spermatozoa ml–1 (range, 350–450 x 106 spermatozoa ml–1). This was achieved by centrifuging (800 x g, 20 min) and thereby concentrating dilute ejaculates (<350 x 106 spermatozoa ml–1) or by diluting concentrated ejaculates (>450 x 106 spermatozoa ml–1) with seminal plasma isolated from excess volume of the same ejaculate by centrifugation (2000 x g, 10 min). Once the concentration was standardized and processed by SSPA, ejaculates were divided into three equal volumes and cryopreserved by one of three cryopreservation methods (CM1, CM2, and CM3) using 0.25- and 0.5-ml straws (Minitube of America). The three cryopreservation methods comprised cryodiluent components that were based on reported methods of dolphin semen cryopreservation [11, 16] and a toxicity trial demonstrating optimum postthaw SMI with final glycerol concentration of 3% (unpublished results). Initial dilutions for all three methods were performed slowly over 5 min at 21°C. After cooling (CM1 to 2°C, CM2 and CM3 to 5°C) and equilibration with glycerolated extender (cryodiluent) for 1 h, prefreeze SMI and acrosome status were determined. For each ejaculate, the sperm suspension was transferred to a minimum of four 0.25-ml and four 0-5 ml straws (per treatment), sealed with ball bearings (4°C; Minitube of America), and cryopreserved at the rate described for each method below using a programmable freezer (Minidigicool; IMV International Corp.). After freezing, straws were plunged into liquid nitrogen and stored until thawing. Final prefreeze concentration for all methods was approximately 100 x 106 spermatozoa ml–1 (range, 88– 113 x 106 spermatozoa ml–1).

The CM1 (a modification of the method previously used in the dolphin [14]) was performed as follows: Semen was slowly diluted over 5 min with Platz Diluent Variant (PDV) [19] without glycerol. The PDV (350 mOsm kg–1, pH 7.2) contained 11% lactose (L2643; Sigma-Aldrich Chemicals, St. Louis, MO), 20% egg yolk (v/v), and 50 µg ml–1 of gentamicin (Sigma). Before use, PDV was centrifuged (15 000 x g, 30 min), filtered (0.22 µm), and stored at –80°C. The sperm suspension was cooled from 21°C to 5°C over 1 h (–0.27°C min–1). Once at 5°C, the sperm suspension was placed into an ice-water bath (2°C) for 1 h (cooling rate, –0.6°C min–1), then diluted 1:1 (v/v) slowly with PDV containing 6% glycerol (G2025; Sigma; 3% final glycerol concentration). Dilutions with glycerolated cryodiluent for all methods (CM1, CM2, and CM3) were made in a stepwise fashion (25%, 25%, and 50% of volume) at 10-min intervals. Straws were frozen as follows: 3°C to –25°C at –100°C min–1, –25°C to –85°C at –15°C min–1, and –85°C to –140°C at –100°C min–1.

The CM2 was performed as follows: Semen was diluted 1:3 (semen: diluent, v/v) with AE and cooled to 15°C over 1 h (–0.1°C min–1). After reaching 15°C, the sperm suspension was centrifuged (15°C, 800 x g, 20 min). The supernatant was removed, and the pellet was resuspended 1:1 (semen:diluent, v/v) to a concentration of 150 x 106 spermatozoa ml–1 with PDV (prepared as in CM1) at 15°C by diluting dropwise over 5 min. After gentle mixing, the sperm suspension was placed at 5°C and cooled over 1 h (–0.17°C min–1). Once at 5°C, the extended semen was diluted 2:1 (semen:diluent, v/v) with cryodiluent (89.5% PDV, 9% glycerol, 1.5% Equex STM [Nova Chemical, Inc., Calgary, AB, Canada] [20]) for a final glycerol concentration of 3% (v/v). Straws were frozen as follows: 5°C to –6°C at –3°C min–1, hold for 1 min at –6°C, and then –6°C to –140°C at –50°C min–1.

The CM3 was performed as follows: Semen was diluted 1:1 (v/v) with a TYB (Refrigeration Media; Irvine Scientific, Santa Ana, CA; 320 mOsm kg–1, pH 7.2) without glycerol slowly over 5 min. The sperm suspension was cooled from 21°C to 5°C over 1 h (–0.27°C min–1). Once at 5°C, the sperm suspension was diluted 1:1 with TYB containing 6% glycerol (Freezing Media; Irvine Scientific; modified from 12% by dilution with Refrigeration Media). Straws were frozen as follows: 5°C to –80°C at –100°C min–1, then –80°C to –140°C at –200°C min–1.

Semen was thawed (n = 3 straws/treatment/ejaculate) using three methods comprising different thawing rates, after which samples were held at 21°C: 1) slow thawing rate (SLOW): 0.25-ml straw, 2°C (ice-water bath) for 50 sec, then 35°C for 30 sec; 0.5-ml straw, 2°C (ice-water bath) for 1 min, then 35°C for 30 sec; 2) medium thawing rate (MEDIUM): 0.25-ml straw: 35°C for 50 sec; 0.5-ml straw: 35°C for 1 min; and 3) fast thawing rate (FAST): 0.25-ml straw, 50°C for 8 sec; 0.5-ml straw, 5 sec in air, then 50°C for 10 sec. Straws (n = 3 per treatment) were shaken vigorously during thawing, combined in a 5-ml polystyrene tube, and then diluted (1: 1, over 5 min) with AE prewarmed to 35°C. After dilution, sperm suspensions were held at 21°C. Aliquots were removed for assessment of SMI at 0-, 3-, and 6-h postthaw and for acrosome integrity at 0- and 6-h postthaw. Thawing rates were determined using a thermocouple probe secured inside an unsealed straw of frozen diluent (n = 4 thaws per treatment). Temperature readings on the thermocouple were recorded every 2 sec over the time course of each thawing treatment. Thawing rates were calculated as the temperature change from storage temperature by the total time (sec) required to reach its final temperature (35°C).

Experiment 2: Effect of 24-h Semen Storage Methodand Sperm Concentration at Freezing on In Vitro Sperm Characteristics

A preliminary trial (unpublished results) on the motility and viability of dolphin spermatozoa (n = 3 ejaculates) held in four storage media (skim milk-glucose extender, AE, TYB, and PDV) at 4°C and 21°C (eight treatments) was performed to determine four optimum storage medium/temperature combinations for use in experiment 2. As described in experiment 1, concentrations of all samples were standardized to 400 x 106 spermatozoa ml–1. Ejaculates were divided into four aliquots for dilution (1:1) and storage (24 h) at 4°C with a skim milk-glucose extender (EP4°C; EquiPro; Minitube of America) or TYB (TYB4°C) and at 21°C with a Hepes-Tyrode balanced salt solution (containing bovine albumin) (TALP) medium, AE (AE21°C), or no dilution (NEAT21°C).

After 24 h, SMI and acrosome status were determined, and samples were processed for cryopreservation at low (LOW; 20 x 106 spermatozoa ml–1) and standard (STD; 100 x 106 spermatozoa ml–1) final sperm concentrations using the optimum method from experiment 1 (CM3). Samples representative of each treatment were centrifuged and resuspended in an appropriate volume of TYB to provide sperm concentrations. For each ejaculate, the sperm suspension was transferred to a minimum of three 0.25-ml straws (per treatment) and cryopreserved as described for CM3. Straws were thawed using MEDIUM and then processed as described in Experiment 1.

Statistical Analysis

Data for SMI, viability, acrosomal status, percentage SMI, and percentage acrosomal status (expressed as the percentage of that obtained for the initial ejaculate) were analyzed using ANOVA (SigmaStat, Vers 2.0; SSPS, Inc., San Rafael, CA). For experiments 1 and 2, the percentage SMI data were normalized by log-transformation before ANOVA. All pairwise multiple-comparison procedures between means were conducted using the Student-Newman-Keuls (SNK) test. A level of P < 0.05 was considered to be significant. Data are presented as the mean ± SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ejaculate Characteristics

Multiple ejaculates were often collected from each male during a collection session, and a total of 49 ejaculates were collected from the three males during the 24 collection sessions for experiments 1 and 2. If an ejaculate with adequate concentration could not be collected during a collection session, the ejaculates were pooled and centrifuged before the initial sample evaluation. These pooled ejaculates (n = 25) were not used to compile the ejaculate characteristics presented in Table 1. The number of ejaculations required to obtain an adequate sample from male 2 (3.0 ± 0.8 ejaculates) was greater (P < 0.05) than that for male 3 (1.3 ± 0.5 ejaculates) but similar to that for male 1 (2.0 ± 0.9 ejaculates). Overall, ejaculates were of high quality, with the sperm motility, viability, normal acrosomes, and normal morphology all greater than 83% (Table 1).


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  		TABLE 1. Characteristics of bottlenose dolphin ejaculatesa

Acrosome Evaluation

The Spermac Stain was effective at differentially staining the bottlenose dolphin acrosome, midpiece, and tail a deep green, whereas the nucleus was stained red (Fig. 1). Exposure time of spermatozoa to the red initial stain was increased from 2 min (as recommended by the manufacturer) to 3 min to improve resolution of the sperm nucleus and ease of acrosome evaluation.



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  		FIG. 1. Light micrograph of bottlenose dolphin spermatozoa after staining with Spermac Stain. Acrosome-intact spermatozoa (a) exhibit a blue- green acrosome, a red nucleus, and a blue-green midpiece and tail. Examples of spermatozoa classified as nonintact (b) include those with membrane vacuolation at the equatorial segment (line) and those without an acrosome (arrow). Magnification x1000

Experiment 1: Effect of Cryopreservation Method, Straw Size, and Thawing Rate on In Vitro Sperm Characteristics

After cooling and equilibration (prefreeze), a significant effect of cryopreservation method and male on %SMI (expressed as a percentage of the initial ejaculate) (Table 2) was observed. Acrosome integrity (percentage of initial acrosome intact value) was similar among treatments before freezing and after thawing, and it remained high during the postthaw incubation period. The prefreeze %SMI for each cryopreservation method in ascending order was CM2 (46.5% ± 26.1%), CM1 (71.1% ± 14.7%, P < 0.003), and CM3 (88.1% ± 10.6%, P < 0.001). Prefreeze %SMI for male 1 (63.3% ± 25.6%) and male 3 (59.5% ± 27.4%) was lower (P < 0.001) than that for male 2 (82.7% ± 14.9%). Significant interactions between cryopreservation method and male were observed for CM1 and CM2 (P < 0.001). For CM1, male 1 (78.9% ± 5.6%, P < 0.04) and male 2 (78.0% ± 6.1%) had a higher (P < 0.04) prefreeze %SMI than male 3 (58.3% ± 19.7%). For CM2, prefreeze %SMI was higher (P < 0.001) for male 2 (78.2% ± 19.5%) than for male 1 (30.1% ± 5.4%) and male 3 (31.0% ± 8.3%).


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  		TABLE 2. Effect of cryopreservation method, straw size, and thawing rate on in vitro sperm characteristics

At 0-h postthaw, differences (P < 0.001) in %SMI among cryopreservation methods and thawing rates were observed, but no effect of straw size was noted. The CM3 yielded the highest (P < 0.005) %SMI after thawing (53.7% ± 9.3%), followed by CM1 (49.6% ± 8.9%) and then CM2 (44.8% ± 10.1%, P < 0.001).

Thawing rates for each method (0.25- or 0.5-ml straw) were as follows: 1) SLOW: 0.25 ml, 3.6°C sec–1; 0.5 ml, 2.7°C sec–1; 2) MEDIUM: 0.25 ml, 12.8°C sec–1; 0.5 ml, 8.3°C sec–1; and 3) FAST: 0.25 ml, 28.8°C sec–1; 0.5 ml, 21.5°C sec–1 (Fig. 2). The %SMI was higher (P < 0.001) for FAST (52.0% ± 9.8%) and MEDIUM (52.1% ± 9.6%) compared to SLOW (44.0% ± 8.7%). The %SMI for FAST and MEDIUM was similar except for CM3, with which FAST (58.0% ± 7.6%) produced a higher (P < 0.012) %SMI compared to MEDIUM (56.0% ± 7.7%).



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  		FIG. 2. Comparison of thawing rates (mean ± SD) from the six thawing methods (SLOW: 0.25-ml straw, 0°C for 50 sec, then 30 sec at 35°C; 0.5-ml straw, 0°C for 60 sec, then 35°C for 30 sec; MEDIUM: 0.25-ml straw, 35°C for 50 sec; 0.5-ml straw, 35°C for 60 sec; FAST: 0.25-ml straw, 50°C for 8 sec; 0.5-ml straw, 50°C for 10 sec)

Although no overall differences in sperm characteristics were observed at 0-h postthaw between the two straw sizes, within CM2 the large straws had higher (P < 0.002) postthaw %SMI values than the small straws. The treatment combinations that resulted in the two highest 0-h postthaw %SMI values were CM3 x small straw x FAST (%SMI, 61.1% ± 6.2%; TM, 67.9% ± 5.4%; PPM, 90%; KR, 4.3 ± 0.2) and CM3 x large straw x MEDIUM (%SMI, 56.6% ± 8.3%; TM, 63.3% ± 6.1%; PPM, 90%; KR, 4.3 ± 0.3).

At 6-h postthaw, differences in %SMI were observed among cryopreservation method (P < 0.001), straw size (P < 0.011), and thawing rate (P < 0.001). In contrast to results at 0-h postthaw, an interaction was observed between straw sizes and thawing rate at 6-h post-thaw (P < 0.005). The %SMI decreased (P < 0.001) across all treatment combinations over time. By 6-h postthaw, the higher %SMI for CM3 compared to CM1 (as observed at 0-h postthaw) was apparent only within the small-straw-size treatment (P < 0.008). Based on %SMI at 6-h postthaw, the optimum treatment combinations were CM3 x large straw x MEDIUM (%SMI, 45.5% ± 8.7%; TM, 53.8% ± 4.8%; PPM, 89.2% ± 1.4%; KR, 4.1 ± 0.2) and CM3 x small straw x FAST (%SMI, 44.8% ± 11.9%; TM, 54.2% ± 9.3%; PPM, 89.6% ± 1.4%; KR, 4.0 ± 0.4)

Experiment 2: Effect of 24-h Semen Storage Methodand Sperm Concentration at Freezing on In Vitro Sperm Characteristics

After storage for 24 h, %SMI was similar across treatments and males (Table 3). After 24 h of storage and equilibration (prefreeze), differences (P < 0.001) in %SMI and acrosome integrity were observed among semen storage treatments. Within storage treatment, prefreeze %SMI was similar for LOW and STD. Among males, prefreeze %SMI varied (P < 0.001), with male 2 exhibiting a higher %SMI than male 1 and male 3 (76.4% ± 30.9%, 55.7% ± 33.2%, and 62.6% ± 32.9%, respectively; data pooled across storage treatments).


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  		TABLE 3. Effects of 24-h semen storage method and sperm concentration at freezing on in vitro sperm characteristics

At 0-h postthaw, %SMI was influenced by method of semen storage (P < 0.001) and sperm concentration at freezing (P < 0.015). The %SMI immediately after thawing was higher for EP4°C and TYB4°C compared with NEAT21°C and AE21°C (41.0% ± 8.4%, 36.7% ± 7.7%, 23.8% ± 8.6%, and 14.8% ± 8.6%, respectively; data pooled for sperm concentration).

Across storage methods, %SMI at 0-h postthaw was similar for both sperm concentrations with the exception of AE21°C (Table 3).

At 3- and 6-h postthaw, %SMI was higher for samples cryopreserved at STD than at LOW. During the 6-h incubation, %SMI decreased (P < 0.05) for all LOW treatments and STD NEAT21°C but was unchanged for the remaining STD treatments. By 6-h postthaw, %SMI was highest (P < 0.018) for samples stored by TYB4°C and cryopreserved using STD (%SMI, 40.8% ± 7.5%; TM, 49.2% ± 7.3%; PPM, 87.9% ± 2.6%; KR, 4.0 ± 0.1). Acrosome integrity was similar for all treatments at every postthaw assessment time.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Genome resource banking and assisted reproductive technology represent important tools for maintaining maximal genetic diversity of captive marine mammal populations. Although AI with liquid-stored spermatozoa has been successful on two occasions [21], AI with cryopreserved spermatozoa has not, to our knowledge, been reported. In the present study, systematic evaluation of characteristics, storage, and cryopreservation of semen was performed. These results represent a significant step toward optimizing the efficiency of AI with liquid-stored or cryopreserved semen in the bottlenose dolphin.

Semen characteristics detailed in the present study represent the most extensive baseline data to date for a dolphin species. The data demonstrate that bottlenose dolphin semen is of superior quality. Ejaculates are large in volume and comprise high numbers of morphologically normal, progressively motile spermatozoa. A previous study regarding ejaculates collected from one bottlenose dolphin [15] found similar high levels of motility but lower mean volume (4.3 ml) and sperm concentration (200 x 106 spermatozoa ml–1) compared to those of the present study. Such differences could be associated with a reported seasonal reproduction for this species, as indicated by slight seasonal variation in sperm production in one male [15] and in testicular size and testosterone production in eight males [22]. In addition to seasonal influences, preliminary evidence suggests that the social status of males housed in groups may also affect total sperm production. Subdominant males of mature age have been observed to produce ejaculates containing low total numbers of spermatozoa compared with the dominant male of the group (unpublished results). Effects of social dynamics need to be considered if a group- housed male is unable to produce ejaculate containing normal numbers of spermatozoa.

For species with relatively larger sperm heads and acrosomes, evaluation can be performed by light microscopy (1000x) of fixed samples with (e.g., Giemsa stain [23]) or without [17, 18] staining. Acrosome evaluation by phase- contrast microscopy of fixed, unstained dolphin spermatozoa is difficult because of the small size of the sperm head and apical ridge of the acrosome (unpublished results). Evaluation of acrosomal status using fluorescent stains (e.g., fluorescein isothiocyanate-conjugated peanut sativum [FITC-PSA]) would be useful for this species, but access to a fluorescent microscope is required. The differential stain for use with light microscopy, Spermac Stain, has been used with canine spermatozoa [24, 25], and the results have been highly correlated with the fluorescent stain FITC-PSA [26]. As described for canine spermatozoa, Spermac Stain was effective at differentially staining the acrosome, nucleus, midpiece, and tail of dolphin spermatozoa.

All cryopreservation methods tested in the present study employed a slow cooling rate from 21°C to 4°C, but they differed in cryodiluent composition and freezing rate. A semen cryopreservation protocol comprising a TYB/glycerol cryodiluent, slow cooling rate, rapid freezing rate, and medium or fast thawing rate maintained high levels of initial sample sperm characteristics. Sperm characteristics remained high during the 6-h postthaw incubation, indicating that samples would be satisfactory for use in AI.

Both CM1 and CM2 relied on essentially the same extender with or without the detergent Equex STM. Equex STM (also called Orvus ES paste) has been used successfully in the pig [27, 28] and with mixed results in a number of exotic animals [29, 30]. When used in egg yolk-containing diluents, Equex STM appears to enhance resistance of spermatozoa to cooling/freezing damage, possibly by solubilizing and making available to spermatozoa the protective egg yolk-derived lipids and lipoproteins [30, 31]. However, the use of Equex STM in CM2 was associated with decreased prefreeze and postthaw motility. Further study is required to determine if the decreased sperm motility was caused by the Equex STM and/or other components of the cryopreservation method.

All published reports on the cryopreservation of dolphin spermatozoa have relied on a pellet method [19, 32] that has remained virtually unchanged for more than 25 years. Pelleting of semen has been used successfully in numerous domestic (sheep [33], pig [34]) and wildlife (elephant [17], giant panda [35], ferret [36]) species. Straws, however, enjoy significant advantages over pellets by providing a sterile packaging system that ensures sample integrity, ease in labeling, and inhibition of pathogenic contamination that could occur during storage of pellets [30, 37, 38]. Although comparison with the results of previous studies is difficult because of the lack of published methodology and initial ejaculate characteristics, our optimum postthaw results achieved using either straw size appear to be comparable to those achieved with pellets [14]. Furthermore, after the 6-h incubation postthaw, high maintenance of initial motility and acrosome integrity was achieved with both straw sizes. Flexibility in choice of sample volume for freezing is advantageous, because the end use of the sample will influence the preferred straw size.

For all cryopreservation treatments, maintenance of initial motility parameters was greater for the medium and fast thawing rates than for the slow thawing rate. These findings are similar to those of other studies in which thawing at body temperature or 65°C resulted in optimum sperm motility in cattle [39, 40], sheep [41], and pigs [42]. Thawing rates faster than freezing rates are generally believed to help minimize ice crystal growth during thawing [43, 44]. Contrary to previous reports in pigs [42] and cattle [45] but in agreement with a report in red deer [46], the slow thawing rate tested in the present study was not associated with decreased acrosome integrity; all methods were associated with maintenance of greater than 90% of initial acrosome integrity.

Dolphin sperm motility parameters after liquid storage were higher for samples stored at 4°C than for those stored at 21°C, whereas acrosome integrity decreased only for the nondiluted semen stored at 21°C. Sperm aging because of inadequate media components and normal biologic processes has been known to affect motility and membrane integrity in vitro [28] and fertility in vivo [47–49]. Susceptibility to aging appears to depend on male or ejaculate quality before storage [28, 50] and can be affected by the storage temperature (<5°C vs. 18–24°C [51]). We found significant increases in postthaw motility when spermatozoa were held at 4°C as compared to 21°C for 24 h before freezing. Despite storage for 24 h (TYB, 4°C) before cryopreservation, sperm motility and acrosome integrity remained high after a 6-h postthaw incubation for samples frozen at the standard concentration (>40% and 88% of initial SMI and acrosome integrity, respectively). These results suggest that it is feasible to transport dolphin spermatozoa to facilities remote from the site of semen collection either for subsequent AI or further processing (e.g., sperm sexing and/or cryopreservation). However, the impact of sperm aging during liquid storage on in vitro and in vivo functional capacity of bottlenose dolphin spermatozoa requires investigation.

Results showed that the concentration of spermatozoa at freezing had a significant effect on postthaw motility of bottlenose dolphin spermatozoa. Decreased motility parameters were observed for samples cryopreserved using the low sperm concentration (20 x 106 spermatozoa ml–1) compared with the standard concentration (100 x 106 spermatozoa ml–1). Samples comprising the low and standard sperm concentrations were derived from a diluted (1:1, storage concentration of 200 x 106 spermatozoa ml–1) sample stored for 24 h at either 4°C or 21°C. The dilution step to obtain the low concentration was performed just before cooling (21°C storage treatments) or addition of cryodiluent (4°C storage treatments), suggesting a short-term dilution effect rather than a long-term effect of exposure to seminal plasma. The detrimental effect of a high dilution rate on postthaw motility has been documented in cattle [52, 53] and sheep [54]. Dilution of semen at high rates has also been shown to decrease the resistance of boar spermatozoa to cold shock during liquid storage [55]. Dilution of semen is believed to reduce the ratio of protective, low-molecular- weight seminal components to spermatozoa [56]. In support of this, recent research has demonstrated that the addition of seminal plasma postthaw improves viability [57] and fertility in sheep [58]. Sexed spermatozoa are usually frozen at 20 x 106 spermatozoa ml–1 [6, 7] because of limitations in the sperm-sorting rate. In light of the reduced quality of dolphin spermatozoa after cryopreservation at this low concentration, evaluation of the effect of the addition of seminal plasma during freezing and/or after thawing on postthaw motility and viability of dolphin spermatozoa is warranted.

For successful application of AI and sperm-sexing technologies to bottlenose dolphin captive management, development and optimization of semen liquid storage and cryopreservation methods were necessary. Having achieved this, further research is now required to efficiently integrate the aforementioned technologies into current population- management strategies. Research concerning the minimum number of spermatozoa necessary for successful fertilization after AI and regarding the development of sperm-sexing procedures represent key areas for future study in the bottlenose dolphin.


   ACKNOWLEDGMENTS
 
The animal care and animal training staff at SeaWorld California are thanked for training the animals for consistent semen collection, and the animal laboratory staff is thanked for technical support. We especially thank Todd Ryan and Melinda Tucker for their assistance with semen collection and acrosome evaluations, respectively. Bob French and Judy St. Ledger are thanked for assistance with photographic figures, as is Peter Thomson (University of Sydney) for statistical advice.


   FOOTNOTES
 
1 This project was supported by SeaWorld Corporation and is SeaWorld Technical Contribution Number 2003-01-T. Back

2 Correspondence: Todd R. Robeck, SeaWorld Texas, 10500 SeaWorld Drive, San Antonio, TX 78251. FAX: 210 523 3299;Todd.Robeck{at}SeaWorld.com Back

Received: 7 November 2003.

First decision: 9 December 2003.

Accepted: 26 December 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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