CSIRO PUBLISHING

Reproduction, Fertility and Development, 2008, 20, 770–783 www.publish.csiro.au/journals/rfd

Semen collection, characterisation and artificialinsemination in the beluga (Delphinapterus leucas)using liquid-stored spermatozoa

J. K. O’BrienA,B,D, K. J. SteinmanA, T. SchmittC and T. R. RobeckA

ASeaWorld and Busch Gardens Reproductive Research Center, 2595 Ingraham St,San Diego, CA 92109, USA.

BCentre for Advanced Technologies in Animal Genetics and Reproduction,Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia.

CSeaWorld California, 500 SeaWorld Dr, San Diego, CA 92109, USA.DCorresponding author. Email: justineo@vetsci.usyd.edu.au

Abstract. Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biologyand develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters andbiochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months.Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentrationand total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peaktestosterone concentrations (P < 0.05) were observed from October toApril (8.0 ± 1.6 ng mL−1).The effects of hyaluronicacid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parametersand viability were improved (P < 0.05) when semen was stored at 5◦C compared with 21◦C. During the first 24 h of storagesperm agglutination was absent only at 5◦C in the presence of HA. A nulliparous 28-year-old female was inseminatedendoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time inthis species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in aspecialised diluent.

Additional keywords: cetacean, hyaluronan, intrauterine insemination, semen storage.

Introduction

Beluga (Delphinapterus leucas) primarily inhabit the arctic andsub-arctic seas and oceans. While estimates vary due to difficul-ties in assessing migratory and non-migratory populations, thetotal wild population is thought to approximate 150 000 animals,including some discrete river populations (NAMMCO 2000).The species was listed as vulnerable just over a decade ago, prin-cipally due to habitat pollution threats (IUCN 1996). The statushas not changed since this assessment and threats to populationscontinue, particularly to those in close proximity to high levelsof human activity (IUCN 2006).

Family Monodontidae includes two genera, Delphinapterusand Monodon, each of which has one species, beluga (D. leu-cas) and the narwhal (M. monoceros). Information on narwhalreproductive physiology is not available and the species hasnever existed in a captive environment. Beluga have bred andgiven birth in captivity since 1981 (Calle et al. 1993) but thecaptive population remains small and fragmented, with 33 ani-mals housed at nine aquaria in NorthAmerica (Beluga studbook,Andrews 2007), and ∼20–25 animals housed in other facilitiesin Japan, Europe and Russia (B. Andrews, pers. comm.). Almosttwo-thirds of the adult population in the collectively-managed

North American population have reproduced (16 of 27, 59%),but the majority of unrepresented individuals (10 of 11, 91%)are non-related founders (Andrews 2007). Traditional geneticmanagement of captive beluga populations has been reliant onanimal breeding loans among institutions (Robeck et al. 2005a).Although this approach has been successful for numerous facil-ities, application of assisted reproductive technologies (ART)such as artificial insemination (AI) with liquid-stored or cryo-preserved spermatozoa offers great potential to aid in managingthe genetic diversity of this small population. Of high priorityfor AI application is the propagation of offspring from unrepre-sented founders who have failed to reproduce naturally. Further,ART can help to overcome potential (albeit low risk) healthand disease problems associated with transport of live animalsfor breeding loans. As such, cooperative research of the captivepopulation is in progress to gain an understanding of male andfemale reproductive physiology (Calle et al. 1993, 1996, 2000;Robeck et al. 2005a) – prerequisites for successful applicationof any ART.

Information on spermatogenesis and sperm biology repre-sents an important component of a species-specific reproduc-tive baseline database, which can be incorporated into health

© CSIRO 2008 10.1071/RD08031 1031-3613/08/070770

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 771

assessments for free-ranging or captive animals. Collection andpreservation of spermatozoa following the death of an animal(‘gamete rescue’), whether in the wild or in captivity, repre-sents an important conservation tool given that valuable geneticmaterial (in the form of spermatozoa) can be stored indefi-nitely, and potentially used to produce offspring through AI longafter the male has died. Indeed, genome storage banks, facil-itating the organised storage of gametes and other biologicaltissues, have been established for numerous endangered wildlifespecies using gamete rescue methodologies (reviewed by Holtet al. 2003). However, successful integration of gamete res-cue samples into species management requires that controlled,species-specific studies are first performed. Systematic optimi-sation of gamete preservation methods will ensure that samplesretain as much of their fertilising capacity as possible followingeither chilled storage or thawing.

Currently, there are no standard semen preservation proto-cols for the beluga, and the effect of season on sperm productionand quality is unclear. Spermatozoa were collected post-mortemfor morphological studies (Miller et al. 2002) but samples werederived from an acutely-ill animal that had been on long-termaminoglycoside therapy. Thus, preliminary motility results fromthat study (34% progressively motile, 3.0 kinetic rating) aremost probably not representative of the species. Short- andlong-term sperm preservation methods have been establishedfor other cetaceans including the bottlenose dolphin (Tursiopstruncatus; Robeck and O’Brien 2004), killer whale (Orca orci-nus; Robeck et al. 2004) and Pacific white-sided dolphin(Lagenorhynchus obliquidens; Robeck et al. 2003). Artificialinsemination has been successful in the aforementioned species,and is contributing significantly to their genetic and reproduc-tive management in captivity (Robeck et al. 2005b; Robeck andO’Brien 2005). Sperm preservation and AI methodologies arenow required for successful application ofART to beluga captivemanagement.

The goal of the present research was to obtain a greaterunderstanding of male reproductive physiology and develop ashort-term sperm preservation method in the beluga for usein AI. Development of an optimum diluent for liquid stor-age of beluga semen will also facilitate future cryopreservationstudies in this species. Specific objectives were to (1) developa semen collection method eliminating salt water contamina-tion, (2) characterise ejaculate parameters, sperm productionand testosterone production over a 21-month period, (3) deter-mine the effects of hyaluronic acid (HA), antioxidants, storagetemperature and time on in vitro sperm characteristics, and(4) validate the in vivo fertility of spermatozoa after chilledstorage and AI.

Materials and methods

All procedures described within were reviewed and approved bythe SeaWorld Institutional Animal Care and Use Committee andwere performed in accordance with the Animal Welfare Act forthe care of Marine Mammals. All samples were collected usingroutine husbandry training and on a voluntary basis. Training ofhusbandry behaviour to facilitate biological sample collection iscurrently viewed as the standard of care for marine mammals.

AnimalsSemen was collected from a wild-born, adult beluga (aged19 years in 2003, weighing 820 kg) over a 21-month period(May 2003 to February 2005). The male was housed at Sea-World San Diego (SWSD) with two adult wild-born females ofnon-proven fertility (Females 1 and 2; aged 24 and ∼40 yearsin 2003 and weighing 580 and 612 kg, respectively). No copula-tions between the male and these females were observed beforeor during the study. The male was transferred to SeaWorld SanAntonio (SWSA) in February 2005, and sired three calves whilsthoused with five adult females ranging in age from 6 to 19 years.The AI trial was performed with Female 1 (SWSD) in February2007 after the male was transferred to SWSA. Animals werehoused in outdoor pools holding from 258 to 1741 m3 of nat-ural processed salt water maintained at 11◦C. Diet consistedof frozen–thawed whole fish (herring (Clupea harengus), andColumbia River smelt (Thaleichthys pacificus)) fed at ∼4 to 5%of bodyweight per day.

Reagents and mediaAll chemicals were of analytical grade and cell culture testedwhere possible by the manufacturer. Unless otherwise stated, allmedia components were purchased from Sigma-Aldrich (Sigma,St Louis, MO, USA) and were prepared with tissue culture-grade water (Sigma or Millipore, Billerica, MA, USA). Diluentscontaining egg yolk (free-range eggs) were prepared by ultra-centrifugation for 1.5 h at 10 000g and 5◦C. The supernatantwas filtered (0.22 µm; Millipore) and frozen at −80◦C for amaximum of 18 months.

Semen collectionThe male was trained for voluntary semen collection over a two-year period using operant conditioning as previously describedfor the bottlenose dolphin with some modifications (Keller 1986;Robeck and O’Brien 2004). A description of the training pro-cess has been published elsewhere (Graack and Grovhoug 2003).Briefly, the animal received various tactile stimulations to elicitvoluntary extrusion of the penis from the genital groove. Oncea complete erection was obtained, the male was conditioned toejaculate by manual stimulation of the penis with a gloved hand(Nitrisoft, Nitrile powder-free examination glove; Sintex, Hous-ton, TX, USA), lubricated sparingly with mineral oil (Sigma),which has since been replaced with Pre-seed lubricant (INGfer-tility, Valleyford, WA, USA). Urination and defecation occurredduring the initial stages of semen-collection training and wereextinguished using standard training techniques.

When semen could be collected on a consistent basis, mod-ifications to the collection process were required to avoidcontamination of semen with saltwater. Once manual stimula-tion had commenced and the male subjectively appeared in apre-ejaculatory state, the distal end of the penis was wiped withsterile cotton gauze, or rinsed with a HEPES-TALP medium,to remove residual saltwater. The penis tip was then quicklydirected into a modified artificial vagina (AV) device comprisedof a circular polystyrene rim (10 cm diameter) covered by a

772 Reproduction, Fertility and Development J. K. O’Brien et al.

(a)

(b)

Fig. 1. (a) Front and (b) back view of the modified artificial vagina devicefor collection of beluga semen.

15 × 20 cm piece of latex AV liner (IMV International Corp.,Maple Grove, MN, USA), which was secured in place by a sec-ond circular rim (12 cm diameter). The penis tip was directedthrough a small opening in the latex liner and the ejaculatewas collected shortly after further manual stimulation of thepenis into a 700-mL WHIRL-PAK bag (NASCO, Fort Atkinson,WI, USA), which was also attached to the polystyrene rim(Fig. 1).

Evaluation of semen and sperm characteristicsEjaculates were held at room temperature (21◦C) and pro-cessed within 30 min of collection. Ejaculate concentration,volume, pH (pH indicator strips; EM Science, Gibbstown, NJ,USA), osmolality (Advanced Instruments Inc., Norwood, MA,USA), as well as sperm motility parameters, viability (plasmamembrane integrity), acrosomal status and morphology weredetermined.

Analysis of seminal plasma biochemistry was performedusing an Olympus Analyser (Olympus AU400E, OlympusAmerica Inc., Melville, NY, USA) with Olympus reagents andstandards. Seminal plasma was isolated from spermatozoa bycentrifugation (2000g for 10 min at 5◦C). The concentrationor activity of the following analytes was measured (ana-lytical method in parentheses): glucose (hexokinase), fruc-tose (modified sorbitol dehydrogenase reaction; Andersonet al. 1979), blood urea nitrogen (urease), creatinine (kineticJaffe reaction), cholesterol (cholesterol esterase), triglycerides(modified GPO/PAP), total protein (biuret), albumin (brom-cresol green), globulins (calculated test: globulins = totalprotein − albumin), alkaline phosphatase (nitrophenylphos-phate), alanine aminotransferase (modiRed IFCC), aspartatetransaminase (modified IFCC-UV without P5P), gamma-glutamyl transferase (Gamma-glutamyl-carboxy-nitroanilide),creatinine kinase (NAC-activated, modified IFCC), lactose dehy-drogenase (lactate to pyruvate, Wacker modification), calcium(arsenazo III), phosphorus (Phosphomolybdate-UV), sodium(ion specific electrodes), potassium (ion specific electrodes),chloride (ion specific electrodes), total CO2 (enzymatic PEPC-phosphoenolpyruvate carboxylase) and iron (TPTZ-no depro-teinisation (2,4,6-Tri(2-pyridyl)-5-triazine)). The analyser wascalibrated daily according to the manufacturer’s instructionsusing two dilutions of assayed, commercially-available multi-analyte external controls. All tests were performed at 37◦C.

Sperm motility was objectively assessed using computer-assisted sperm analysis (CASA, Hamilton–Thorne, HTM-IVOSVersion 12.2). Semen was diluted to a concentration of 15to 25 × 106 spermatozoa mL−1 and a 2-µL aliquot was trans-ferred to a 4-chamber 20-µm linear-flow slide (Leja ProductsB. V., Nieuw Vennep, Amsterdam, The Netherlands) at 35◦C.Five randomly-selected microscopic fields were scanned tocalculate the following motility parameters: average pathwayvelocity (VAP, µm s−1), straight-line velocity (VSL, µm s−1),the amplitude of lateral head displacement (ALH, µm), beatcross frequency (BCF, Hz), straightness of sperm movement(STR (VSL/VAP), %), and percentage of progressively-motilespermatozoa (ProgMot, %; VAP >25 µm s−1). The instrumentsettings were 30 frames at a frame rate of 60 frames s−1, min-imum contrast was 40, minimum cell size (pixels) was 5; forprogressive cells the VAP was >15 µm s−1 and STR was >70%.Spermatozoa with aVAP of more than 5 µm s−1 were consideredmotile. For Study 4, three additional groups based on velocity ofmovement were also determined: rapid (RAP, VAP >25 µm s−1,%), medium (MED, 5 µm s−1 <VAP <25 µm s−1, %) and slow(SLOW, VAP <5 µm s−1, %).

For assessment of viability, 10 µL of sample were mixedwith 30 µL of a live–dead exclusion stain (eosin-nigrosin; IMVInternational Corp.) for 30 s. A smear was made and allowed toair-dry for evaluation within 30 min (100 spermatozoa per sam-ple, ×1000). Spermatozoa were classified as viable (no stainuptake) or non-viable (partial or complete stain uptake).

For analysis of acrosomal status and sperm morphology thesperm sample was smeared on a glass slide and allowed to air-dry for 5 min. The slide was then fixed with formal saline andstained (Spermac Stain; Minitube ofAmerica,Verona, WI, USA)within 2 weeks of fixation. Acrosomes were classified as normal

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 773

Table 1. Diluents used in a beluga semen storage pilot trialAll diluents were prepared in-house with the exception of Andromed and EquiPro (315 ± 10 mOsm kg−1, pH: 7.0 ± 0.3).

In-house diluent osmolality and pH were 360 ± 5 mOsm kg−1 and 7.0 ± 0.1, respectively

Diluent Components Reference

AndromedA Soyabean lecithin (proprietary recipe) Aires et al. (2003)Beltsville extender (BF5F) TES, Tris, glucose, fructose, egg yolk Modified from Pursel and Johnson (1975)EquiProA Selective milk proteins, sugars, glycine Kenney et al. (1975); Pagl et al. (2006)

(proprietary recipe)Platz Diluent Varient (PDV) Na-citrate, lactose, egg yolk Modified from Platz et al. (1978)TEST-yolk buffer (TYB) TES, Tris, fructose, egg yolk Modified from Graham et al. (1972)Whole milk Heat-treated whole milk Foote et al. (1993)

AMinitube of America, Verona, WI, USA.

or abnormal (100 spermatozoa per sample, ×1000). A normalacrosome had a distinct outline and was stained blue-green.Acrosomes were classified as abnormal if they were partiallyor completely lost or if obvious membrane irregularities (pit-ting, vacuolation) were present (Robeck and O’Brien 2004). Formorphology, spermatozoa were evaluated for gross structuralabnormalities (200 spermatozoa per sample, ×1000).

Characterisation of ejaculate parameters, semenproduction and testosterone productionSemen collection was attempted at least once per week duringthe 21-month study period to characterise ejaculate param-eters (including sperm viability, morphology and acrosomeintegrity) and examine the effects of season on sperm pro-duction and quality. Seminal plasma biochemistry was deter-mined for 21 non-contaminated, 10 urine-contaminated and9 saltwater-contaminated ejaculates collected during April toOctober.

Serum testosterone concentration was measured at monthlyor bi-monthly intervals throughout the study period. Concentra-tions were determined by a commercial laboratory (The TexasVeterinary Medical Diagnostic Laboratories College Station,TX, USA).

Effect of hyaluronic acid and antioxidant supplementationon in vitro sperm characteristics of liquid-stored semenDifferent semen storage diluents (Table 1) were examined in apreliminary trial. All diluents were supplemented with gentam-icin sulfate (50 µg mL−1). A pilot trial using three ejaculateswas performed using all diluents except heat-treated wholemilk, as the latter was associated with high levels of spermhead–head agglutination and an immediate decrease in motil-ity. Semen was diluted 1 : 4 (semen : diluent) before storage at5◦C or 21◦C. Based on in vitro sperm characteristics (motil-ity, viability and agglutination rating) after storage for 6 h,BF5F medium was selected as the optimum liquid storage basemedium for beluga semen (0 h progressive motility and viability65.2 ± 5.9%, 82.0 ± 2.6%; 6 h progressive motility after stor-age at 5◦C and 21◦C, respectively: non-diluted 18.5 ± 12.1%and 24.8 ± 13.0%; Andromed 6.9 ± 6.4% and 6.0 ± 5.2%;BF5F 54.0 ± 7.8% and 53.6 ± 8.4%; EquiPro 27.0 ± 4.0%

and 22.2 ± 20.7%; PDV 35.0 ± 13.0% and 26.8 ± 15.5%;TYB 38.3 ± 3.8% and 43.3 ± 4.5%; 6 h viability after stor-age at 5◦C and 21◦C, respectively: non-diluted 76.7 ± 2.5%and 81.3 ± 3.8%; Andromed 58.5 ± 26.2% and 65.5 ± 2.1%;BF5F 77.3 ± 4.7% and 77.7 ± 9.5%; EquiPro 73.0 ± 8.5%and 76.0 ± 10.5%; PDV 73.7 ± 5.1% and 77.3 ± 1.5%; TYB83.3 ± 11.9% and 77.0 ± 12.2%). Sperm head–head agglutina-tion occurred regardless of diluent type.

BF5F was modified from the original recipe by omittingsodium triethanolamine lauryl sulfate and increasing the concen-tration of fructose and glucose. BF5F contained 52.3 mm TES,16.5 mmTris, 105.4 mm fructose, 105.4 mm glucose and 20% v/vegg yolk. The osmolality and pH of BF5F (360 ± 5 mOsm kg−1

and 7.0 ± 0.1, respectively) were designed to simulate the osmo-lality and pH of non-contaminated beluga semen. BF5F wassupplemented with hyaluronic acid (HA; sodium hyaluronate,Bioniche Animal Health, Bogart, GA, USA), and/or the anti-oxidants catalase and superoxide dismutase (AO, Sigma), todetermine the optimum diluent for short-term storage of semen(refer to the following paragraph). Diluent treatment groups werestored at 21◦C or 5◦C to evaluate the effect of temperature onin vitro sperm characteristics.

Four non-contaminated ejaculates were used to examine theeffect of two storage temperatures and four storage diluents onin vitro sperm characteristics (2 × 4 factorial). Each ejaculatewas divided into four aliquots and diluted (1 : 3; semen : diluent,v/v) over 5 min (semen and diluent were both 21◦C) with:(1) BF5F (−HA/−AO), (2) BF5F plus HA (+HA/−AO;final concentration of HA was 1 mg mL−1), (3) BF5F plusantioxidants (−HA/+AO; 50 U mL−1 catalase and 100 U mL−1

final superoxide dismutase) or (4) BF5F plus HA and AO(+HA/+AO). Each aliquot was then divided in half and heldat 5◦C or 21◦C. The chilled storage treatment group was cooledfrom 21◦C to 5◦C at ∼−0.15◦C min−1. Sperm motility (evalu-ated by CASA), viability and acrosomal status were assessed at0, 6, 24, 48, 72, 96 and 120 h after dilution. Each treatment groupwas assessed in triplicate (total of 12 replicates per treatment).

Artificial insemination using liquid-stored semenOestrous behaviour (rubbing the genital slit on structures in theunderwater habitat) by Female 1 was observed on February 3,

774 Reproduction, Fertility and Development J. K. O’Brien et al.

2007. From this date onwards, ovarian activity was monitored bytransabdominal ultrasonography (GE Logiq Book, GE MedicalSystems, Milwaukee, WI, USA; 3.5 mHz convex probe). Aseparate study (Steinman et al. 2007) indicated beluga werefacultative-induced ovulators, so ovulation induction was neces-sary in the present trial once the follicle reached a preovulatorysize (>8 cm in circumference). Three injections of a GnRHanalogue (Cystorelin, Merial, Duluth, GA, USA; 250 µg perinjection), administered intravenously every 2 h, were used toinduce ovulation (Steinman et al. 2007). The insemination wasperformed at 2100 hours on February 15, 2007 (28 h after thefirst GnRH injection) using a flexible endoscope (9 mm exter-nal diameter and 200 cm long, Olympus America) equippedwith a sterile, custom-made catheter (2.2 mm external diameter[7 Fr], 350 cm long, Smiths Medical PM, Inc., Waukesha, WI,USA).

At the time of the AI, the male was housed at SWSA. Twonon-contaminated ejaculates were collected on February 15,2007, one at 0625 hours and one at 0640 hours (PST). Ejacu-lates were immediately diluted (1 : 3; semen : BF5F+HA, v/v)over 5 min (final concentration of HA was 1 mg mL−1), andslowly cooled to 7–8◦C (∼−0.15◦C min−1) in a modified Equi-tainer (Hamilton Research, South Hamilton, MA, USA; thetwo coolant cans were placed in the Equitainer after storageat −20◦C and 19–21◦C, respectively) placed inside a foam-insulated box during transport to SeaWorld California. Theanimal was removed from the water for the AI, and placed in lat-eral recumbency on multiple cell foam pads arranged to allowelevation of the posterior body. The female was kept wet dur-ing the procedure and monitored for respiration rate. A total of60.7 × 107 progressively-motile spermatozoa (in 7.2 mL) wereplaced in the uterus (70% and 30% of total spermatozoa placedin the right and left uterine horn, respectively) at 2100 hours(14.5 h after collection of semen).An additional 12 mL of diluent(BF5F) was also transferred to the uterus to aid in the placementand flushing of the catheter. The AI procedure took 11 min.

For luteal phase monitoring and pregnancy diagnosis, bloodsample collections and transabdominal ultrasounds were per-formed weekly after the AI. Progesterone concentrations inblood plasma were determined by a commercial laboratory(Quest Diagnostics, Irving, TX, USA). Progesterone concen-trations greater than 5 ng mL−1 (baseline concentration = 0.1 to1 ng mL−1) on Day 30 after insemination are indicative of preg-nancy. Thereafter, confirmation of pregnancy was achieved bytransabdominal ultrasound for detection of uterine fluid and afetus.

Statistical analysisData for sperm motility characteristics, viability and acrosomalstatus were analysed using ANOVA (SigmaStat, Version 2.0;SSPS, San Rafael, CA, USA). Prior to ANOVA, motility datawere normalised, when necessary, by arc-sin transformations.All pairwise multiple-comparison procedures between meanswere conducted using the Tukey test. P < 0.05 was consid-ered to be significant. Data are presented as the untransformedmean ± s.d.

Table 2. Characteristics of non-contaminated beluga ejaculates(n = 124 ejaculates, unless specified)

Parameter Mean ± s.d.

Semen characteristicsEjaculate volume (mL) 1.7 ± 1.2Osmolality (mOsm kg−1)A 356.3 ± 9.4pH 7.1 ± 0.2Sperm concentration (×107 mL−1) 29.5 ± 16.3Total spermatozoa per ejaculate (×107 mL−1) 53.7 ± 48.6

Sperm characteristicsTotal motilityB 58.7 ± 8.8Progressive motilityB 44.9 ± 5.2VAP (µm s−1)B 69.3 ± 8.2VSL (µm s−1)B 66.1 ± 9.4VCL (µm s−1)B 84.2 ± 5.9ALH (µm)B 2.2 ± 0.3BCF (Hz)B 37.7 ± 1.6STR (%)B 92.9 ± 3.0Viability (%) 83.0 ± 6.3Acrosome normal and intact (%) 81.7 ± 6.1Morphologically normal (%) 60.3 ± 6.3Morphologically abnormal (%) 39.7 ± 18.4

HeadMicrocephalic 4.2 ± 1.9Macrocephalic 0.4 ± 0.8Bicephalic 0.2 ± 0.6Tapered 24.8 ± 5.7Amorphous 3.6 ± 2.3Missing 3.1 ± 2.2Detached 0.1 ± 0.4

MidpieceDegenerate 0.2 ± 0.5Abaxial 1.7 ± 1.5

FlagellumBent 0.4 ± 0.8Coiled 0.3 ± 0.6Double 0.6 ± 0.3

Cytoplasmic droplet (proximal) 0.1 ± 0.2

ADetermined from analysis of 68 ejaculates.BDetermined from analysis of 12 ejaculates.

ResultsCharacterisation of ejaculate parameters, semenproduction and testosterone productionA total of 124 non-contaminated ejaculates were collected overthe study period (Table 2). Sperm viability, acrosome integrityand morphology did not change during the study but ejaculatevolume, sperm concentration and total spermatozoa per ejaculatewere higher from January to June than from July to December(Fig. 2, P < 0.05). The predominant type of abnormal morpho-logical form was a tapered head (Fig. 3), and the frequencyof this and other forms did not change throughout the study(P > 0.05).

Annual serum testosterone concentration remained above1 ng mL−1 (Fig. 2), but concentrations were higher (P < 0.05)during the months of October to April (8.0 ± 1.6 ng mL−1) thanthe nadir months of May to September (2.7 ± 1.2 ng mL−1).

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 775

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Fig. 2. Profiles of sperm production, ejaculate volume (n = 124) and serum testosterone concentration (n = 35) throughout the 21-monthstudy period.

Fig. 3. Light micrograph of beluga spermatozoa after fixation and staining with Spermac Stain showing a normal spermatozoon and aspermatozoon with a tapered head (arrow). Both spermatozoa possess an intact acrosome (stained blue-green). Original magnification×1000.

Seminal plasma chemistry parameters are shown in Table 3.Ejaculates with urine or salt water contamination displayed ele-vated osmolality (P < 0.05), but pH remained similar (P > 0.05)to non-contaminated samples. Progressive motility was higher(P < 0.05) for clean compared with contaminated ejaculates,

but viability remained the same among all groups. Creatinine,BUN and phosphorus concentrations were elevated (P < 0.05)in samples containing urine compared with non-contaminatedand salt-water contaminated samples. Sodium and chloride ionconcentrations were higher in ejaculates containing saltwater

776 Reproduction, Fertility and Development J. K. O’Brien et al.

Table 3. Biochemistry of beluga seminal plasma from non-contaminated and contaminated ejaculatesa,bValues with different superscripts within the same row are significantly different (P < 0.05)

Parameter Non-contaminated Contaminated (urine) Contaminated (saltwater)

Number of samples 21 10 9Ejaculate volume (mL) 2.2 ± 1.1 1.7 ± 0.9 2.1 ± 1.3Osmolality (mOsm kg−1)a 352.3 ± 11.9a 418.9 ± 76.8b 528.8 ± 146.6b

pH 7.1 ± 0.2 6.9 ± 0.1 7.1 ± 0.2Sperm concentration (×107 mL−1) 25.6 ± 16.5 16.6 ± 12.5 15.0 ± 12.8Progressive motility (%) 49.0 ± 4.9a 34.8 ± 20.0b 8.5 ± 10.1c

Viability (%) 80.9 ± 6.7 79.2 ± 5.7 83.0 ± 3.2Glucose (mmol L−1) <0.6 <0.6 <0.6Fructose (mmol L−1) 2.3 ± 1.8A 2.5 ± 1.5B n/dBUN (mg mL−1) 0.4 ± 0.3a 2.0 ± 1.2b 0.5 ± 0.2a

Creatinine (mg mL−1) 0.02 ± 0.008a 0.1 ± 0.1b 0.01 ± 0.01a

Cholesterol (mg dL−1) 30.4 ± 4.8 23.0 ± 6.4 20.0 ± 2.9Triglycerides (mg dL−1) 18.5 ± 4.5 11.3 ± 7.6 11.3 ± 0.6Total protein (g dL−1) <3.0 <3.0 <3.0Albumin (g dL−1) <1.5 <1.5 <1.5Globulins (g dL−1) <1.5 <1.5 <1.5Alk phosphatase (U L−1) >30 000 >30 000 >30 000Alt (U L−1) 5.0 ± 1.2 6.0 ± 3.5 4.0 ± 1.2Ast (U L−1) 74.8 ± 9.6 61.7 ± 11.5 44.3 ± 27.4GGT (U L−1) >24 000 >24 000 >24 000Creatine kinase (U L−1) 310.3 ± 66.3a 174.8 ± 86.6ab 164.0 ± 114.8b

LDH (U L−1) 680.0 ± 129.8 556.3 ± 120.4 454.8 ± 187.3Calcium (mmol L−1) 1.2 ± 0.2 1.4 ± 0.3 2.5 ± 2.9Phosphorus (mmol L−1) 1.2 ± 1.1a 6.1 ± 3.8b 0.5 ± 0.4a

Sodium (mmol L−1) 100.6 ± 9.5a 108.7 ± 11.2a 192.3 ± 78.2b

Potassium (mmol L−1) 17.7 ± 1.9 19.8 ± 5.1 11.9 ± 1.7Chloride (mmol L−1) 40.5 ± 18.0a 54.2 ± 22.1a 160.0 ± 103.4b

TCO2 (mmol L−1) 4.7 ± 1.9 3.9 ± 1.3 4.8 ± 3.3Iron (µg dL−1) 26.5 ± 7.4a 16.7 ± 5.6b 10.8 ± 2.9b

Total bilirubin (mg dL−1) 0.07 ± 0.03 0.09 ± 0.02 0.06 ± 0.03

ADetermined from analysis of three ejaculates.BDetermined from analysis of two ejaculates.

compared with other sample types. The concentration of ironwas higher (P < 0.05) for non-contaminated compared withcontaminated samples.

Effect of temperature, hyaluronic acid and antioxidantsupplementation on in vitro sperm characteristicsof liquid-stored semenThere was a significant effect of diluent and temperature ondegree of sperm head–head agglutination (Fig. 4). Agglutinationoccurred at both storage temperatures when HA was absent fromthe storage diluent (−HA/−AO, −HA/+AO).Agglutination wasabsent in treatments containing HA throughout the trial whensamples were stored at 5◦C. At 21◦C, agglutination occurred inall treatment groups from 24 h of storage.

Regardless of storage temperature, sperm samples stored indiluents containing HA displayed reduced (P < 0.05) VAP, VSLandALH, increased (P < 0.05) STR, and similar (P > 0.05) BCFcompared with those not containing HA during the first 6 h ofstorage (VAP 64.2 ± 5.8 and 73.8 ± 7.0 µm s−1; VSL 61.4 ± 6.3and 70.0 ± 7.5 µm s−1; VCL 79.1 ± 5.4 and 93.6 ± 6.9 µm s−1;

ALH 2.2 ± 0.3 and 2.6 ± 0.3 µm; STR 93.2 ± 1.8% and91.6 ± 2.6%; BCF 38.1 ± 1.8 and 37.8 ± 1.7 Hz; data pooledacross storage temperature and time (0 h, 6 h), with HA and with-out HA, respectively). After 6 h of storage, motility parameterswere similar (P > 0.05) for sperm samples stored in the presenceor absence of HA.

With the exception of HA effects on velocity parameters dur-ing the first 6 h of storage, and on sperm head–head agglutinationthroughout the entire storage period, neither HA nor AO influ-enced in vitro sperm characteristics (P > 0.05). Thus, resultswere pooled across diluent treatment groups for further anal-yses. There was a significant effect of temperature on all spermcharacteristics except for acrosome integrity during the five-daystorage period (Fig. 5). By 24 h, progressive motility was higherfor 5◦C than 21◦C samples (P < 0.05). By 48 h, motility param-eters VSL, VAP, BCF and STR were improved (P < 0.05) whensemen was held at 5◦C compared with 21◦C. ALH and viabilitywere not negatively impacted by the higher storage temperatureuntil 96 h of storage. Overall, sperm characteristics of samplesstored at 5◦C, excluding acrosome integrity and viability, startedto decrease (P < 0.05) by 48 h of storage.

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 777

Time (h)0 6 24 48 72 96 120

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Fig. 4. Sperm agglutination index of samples stored at (a) 5◦C or (b) 21◦C. (Data are mean ± s.d.)

Time (h)

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Fig. 5. In vitro sperm characteristics of samples stored at 5◦C (solid line) or 21◦C (dotted line): (a) progressive motility (PMot) and viability (Viab),(b) acrosome integrity (NAcr), (c) average path velocity (VAP), straight line velocity (VSL) and straightness (STR), and (d) beat cross frequency (BCF)and amplitude of lateral head displacement (ALH). (Data pooled across HA and AO treatment groups; data are mean ± s.d.)

778 Reproduction, Fertility and Development J. K. O’Brien et al.

Artificial insemination using liquid-stored semenAt the time of AI, the preovulatory follicle (right ovary)had a circumference of 9.06 cm (2.49 × 3.26 cm in diameter).Ultrasound monitoring confirmed that ovulation occurred within11.5 h of insemination. Sperm characteristics at insemina-tion were: progressive motility 45%; VAP 63.3 ± 29.9 µm s−1;VSL 58.2 ± 31.2 µm s−1; VCL 80.8 ± 32.0 µm s−1; ALH3.2 ± 1.7 µm; BCF 37.9 ± 10.9 Hz; STR 89.0 ± 16.0%; viability53.7 ± 5.1%; acrosome integrity 84%.

After insemination, progesterone concentrations for Female 1underwent a gradual increase and were elevated at Day 21(9.9 ng mL−1), the time in non-conceptive cycles when proges-terone concentrations are at baseline levels (0.1 to 1.0 ng mL−1)due to regression of the corpus luteum. Thereafter, proges-terone concentrations remained high (Day 29 to Day 463:21.1 ± 9.5 ng mL−1). Pregnancy was first confirmed using ultra-sound by detection of uterine fluid on Day 41 after insemination(Fig. 6a). A fetus was first visualised by ultrasound on Day 76(Fig. 6b). A female calf weighing 43.6 kg was born on 27 May2008 after 467 days of gestation (within the normal range for thespecies, 479 ± 20 days, Robeck et al. 2005a).

Discussion

There exists great variation in physiological mechanisms amongcetaceans and reproductive physiology is no exception. Resultsof the present study have shown that beluga spermatozoa col-lected from the male herein required a short-term preservationmethod that differs from those developed in other cetaceans.Ejaculates prepared and transported using this method were usedto achieve the first pregnancy after AI in this species; a culmi-nation of research on several facets of reproductive physiologyin male and female beluga.

The initial stages of semen collection training and spermevaluations produced clear evidence of the detrimental effectof saltwater and urine on in vitro sperm motility. Assessmentof ejaculate osmolality and pH were essential to establish nor-mal parameters for beluga semen and enable refinement of thecollection process. Contamination of semen with saltwater orurine led to exposure of spermatozoa to detrimental hyperos-motic conditions (osmolality of urine and salt water is typicallygreater than 1000 mOsm kg−1). Biochemical analysis of semi-nal plasma also allowed discrimination of contamination type.Contamination of semen with urine was typically only observedduring the initial stages of training but was important to detectso its occurrence could be extinguished from semen collectiontraining sessions. The optimised method incorporating rinsingor wiping of the penis tip (for removal of saltwater before ejacu-lation) and a modified artificial vagina (to avoid introduction ofsaltwater during or after ejaculation) facilitated the collection ofnon-contaminated ejaculates having physiologically normal bio-chemistry, and maximum in vitro sperm quality. This collectionmethod will be used in future studies on sperm cryopreservationand sperm sorting where it is imperative to avoid any degreeof damage due to cumulative stress on sperm membrane andfunction associated with these procedures (O’Brien and Robeck2006).

Captive and wild beluga exhibit reproductive seasonality,with females undergoing oestrous cycles during spring months(typically March–May, with slight geographical variation), coin-ciding with maximal testosterone production in males (Brodie1971; Heide-Jørgensen and Teilmann 1994; Robeck et al.2005a). Whilst a longitudinal study on adult males demonstratedthat testosterone always remained above 1 ng mL−1, therebyimplying that some degree of spermatogenesis could be sup-ported outside the breeding season (Robeck et al. 2005a), it wasnot known if mature spermatozoa could be produced through-out the entire year. In fact, histology results from two wildadult males in Greenland indicated that epididymal sperma-tozoa were completely absent during the non-breeding season(October; Heide-Jørgensen and Teilmann 1994). Results hereinclearly demonstrate that spermatozoa were produced during allmonths of the year, but production was seasonal with maximumsperm numbers coinciding with female oestrous cycles. Simi-lar to the previous report by Robeck et al. (2005a), testosteroneproduction by the male in the present study remained above1 ng mL−1 regardless of month, but exhibited seasonal varia-tion that could be correlated with changes in sperm production.Certainly, data from additional males, both captive and wild,are required to compare the seasonality of sperm productionin the two groups, although data from the latter would need tobe collected post-mortem unless adequate sedation and elec-troejaculation methods are developed. Whilst sperm productionvaried throughout the year in the captive male, sperm quality,as assessed by membrane viability and sperm morphology, wasunchanged. It is reasonable to recommend that semen collectiontraining sessions commence during late winter months (January–February) when testosterone production is maximal and sexualbehaviour is frequently observed.

Ejaculates from the beluga in the present study had reducedvolume and sperm concentration compared with other cetaceansstudied to date. Consequently, total sperm numbers per ejacu-late, using data from months having maximal sperm production(January–May; 995 ± 559 million spermatozoa per ejaculate),were ∼5 to 14-fold less for the beluga compared with the bot-tlenose dolphin (14 ± 8 billion spermatozoa per ejaculate, n = 7males; Robeck and O’Brien 2004; O’Brien and Robeck 2005),Pacific white-sided dolphin (5 ± 3 billion spermatozoa per ejac-ulate, n = 2 males; Robeck et al. 2003) and killer whale (5 ± 4billion spermatozoa per ejaculate, n = 4 males; T. R. Robeckand J. K. O’Brien, unpubl. data; Robeck and Monfort 2006). It ispossible that the comparatively low numbers of spermatozoa col-lected from the beluga in the present study are not representativeof the species. However, post-mortem samples from adult wildbeluga as part of Alaskan subsistence hunting practices, duringthe breeding season (April–May), contained only slightly greaternumbers of spermatozoa (1.6 ± 0.9 billion spermatozoa per maleafter flushing of caudae epididymides and vas deferens; n = 3males; T. R. Robeck and J. K. O’Brien, unpubl. data). Belugaexist in social groups of varying size which are often segregatedby sex and age (Ognetev 1981; Heide-Jørgensen and Teilmann1994). The beluga mating system is likely polygamous withapparent intense competition among males for breeding oppor-tunities, but the extent of sperm competition and other facets oftheir mating system are unknown (O’Corry-Crowe et al. 1997).

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 779

(a)

(b)

Fig. 6. Transabdominal ultrasonographic images (4 MHz convex transducer) of early pregnancy in the beluga(Female 1). (a) Day 41 after insemination, fluid (F) is visualised in the uterine horn; (b) Day 147 after inseminationshowing a fetus with a distinguishable skull (S), flipper (F) and tail flukes (T).

780 Reproduction, Fertility and Development J. K. O’Brien et al.

Across the cetacean species described in the previous para-graph, the percentage of spermatozoa with normal morphologyis typically greater than 80%, which is substantially higher thanthat for the beluga in the present study (∼60%), whose maindefect was a tapered head (representing ∼25% of all cells). Incattle, spermatozoa with tapered heads have similar motility tonormal forms, and are able to traverse the reproductive tract andreach the oviduct (Mitchell et al. 1985). Increased incidence oftapered heads in ejaculates of sub-fertile cattle (Blom 1973) andhumans (Rousso et al. 2002) indicates a potential genetic abnor-mality, but it is unknown whether beluga spermatozoa with atapered head represent a deleterious morphological form. Basedon the conception and successful pregnancy achieved after AI inthe present study, as well as the natural conceptions achievedby the male shortly after completion of the present study ata different SeaWorld facility (n = 3 calves born to date), theobserved frequency of spermatozoa with tapered heads does notappear to impact fertility. Clearly, ejaculates from additionalbeluga are required to establish a comprehensive descriptionof ejaculate parameters for this species. Objective assessmentof motility using CASA was useful for comparing treatmentsand the database developed thus far may be built upon in futurestudies using additional males. However, those studies wouldbe strengthened by examination of potential sperm subpopula-tions and use of appropriate non-parametric tests (e.g. Holt et al.2007), neither of which was performed in the present study.

Biochemical analysis of ejaculates revealed that fructose con-centrations are low compared with other species. This resultis not unexpected since beluga lack seminal vesicles, and thisaccessory gland is the main source of fructose found in ejac-ulates from numerous mammalian species. The concentrationof fructose in beluga ejaculates was similar to that determinedin the dog, and this species also lacks seminal vesicles (Mann1964; Anderson et al. 1979). The similarly-low concentrationsof glucose observed in beluga seminal plasma indicates thatsuch concentrations are adequate for sperm metabolic require-ments or that spermatozoa have an alternate source of energyin seminal plasma or female reproductive tract secretions. Indogs, the enzyme alkaline phosphatase, produced predominantlyin the cauda epididymis, is used to characterise ejaculationas being complete (combined concentration of alkaline phos-phatase in first and second fraction is >10 000 U L−1) orincomplete (combined concentration is <5000 U L−1; Kutzleret al. 2003). Preliminary data in dolphins indicate that alkalinephosphatase in seminal plasma is derived from the epididymis,since males producing ejaculates with low sperm concentrations(<10 million mL−1) have reduced alkaline phosphatase con-centrations (<1000 U L−1) compared with concentrated ejac-ulates (>100 million spermatozoa mL−1; >5000 U L−1) (J. K.O’Brien, unpubl. data). Concentrations of alkaline phosphatasein beluga seminal plasma exceeded the limit of the assay(30 000 U L−1) thereby suggesting that samples collected in thepresent study represent complete ejaculates. Determination ofthe source of seminal plasma alkaline phosphatase in ejacu-lates from beluga and other cetaceans is warranted since itmay be a useful parameter to include in comprehensive fertilityassessments.

Sperm head–head agglutination was observed in all ejac-ulates, regardless of whether they were contaminated withsaltwater or urine. Agglutination has been observed in dilutedboar semen and does not appear to influence fertility of freshejaculates (Bollwein et al. 2004).Agglutination is also a commonobservation during IVF in numerous species, and is indicativeof capacitated, or partially capacitated spermatozoa (Harayamaet al. 2000).Although these data indicate that agglutination is notdetrimental to in vitro sperm parameters, it is unknown whetheragglutinated beluga spermatozoa can migrate within the femalereproductive tract as efficiently as non-agglutinated spermato-zoa following insemination. The agglutination may be specificto the male of the present study, but it has been observed inbottlenose dolphin samples after systemic treatment with anti-fungal medications, or after direct exposure of sperm to urine orethanol (unpubl. data). During the present study the male wasnot receiving any medication, and sperm were not exposed toethanol (based on known laboratory quality control measures) orurine (based on creatinine analysis). Taken together, it was con-sidered pertinent to direct efforts towards developing a diluentthat inhibited sperm agglutination.

Initially it was hypothesised that the occurrence of agglutina-tion in the present study could be due to an effect of egg yolk, ashas been observed in goats, due to the production of toxic compo-nents following the reaction of seminal plasma enzymes and eggyolk (Ritar and Salamon 1982). However, reduced concentra-tions of egg yolk tested in preliminary trials did not decrease theincidence of agglutination, nor did egg yolk-free diluents (datanot shown). Ultimately, inclusion of HA in the storage diluentwas effective in preventing beluga sperm head–head agglutina-tion. All in vitro sperm parameters across time were unaffectedby HA (and degree of agglutination) with the exception of veloc-ity parameters during the first 6 h of liquid storage, which werereduced for HA-containing groups, probably owing to its highviscosity. This is in contrast to studies in humans on fresh sper-matozoa where HA actually improved sperm velocity (Huszaret al. 1990). However, the concentration of HA used in that studywas half of that used here. Interestingly, supplementation of pig-freezing diluents with HA at the same concentration used inthe present study also resulted in increased sperm velocity afterthawing (Pena et al. 2004). It should be noted that the manufac-turer of HA used in those studies differed from that in the presentstudy.

An explanation for the inhibitory effect of HA on agglutina-tion may reside in the actions of HA throughout the reproductivesystem. HA is a non-sulfated glycosaminoglycan (GAG) thatoccurs in various reproductive (and non-reproductive) tissuesand fluids. In the reproductive system of some livestock andlaboratory species, HA has been identified in cervical mucus,cumulus cell matrix (Eppig 1979), follicular fluid (Grimek et al.1984), oviducal fluid (Rodriguez-Martinez et al. 1998; Tien-thai et al. 2000), oviducal epithelium (Tienthai et al. 2000;Rodriguez-Martinez et al. 2001) and seminal fluid (Lee et al.1989). Evidence also suggests that spermatozoa possess HAreceptors (Kornovski et al. 1994; Ranganathan et al. 1994),and mature human spermatozoa bind to HA coated onto slidesor Petri dishes (Huszar et al. 2003). Mature HA-bound human

Beluga short-term sperm preservation and AI Reproduction, Fertility and Development 781

spermatozoa have reduced frequencies of chromosomal aneu-ploidies and are devoid of chromosome fragmentation, leadingto the use of HA-binding ability for selection of spermatozoasuitable for use in ICSI procedures (Jakab et al. 2005; Huszaret al. 2007). HA improved in vitro sperm velocity and longevitywhen added to fresh human semen (Huszar et al. 1990), andin vitro sperm motility and longevity when added to samplesafter thawing (Sbracia et al. 1997). Whilst there were no effectsof HA on percentage motility in the present study, an inhibitoryeffect on agglutination, as discussed, suggests that HA may mod-ulate sperm plasma membrane structure in the beluga. Pena et al.(2004) reported that frozen–thawed pig spermatozoa previouslyincubated with HA displayed merocyanine staining patternscharacteristic of non-capacitated cells, and hypothesised that HAmay delay or inhibit capacitation-like events. It remains to bedetermined if the binding of HA to a beluga sperm membranereceptor(s) also acts to block the same receptor(s) involved inmembrane changes leading to capacitation or agglutination.

Reactive oxygen species (ROS) are produced by metabolisingspermatozoa and accumulate during liquid storage.Antioxidantsare commonly included in domestic bull liquid storage dilu-ents to attenuate lipid membrane peroxidation (occurring underthe influence of ROS) and reduce the detrimental impact ofROS on sperm function (Shannon and Curson 1982; Vishwanathet al. 1994; Vishwanath and Shannon 1997). Inclusion of theantioxidants catalase and superoxide dismutase (reducers of thesuperoxide anion (Alvarez et al. 1987) and hydrogen peroxide(Shannon 1964), respectively) in ram diluents increases the fer-tile lifespan of sperm destined for use in IVF (Maxwell andStojanov 1996). In the present study, catalase and superoxidedismutase, supplemented at the same concentrations used in theformer sheep study, had no effect on beluga sperm motility ormembrane parameters. It is possible that the five-day storageperiod was too short to detect any differences, that antioxi-dant concentrations were not optimal, or that evaluation testswere not of adequate sensitivity to detect functional changesin spermatozoa. Regardless, the absence of a negative effectat the concentrations tested indicate it is reasonable to includethese antioxidants in beluga liquid storage diluents until furtherresearch into their effect on sperm function has been conducted.

Motility and membrane parameters were well-maintainedduring the five-day storage period at 5◦C, but the extent towhich fertility of beluga spermatozoa is maintained over timeis unknown. In domesticated species, fertility of liquid-storedsemen can typically be maintained for 2–5 days following col-lection (Vishwanath and Shannon 1997; Johnson et al. 2000;Salamon and Maxwell 2000; López-Gatius et al. 2005). Whilelarge-scale AI trials to determine the fertile lifespan of belugaspermatozoa are not possible, in vitro results of the present studysupport the use of beluga spermatozoa for AI within 24 h of col-lection when semen is stored in a specialised diluent at 5◦C.Certainly, the production of a calf using AI demonstrates thatit is feasible to transport beluga spermatozoa in chilled, liquidform to facilities within 15 h of the site of semen collection forsubsequent AI. The present study has provided new informa-tion on sperm biology, and methodologies for semen collection,short-term semen storage and AI. This knowledge represents an

important step towards cooperative management practices incor-porating ART and maintaining maximal genetic diversity withinthe captive beluga population.

AcknowledgementsAnimal care, animal training, curatorial and veterinary staff at SeaWorld Cal-ifornia andTexas are thanked for assistance with animal handling and samplecollection. Mrs Mitzi Synnott and Mrs Nicole Grovhoug are particularlythanked for the initial training which led to successful collection of belugasemen, and we are grateful to all Wild Arctic Staff for ongoing collectionsand assistance with AI procedures. Animal Care Laboratory staff (SeaWorldCalifornia) including Ms Whitney Coleman, Mrs LeighAnn Smith, Mrs PamThomas and Mrs Melinda Tucker are thanked for technical support, as is MsSherry Dickerson, Dr Steve Osborn and Mr Chris White (SeaWorld Texas)for sample collection and preparation. Dr Robert Anderson (Obstetrical andGynaecology Research, Rush Medical Center, Chicago) performed the sem-inal plasma fructose analyses, for which we are grateful. Mr Brad Andrewsis also thanked for invaluable support of this research. This project wasfunded by SeaWorld Corporation and is a SeaWorld Technical Contributionno. 2008–01-T.

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Manuscript received 17 February 2008, accepted 4 June 2008