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Theriogenology 74 (2010) 989–1001

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Deep intra-uterine artificial inseminations using cryopreservedspermatozoa in beluga (Delphinapterus leucas)

T.R. Robecka,*, K.J. Steinmana, G.A. Montanoa,b, E. Katsumatac, S. Osbornd,L. Daltond, J.L. Dunne, T. Schmittf, T. Reidarsonf, J.K. O’Briena,g

a SeaWorld and Busch Gardens Reproductive Research Center, SeaWorld Parks and Entertainment Corporation, San Diego, CA 92109, USAb Department of Animal Science, Texas A&M University, College Station, Texas 77840, USA

c Kamogawa SeaWorld, Kamogawa, Chiba 296-0041, Japand SeaWorld San Antonio, 10500 SeaWorld Drive, San Antonio, TX 78251, USA

e Mystic Aquarium and Institute for Exploration, Mystic, CT 06355 USAf SeaWorld San Diego, 500 SeaWorld Drive, San Diego, CA 92109, USA

g Faculty of Veterinary Science, University of Sydney, NSW 2006, Australia

Received 20 November 2009; received in revised form 24 April 2010; accepted 25 April 2010

bstract

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) haveeen successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawedemen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10nsemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean � SD)3.0 � 12.2, 38.4 � 8.8, 88.0 � 0.1, and 59.3 � 15.7%, respectively. A series of GnRH injections (3 x 250 �g, IV, 1.5 to 2 hpart) were used to induce ovulation, once a growing follicle �2.5 cm in diameter was visualized via trans-abdominalltrasonography. Artificial insemination was performed at 30.1 � 3.8 h post-initial GnRH injection with semen deposited in theterine horn, 92.6 � 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was locatedeyond a series of 5 to 10 vaginal rings, 44.8 � 9.3 cm from the external genital opening. The internal bifurcation of the uterusas 27 � 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 � 7.6 h post-AI. Two of 10 inseminations (20%) resulted inregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancys ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are importantxamples of how assisted reproductive technologies can provide tools for the global management of threatened species.

2010 Elsevier Inc. All rights reserved.

eywords: Artificial insemination; Conservation biology; Directional semen freezing; Genome resource banking; Ovulation induction

www.theriojournal.com

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. Introduction

Conservation of wild populations requires a mul-idiscipline approach aimed at describing both the

* Corresponding author: Tel.: �1 619 225 3177; Fax: �1 210 225178.

aE-mail address: [email protected] (T.R. Robeck).

093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.theriogenology.2010.04.028

opulation biology of a species and the reproductiveiology of their ex-situ representatives. Beluga areurrently listed as near-threatened by the Internationalnion for the Conservation of Nature (IUCN); how-

ver, the Cook Inlet sub-population is listed as criticallyndangered [1]. Ongoing environmental changes in therctic due to global warming have already increased
nthropogenic beluga habitat encroachment and may
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990 T.R. Robeck et al. / Theriogenology 74 (2010) 989–1001

ave adverse effects on food sources. Since these envi-onmental changes are not likely to subside, and untilurther population data can be collected, the IUCN cau-ions that beluga are a conservation-dependent species [1].

Currently, 37 beluga are collectively managed byeven facilities within North America [2]. Although therst successful zoologically born beluga occurred in981 [3], the fractured ex situ population has madestablishment of sustainable beluga populations prob-ematic. Whereas problems facing wild populations ofeluga differ from those in aquaria, it is clear thatithout sound management practices and increased re-

earch efforts, ex situ populations are also threatened.hus, collaborative multi-institutional management and

esearch projects have been conducted to improve theeproductive success of those members of this speciesn human care, while concurrently improving ournowledge of the reproductive physiology of the spe-ies in general [3–7]. These efforts have helped tomprove neonatal survival, and have identified inequal-ties in founder representation, with only half of thedult founders (10/18, 56%) having successfully repro-uced. Efforts to increase genetic representation of allounder animals are required for long-term viability ofhe population.

Once developed, assisted reproductive technologiesART) provide management tools, including long-termemen cryopreservation/storage and AI, that aid inaintaining genetic diversification of ex situ popula-

ions. Although rapid advances in applying these tech-ologies to other cetacean species have occurred8–13], variations in beluga reproductive biology andifficulties in storing spermatozoa have slowedrogress in this species [14,15]. Steinman et al [16]emonstrated with endocrinological and ultrasono-raphic evidence that beluga were facultative inducedvulators and then developed an ovulation inductionrotocol using GnRH. Anatomical information of theeluga reproductive tract, important for developing annsemination methodology, has only been describedrom post-mortem samples [17].

The objective of this research was to develop andmplement methodology for AI, utilizing frozen-hawed spermatozoa in the beluga. Initial attempts toevelop a genome resource bank with semen collectedrom wild animals harvested during native subsistenceunts was not successful due to the fragility of belugapermatozoa [15,18]. Once regular access to semenollected voluntarily from a trained captive beluga wasvailable, a short-term liquid storage method was de-
eloped, followed by the first successful AI with liquid- s
tored semen [14]. However, to effectively managelobal beluga populations and develop a genome re-ource bank, methods for sperm cryopreservation wereecessary. Through the use of a novel cryopreservationethodology, directional freezing, combined with a

nique extender-cryoprotectant combination, a methodhich produced satisfactory post-thaw in vitro sperm

haracteristics, was developed [15]. Demonstrating theertility of this frozen-thawed semen through the appli-ation of AI was the next logical step.

. Materials and methods

.1. Reagents and media

All chemicals were of analytical grade and cell cultureested, where possible, by the manufacturer. Unless oth-rwise stated, all media components were purchased fromigma-Aldrich (Sigma, St Louis, MO, USA) and wererepared with tissue culture grade water (Sigma or Milli-ore, Billerica, MA, USA). Diluents containing egg yolkfree range eggs) were prepared by ultracentrifugation for.5 h at 10,000 g. The supernatant was filtered (0.22 �m)nd frozen at �80 °C for a maximum of 18 mo.

.2. Animals

The seven females used in the AI trials (aged 6–30 y)ere housed at four institutions: Kamogawa Sea World

KSW: Kamogawa, Chiba, Japan); Mystic Aquariumnd Institute for Exploration (MA-IFE: Mystic, CT,SA); SeaWorld San Antonio (SWSA, San Antonio,X, USA); and SeaWorld San Diego (SWSD, Saniego, CA, USA; Table 1). The animals were housed invariety of indoor (KSW) or outdoor pools (SWSA,

WSD, MA-IFE), ranging in size from 258 to 1741 m3.or all AI trials, semen was collected from a wild born,roven adult beluga (aged 21 y in 2005, weighing 820g) from Feb 2005 to April 2009 (Table 1). The maleas housed at SWSA from February 2005 to January008, at which time he was moved to SWSD. The dietonsisted of frozen-thawed whole fish (herring, Clupeaarengus, and/or Columbia River smelt, Thaleichthysacificus) fed at approximately 4 to 5% of body weighter day.

.3. Endocrine monitoring

Voluntary urine samples were collected from two ofhe seven female beluga, as previously described forottlenose dolphins (Robeck et al 2005). Briefly, thenimals were trained to lie in dorsal recumbency in
hallow water (�1.5 m) with their flukes and peduncle

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991T.R. Robeck et al. / Theriogenology 74 (2010) 989–1001

eld at the water surface by a trainer who was standingn the pool. A second trainer would apply firm, steadyressure on the abdomen, directly over the urinaryladder. The animals would urinate in response to theressure and eventually became conditioned to urinateith only a slight touch in the same location. The urineas aspirated from the genital opening with a 20 mL

yringe or caught midstream directly into a sterile col-ection cup (118 mL, Fisher Scientific, Pittsburg, PA,SA). Urine samples (n � 281) were collected dailyntil 10 d after synchronization, then three times a dayuring the estimated peri-ovulatory period. Samplesere stored in duplicate at �80 oC pending analysis.on-extracted urine samples were analyzed for deter-ination of total immunoreactive concentrations of es-

rogen conjugates (EC) by a single antibody (estronelucuronide), direct enzyme immunoassay (EIA) asreviously described in beluga and other cetacean spe-ies [8–10,16]. Urinary EC concentrations were in-exed against creatinine (Cr) and expressed as ng/mgr. Creatinine concentrations were determined byethods previously described [16,19]. In addition, a

apid LH semi-quantitative canine LH (cLH) kit (Wit-ess® Synbiotics Corp. Kansas City, MO, USA), pre-iously validated for cetaceans [10], was used to detectrinary or serum LH in response to the ovulation in-uction protocol. Urinary LH profiles were obtainedsing a single antibody, direct EIA which allowed LHoncentration determination within 2.5 h [10,12,16].he EIA for LH was modified from the double antibody

able 1escription of belugas used and samples collected during the study.

nimal Facilitya Sex Birth date Body leng

KSW F 1987c 395

MA-IFE F 1981c 362SWSA F July 24, 2000d 328

SWSA F 1985c 337

SWSA F July 25, 1999d 315SWSD F 1984c 340SWSD F 1979c 350SWSA/SWSD

M 1984c 416

a MA-IFE: Mystic Aquarium and Institute for Exploration; SWSDSea World.

b Reproductive history prior to the start of the artificial inseminatic Estimated age of wild caught animals was based on length at cad Captive born.e P: serum progesterone; E2: serum estradiol; Ult: ultrasound eval

IA previously developed [20]. Intra-assay variation K

as �10% and inter-assay variation was 10.3 and0.9% at 30 and 60% binding (n � 24). Serial dilutionsf beluga urine yielded displacement curves that wereimilar to the standard curve (R2 � 0.99). The meanecovery of LH added to a pool of beluga urine was8.4 � 22.8% (y � 0.67x – 0.19, R2 � 0.99).

In one of the four animals (Female 1) not trained forrine collection, serum samples were collected daily orvery other day after detecting a large (�2.0 cm iniameter) follicle by trans-abdominal ultrasonography.erum was obtained after centrifugation at 1000 g for5 min and stored as 1 mL aliquots at �80 °C untilnalysis. Estradiol (E2) concentrations were deter-ined in Female 1 by a commercial laboratory (Ka-eda Medical Center, Kamogawa, Chiba, Japan) usingcommercially available E2 EIA kit (Cobias®, Rocheiagnostics, Indianapolis, IN, USA). The assay had a

ensitivity of 5 pg/mL and the E2 antibody major cross-eactivity was to estrone (51.5%), 17�-estradiol-3,17ulfate (41.1%), 17�-ethinyl-estradiol (30.9%), 17�-stradiol-17-valerate (29.4%), estriol (7.7%), 2-me-hoxy-estradiol (5.4%), and �5% for all others tested.amples for progesterone analysis were collected fromll animals after each AI (n � 7 animals; n � 10 AIs)n Day 0 (Day of insemination), then weekly until Day2 post-AI for pregnancy determination. Progesteroneoncentrations were determined using an immunof-ourescence assay (IFA) at a commercial laboratory asreviously validated for beluga ([3–5,7] SWSA,WSD, MA: Quest Diagnostics, Irving, TX, USA;

Weight (kg) Reproductivehistoryb

No. AIAttempts

Samplescollectede

865 Nulliparous 3 P, E2,Ult

535 Nulliparous 1 P, Ult580 1 abortion 2 EC, LH,

P, Ult619 2 calves 1 EC, LH,

P, Ult481 1 calf 1 P, Ult623 Nulliparous 1 P, Ult571 1 calf 1 P, Ult1004 Sired 4

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.4. Synchronization of estrus

For estrus synchronization, animals were eitheriven 0.044 mg/kg po of altrenogest (Regu-Mate®,ntervet Inc., Millsboro, DE, USA) or 0.05 mg/kg poedroxyprogesterone acetate (Provera®, Pfizer Inc.,okyo, Japan) once a day for 30 d. The drug wasdministered by either filling gel capsules then insertingr by directly injecting the hormone into the coelomicavity of a herring just prior to feeding.

.5. Ovarian ultrasonography

Trans-abdominal ultrasonography was used to helpefine the timing of ovulation post-GnRH inductionuring natural or following altrenogest-induced cycles,nd to confirm pregnancy as previously described forottlenose dolphins, Pacific white-sided dolphins, andiller whales [8–10,21]. Ultrasonographic examina-ions were performed using either a Sonosite Micro-axx (Kabushiki Kaisha Sonosite Japan, Tokyo, Ja-

an), Aloka 900 (MA, KSW: Corimetrics Medicalystems, Tokyo, Japan), or a GE Logiqbook™,SWSA, SWSD: GE LogiqGE Medical Systems, Mil-aukee, WI, USA) using a 3.5 MHz transducer (wide

ootprint convex linear transducer). Females were ex-mined once on Days 0 and 10 post-altrenogest, thenaily to thrice daily from Day 11 to detection of ovu-ation. Once a preovulatory follicle (POF) was ob-erved, ovaries were visualized ultrasonographically ateast once daily. Ovarian follicular diameter, follicularircumference, and the time and side of ovulation wereetermined as previously described [9].

.6. Semen collection

The male was trained for voluntary semen collectionver a 2 y interval using operant conditioning, as pre-iously described for beluga [10,14,22]. Briefly, thenimal received a variety of tactile stimuli to elicitoluntary extrusion of the penis from the genitalroove. Once a complete erection was obtained, theale was conditioned to ejaculate by manual stimula-

ion of the penis with a gloved hand (Nitrisoft, Nitrileowder-free examination glove; Sintex, Houston, TX,SA), lubricated sparingly with Pre-seed® lubricant

INGfertility™, Valleyford, WA, USA). Once manualtimulation commenced and the male subjectively ap-eared in a pre-ejaculatory state, the distal end of theenis was wiped with sterile cotton gauze, or rinsedith a HEPES-TALP medium, to remove residual salt-ater. The penis tip was then quickly directed into a
odified artificial vagina [14], and the ejaculate was H
ollected shortly after further manual stimulation of theenis into a WHIRL-PAK bag (2041 mL, NASCO, Forttkinson, WI, USA).

.7. Evaluation of raw semen

Raw ejaculates were held at room temperature (21C) and processed immediately after collection. Ejacu-ates were diluted 1:1 (v:v) with Beltsville extenderBF5F [23]) that had been modified for liquid storage ofeluga semen (52.3 mM TES, 16.5 mM Tris, 105.4 mMructose, 105.4 mM glucose, 20% v:v egg yolk, and 50g/mL gentamicin sulfate; 360 � 5 mOsm/kg and pH.0 � 0.1 [14]). The BF5F extender was supplementedith hyaluronic acid (HA; sodium hyaluronate,ioniche Animal Health, Bogart, GA, USA, final con-entration 1 mg/mL) to prevent sperm head-to-headgglutination [14].

Ejaculate concentration, volume, pH (pH indicatortrips; EM Science, Gibbstown, NJ, USA), osmolalityAdvanced Instruments Inc., Norwood, MA, USA), asell as viability (plasma membrane integrity), acroso-al status and morphology were determined as previ-

usly described for beluga [14]. Motility was evaluatedbjectively using computer assisted sperm analysisCASA, HTM-IVOS Version 12.2, Hamilton-Thorneiosciences, Beverly, MA, USA) as previously de-

cribed for beluga [14,15]. Briefly, five randomly se-ected microscopic fields were scanned to determineverage pathway velocity (VAP, �m/s) and straight-ine velocity (VSL, �m/s). The instrument settingsere 30 frames at a frame rate of 60 frames/s, mini-um contrast was 40, minimum cell size (pixels) was

; for progressive cells the VAP was �15 �m/s andTR (straightness of sperm movement) was �70%.permatozoa with a VAP �5 �m/s were consideredotile.Only ejaculates free of contamination with saltwater

r urine (osmolality �365 mOsm/kg) were used. Via-ility for fresh and post-thaw samples was analyzed byixing 10 �L of sample with 10 �L of a live-dead

xclusion stain (eosin-nigrosin; IMV TechnologiesSA, Maple Grove, MN, USA) for 30 s. A smear wasade and allowed to air dry for evaluation within 30in (100 spermatozoa per sample, 1000x). Spermato-

oa were classified as viable (no stain uptake) or non-iable (partial or complete stain uptake).

.8. Semen cryopreservation

Ejaculates (n � 42) were cryopreserved as previ-usly described [15]. Semen was diluted with BF5F �
A, cooled to 5 °C over 1.5 h (� �0.2 °C/min) and

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esuspended in the cryodiluent BF5F � 182 mM tre-alose (96 mM, final trehalose concentration) for a finaloncentration of 60 to 100 x 106 spermatozoa/mL.perm samples were transferred to 9 mL hollow tubesIMT International Ltd, Chester, UK) for cryopreserva-ion using a directional solidification (“directionalreezing”) machine (MTG-516, IMT International Ltd).he hollow tube was moved through the first block (5C) for 45 s at a constant velocity (3 mm/s) beforeeaching a distance of 2 mm into the opening of aecond block (�50 °C) and held for 60 s for initiationf seeding (rapid induction of ice nucleation from theeeding point throughout the length of the glass tube).he tube was then moved at 3 mm/s across the secondlock for 5 min before entering the collection chamber�100 to �110 °C), followed by immediate transfer toiquid nitrogen. Hollow tubes were thawed in air for0 s then transferred to a 35 °C water bath equippedith modifications to enable uniform sample thawingver 50 s (Harmony CryoCare Activator™, IMT Inter-ational Ltd). An aliquot (100 �L) of the sample wasemoved and diluted (1:1 over 5 min) with BF5F-HAwarmed to 35 oC) for post-thaw analysis of spermharacteristics, as described for raw ejaculates. Motilityarameters of post-thaw ejaculates were evaluated inemote locations without access to a CASA machinend thus, were subjectively analyzed for progressiveotility (PM) and kinetic rating (KR, 0–5 scale, 0 � no

orward movement, 5 � rapid forward progressiveovement). The remainder of the sample was stored at

1 oC until AI was performed.

.9. Ovulation induction and AI

A total of 10 AIs were performed with frozen-thawedemen in seven females from 2005 to 2009. Separatetudies indicated that beluga were facultative inducedvulators, indicating that an exogenous source of GnRHust be administered to ensure ovulation [14,16]. Conse-

uently, females with growing follicles that had reached areovulatory size (�2.5 cm in diameter) were givennRH (Cystorelin®, Merial, Duluth, GA, USA; 3 x 250g iv, q. 1.5 to 2 h) to induce ovulation. The inseminationas done 30 h after the first GnRH injection.One to 2 h prior to each procedure, all females were

re-medicated with valium (Diazepam, Abbott Lab,hicago, IL, USA; 0.1 to 0.2 mg/kg). The females were

emoved from the water and placed in lateral recum-ency on multiple 10 cm thick closed cell foam pads.he females were placed on their side, kept wet during

he procedure and vital signs were monitored through-
ut. According to previous anatomical studies, beluga c
ave a relatively long vagina (25 to 50 cm) that con-ains 10 to 15 circumferential vaginal folds or rings thatecome more pronounced as they approach the cervix17]. The length of the vagina and its folds, and theepth of the external cervical os (cEOS), uterine bifur-ation and semen placement, were all recorded. Semenas deposited using a 9 mm o.d., 200 cm long flexible

ndoscope (Olympus America, Melville, NY, USA)quipped with a custom-made catheter in the workinghannel (2.7 mm o.d. [7 Fr], 350 cm long, Smithsedical PM, Inc., Waukesha, WI, USA).

.10. Pregnancy diagnosis

Pregnancy diagnosis was accomplished on unre-trained animals by a combination of serum proges-erone monitoring on a weekly basis post-AI, andrans-abdominal ultrasonography (GE Logiqbook™)imonthly from Day 21 post-AI. Fetal measure-ents, biparietal and cross-sectional thorax at the

evel of the heart were determined as described forottlenose dolphins [24].

.11. Statistical analysis

All statistical analyses were performed using Sig-aStat (Version 3.5; SSPS, San Rafael, CA, USA).tudent’s t-tests were used to compare progesteroneoncentrations post-AI between pregnant and non-regnant animals. Data are expressed as means � SD.

. Results

.1. Semen collection and cryopreservation

Due to the relatively low ejaculate volume andperm concentration, a total of 42 ejaculates were re-uired for use during inseminations (Table 2). Theean ejaculate volume, sperm concentration and total

permatozoa were 1.6 � 0.9 mL, 361.0 � 128.9 x 106

permatozoa/mL, and 594.4 � 362.2 x 106 spermato-oa, respectively. Overall, post-thaw samples cryopre-erved in 91 mM trehalose using directional freezingetained 52.6, 95.1, and 67.4% of their raw progressiveotility, kinetic rating and viability, respectively.

.2. Estrus synchronization, ovulation induction andndocrinology

In all but one AI attempt (n � 9/10), estrus wasynchronized using Regu-mate® (n � 7) or Provera®n � 2). The animal (Female 6) which did not receiveprogestagen was artificially inseminated on a natural

ycle after a growing follicle was detected by routine

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ltrasonographic examination. All animals placed on arogestagen responded with follicular development.or nine of the 10 inseminations, animals were given

ntravenous GnRH in an attempt to induce ovulation at1.3 � 5.5 d post-progestagen withdrawal. Female 1as synchronized four times; twice with Provera® and

wice with Regu-mate®. For her first AI attempt, shepontaneously ovulated 18 d after Provera® with-rawal, before she could be artificially inseminated.or her second AI attempt, ovulation was inducedith GnRH at Day 12 following Provera® with-rawal. For the remaining two attempts using Regu-ate®, ovulation was induced with GnRH at 16 and

7 d post-Regu-mate®, respectively. All four AIsith Female 1 were unsuccessful. Female 3 beganer LH surge spontaneously 25 d post regumate®nd was inseminated 30 h post-LH surge initiation,ut she did not conceive.

In the nine (of 10) AI attempts when females wereiven GnRH, the test line on the cLH kit was negativecolorless) for serum LH immediately before the firstnjection. The cLH kit test line became positive (coloras similar or darker than the control line) just prior to

he second GnRH injection 1.5 to 2 h later. Ovulationccurred in eight of the nine induction attempts. Al-hough ovulation could be confirmed or denied vialtrasonography during all attempts within 24 h post-I, more precise timing of ovulation (to within a few h)

able 2haracteristics (means � SD) of beluga semen from Male 1 used fo

nd point Raw ej(n � 4

emen characteristicsVolume (mL) 1.6 �Osmolality (mOsm/kg) 364.4 �Sperm concentration (x 107/mL) 36.1 �Total spermatozoa per ejaculate (x 107) 59.4 �

perm characteristicsSubjective total motility (%, n � 26)c 83.8 �Objective total motility (%, n � 16)d 88.0 �Subjective progressive motility (%)c 73.0 �Objective progressive motility (%)d 71.0 �Kinetic rating (0-5)e 4.1 �VAP (�m/s, n �16) 85.8 �VSL (�m/s, n � 16) 104.0 �Viability (%) 88.0 �

AP: average pathway velocity; VSL: straight-line velocity.a Final volume of insemination dose.b Total number of spermatozoa per insemination.c Estimated by examining four to five fields of diluted (�25 x 106

d Determined by computer assisted sperm analysis.e Kinetic rating of spermatozoa graded subjectively: 0 � no move

as obtained in only four attempts within 12 h post-AI. w

or those attempts, ovulation occurred 36.6 � 2.6 hn � 4) post-initial GnRH injection. The mean diameternd circumference of the largest follicle at the time ofnRH injection from the eight animals that ovulatedost induction was 2.8 � 0.6 cm and 8.3 � 1.8 cm,espectively. In the one instance where ovulation failedo occur within 24 h post-GnRH administration (Fe-ale 4), the ovulation induction protocol was errone-

usly administered when her follicle was only 1.78 cmn diameter (5.49 cm in circumference). Trans-abdom-nal ultrasonographic images obtained before the GnRHnjections were inaccurate and it was falsely believedhat the POF was at least 2.5 cm in diameter; it wasnly during the ultrasnographic examination just prioro the AI that an accurate measurement obtained. Sec-ndary follicles, 2.2 � 0.5 cm in diameter, were ob-erved in 7 of 10 inseminations. Five of seven second-ry follicles ovulated within 12 h of the primaryollicles. The primary follicle was located on the leftvary 50% of the time, whereas the secondary folliclesere located on the contra-lateral ovary to the primary

ollicle 57% of the time.In the three AI attempts with the two animals that

ould be trained for urine collection (Females 3–4),eak urinary EC values were 83.8 � 9.1 ng/mg cr (n �; Fig. 1). Peak EC at time of GnRH injection (n � 2),eak LH for natural (n � 1) and induced cycles (n � 1)

ial insemination.

Pre-freeze Post-thaw

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g/mg cr, respectively. Peak estrogens (urinary EC orerum E2) occurred 18 � 15.9 h (n � 4) prior to GnRHnjection. For the one animal with a natural cycle, peakC (93.7 ng/mg cr) occurred 12 h prior to peak LH. Forll animals injected with GnRH (n � 9), the cLH kit

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ig. 1. Hormone profiles of two beluga during AI attempts afteremale 1) AI. Top graph shows urinary LH and EC (estrone conjurge in response to GnRH administration. Also note the continualas serum estrogen (E2) and progesterone profiles. Note that thomposite serum progesterone profiles (box in upper left of lowend non-conceptive (n � 8) AIs. Means from Days 14 and 21 werack of samples from conceptive groups).

est lines were negative at the time of the first injection, w

nd strongly positive just prior to the second and thirdnjections. Progesterone concentrations at the time ofhe first GnRH injection and at the time of the AI were.34 � 0.18 ng/mL and 1.41 � 1.03 ng/mL, respec-ively (Fig. 1). For the attempt when the female (4)

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ssful (top graph; Female 3) and a non-successful (lower graph;and serum progesterone profiles. Note the occurrence of the LHted serum progesterone following the LH surge. The lower graph

progesterone post-AI did not remain elevated. Also note the) comparing weekly progesterone post-AI in conceptive (n � 3)rent (*; P � 0.05) between groups (Day 28 was not tested due to

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ping a POF of at least 2.5 cm, peak EC concentra-ion was 24.3 ng/mg cr. Retrospectively, this loweralue was compatible with the smaller POF size1.78 cm in diameter). In Female 1, serum E2 con-entrations peaked at 72.0 pg/mL prior to spontane-usly ovulating a 2.9 cm diameter follicle in March008. These values were subsequently used to help de-

ermine the appropriate timing for three AI attempts. Dur-ng these attempts, her mean POF diameter and peak E2oncentrations were 3.4 � 0.7 cm and 111.9 � 40.4g/mL, respectively (n � 3; Fig. 1).

.3. Artificial insemination and pregnancy monitoring

The number of progressively motile spermatozoaPMS) that were used for the inseminations rangedrom 285 x 106 to 3291 x 106. The two conceptionsccurred after insemination of 525 x 106 spermatozoaMED, minimum effective dose) and 3291 x 106 PMS.he first two inseminations were below the MED (301106 and 285 x 106 PMS, respectively), with the

emaining AIs utilizing numbers which were 1.5- to

ig. 2. Beluga hysteroscopic anatomical characteristics. A: Smooth loo circumferential folds (CF) of the mid-vagina; C: CF in the mid-vs located centrally.

.2-fold higher than the MED. e

The first insemination attempt (Female 7) occurredt an estimated 3 h after ovulation. When time ofvulation could be determined, the remaining insemi-ations occurred 8.5 � 7.6 h prior to ovulation. Theistance from the genital opening to the cEOS, thenternal uterine bifurcation, and final semen depositionas 44.8 � 9.3, 69.1 � 13.2, and 92.6 � 16.2 cm,

espectively. The prominent vaginal rings, estimatedrom five to seven in total, presented an initial imped-ment, particularly when the vagina was not sufficientlynsufflated. After becoming familiar with beluga vagi-al and cervical anatomy through multiple endoscopicxams, subsequent attempts to reach the cervix becameonsiderably easier (Fig. 2). After identifying the cer-ix, the scope was aligned with the transverse cervicalolds to help identify the cEOS, and then passed intohe uterus (Fig. 3). During AI procedures, the internalifurcation of the uterus had to be identified by slowlyetracting the endoscope until the opening of the secondorn was seen. Using this method, the bifurcation wasdentified in eight of the last nine attempts. During the

nal folds of the caudal vagina; B: Transition from longitudinal foldsontinuing cranially; D: Distal vaginal CF with the external cervical

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nly be passed just beyond the internal cervical Os andhus the uterine bifurcation was not identified.

During the course of developing this inseminationrocedure, it was hypothesized that when the animal iseld out of the water in lateral recumbency, gravita-ional effects on the body mass likely compress theownside uterine horn. Therefore, the horn with theeast compression was the first to be insufflated and,ost likely, was the first one entered after passing

eyond the cervix. With the exception of the first AIrocedure, the animal was placed in lateral recum-ency, with the side ipsilateral to the ovary containinghe POF positioned dorsally. Once the endoscope wasassed as deep in the uterine horn as visually possible,he catheter was advanced until slight resistance waset (Fig. 3). At that point, semen was slowly intro-

uced into the uterine horn, followed by 5-20 mL ofF5F to rinse residual spermatozoa from the catheter.he air source was discontinued once the insemination

ig. 3. Beluga hysteroscopic anatomical characteristics during deeprrow). Note the longitudinal folds of the cervix. (B) Entry of the enight quadrant (white arrow head). (C) uterine body and the beginninip of the insemination catheter (white tube) at the presumptive uter

ad begun, to minimize air accumulation in the uterus, u

nd to allow for air to be expelled during normal uterineontractions. After AI, semen was visualized to haveooled in the uterine horn, and the endoscope waslowly removed.

Of the ten AI attempts, two resulted in pregnancyFemales 3 and 5). Frozen-thawed semen was depositedn one uterine horn for seven inseminations, and in bothorns for the remaining three. Of the two animals thatecame pregnant, one had semen deposited in bothorns (Female 5) and the other in one horn (Female 3).he total volume of fluid (frozen-thawed semen andF5F from flushing of the catheter) introduced into theterus across all AIs was 45 � 18 mL. The timeequired to complete the inseminations (time from en-oscope introduction to withdrawal) was 14 � 3 min.he weekly post-AI progesterone samples revealed aormal luteal rise in progesterone of 4.4 � 3.0 ng/mLy Day 14. The progesterone profiles to Day 30 post-AIrom three conceptive AIs (two from the present study

terine AI. (A) Entry of the endoscope in the cervical lumen (whitein the cervical lumen with the opening into the uterus in the upper

e uterine horn (white arrow). (D) Apex of the uterine horn, with thejunction.

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tudy [14] using liquid-stored semen) were compared toight non-conceptive AIs. Progesterone concentrationsrom non-pregnant animals diverged (P � 0.05) fromregnant animals after Day 14 post-AI (Fig. 1). Therst pregnancy (Female 5) resulted in twin calves beingorn at 442 d post-AI. Only the larger of the twin calvesas alive at birth and is currently growing normally

Fig. 4). For the second pregnancy (Female 3), fetalross-sectional thorax and biparietal measurementsere 5.3 cm and 4.0 cm by Day 156 of gestation. Thisregnancy is ongoing and parturition is anticipated inuly 2010.

. Discussion

The production of beluga calves reported hereinsing assisted reproductive technology is a milestonerom 16 y of research on reproductive biology of thispecies. An important discovery during this researchas that unlike other odontocetes examined thus far,eluga are facultatively induced ovulators [16]. Con-equently, intensive endocrine monitoring and ovar-an ultrasonographic assessments were necessary toevise a protocol for ovulation induction, therebynabling determination of ovulation timing and de-elopment of AI.

Research in beluga sperm biology revealed another

ig. 4. The first successful beluga born from artificial inseminationhite Whale and Dolphin Staff (SWSA).

spect of the species’ divergent reproductive physiol- m

gy compared to other odontocetes. Since beluga sper-atozoa were shown to be highly susceptible to dam-

ge during conventional freeze-thaw processes [15], aovel trehalose-based cryodiluent, combined with di-ectional freezing technology, was used to achieveost-thaw sperm characteristics satisfactory for AI. Noturprisingly, using this cryopreservation method, ouremen freezing efforts in the present study producedimilar post-thaw results (38% progressive motility) tohose previously reported [15]. Demonstration of theertility of cryopreserved spermatozoa provided justifi-ation for the development of a beluga gamete resourceank which will allow for global exchange of belugapermatozoa in conjunction with AI. In addition, ex situI programs may now be expanded to include sperma-

ozoa collected post-mortem from incidental deaths ofaptive animals, or from wild animals killed duringubsistence hunting in the arctic.

In contrast to bottlenose dolphins [9,21], Pacifichite-sided dolphins [10], killer whales [8], and indo-acific humpback dolphins [25] where ovulation occursn the left ovary greater than 70% of the time, based onhe present study, beluga ovulate equally (50%) fromach ovary. The observed lack of ovulation bias ineluga ovaries was consistent with post-mortem find-ngs in wild beluga, where there is equal distribution oforpora lutea on both ovaries [17]. Furthermore, ani-

zen-thawed semen (SeaWorld San Antonio, 2009). Photo Credit to
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als in the present study exhibited double ovulations

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0% of the time, a unique finding which has not beenbserved in other odontocete species. Post-mortem re-orts of wild beluga demonstrate that animals fre-uently possess two CLs during early pregnancy, whichhen combined with our results, provided conclusive

vidence for the occurrence of double ovulations in thispecies [17].

Estrus synchronization in the beluga using Regu-ate® has not been previously reported. The period

f time from withdrawing Regu-mate® to follicularecruitment and POF development (21 d) was similaro other odontocetes [8 –10]. Additionally this washe first report on the use of Provera® as a substituteor Regu-mate® for estrus synchronization in anydontocete, which produced similar results with bothompounds in the same animal on different occa-ions.

All cetaceans appear to have unique vaginal andervical folds that probably function to inhibit saltater contamination during copulation [26 –27].eluga have been described as having a series of0 –15 vaginal folds, beginning midway in the vaginand moving cranially toward the cervix [17]. In theurrent study, adequate vaginal insufflation was es-ential to allow passage of the endoscope beyond theaginal folds to the cervical opening. Due to themall and constricted vaginal and rectal openings inetaceans [28], IU placement of the endoscope andatheter could not be verified by palpation or ultra-onography. However, the lengths documentederein for the vagina, cervix and internal uterineifurcation seemed consistent with post-mortem re-roductive tract descriptions by Kleineberg et al17]. These post-mortem data, which described aean total reproductive tract length of 80 cm, and

he mean insemination depth of 93 cm during IUnsemination attempts conducted in the presenttudy, substantiated our belief that semen was depos-ted in the proximal uterine horn.

Despite the use of a deep IU insemination method,he conception rate for the beluga was poor (20%) asompared with killer whales (40%; [8]), bottlenoseolphins (60%; [9]) and Pacific white-sided dolphins50%; [10]). Deep IU insemination has been used suc-essfully in other species to reduce the inseminationose [29–33], increase fertility [34,35], and in somepecies increase the success of AI using semen fromubfertile males [36]. In an attempt to optimize theonception rate in beluga, deep IU insemination usingigh numbers of spermatozoa was employed. Despite
umerous inseminations (n � 7), where the total num- t
er of progressively motile spermatozoa was 1.3 to 6.3imes the minimum effective insemination dose of 524illion spermatozoa, and that inseminations occurredithin 12 h of ovulation, only one additional concep-

ion was achieved. A possible explanation for the lowonception rate relates to the fact that the majority7/10) of inseminations were unicornual, presumably inhe horn ipsilateral to the ovary destined to ovulate.nitially, we hypothesized that even if spermatozoaere deposited into the contralateral horn of the ovu-

ation, sperm migration to the opposite horn wouldccur, as it does in other species [37–40]. Since theterine side of sperm deposition can not be confirmeduring AI in beluga, it is possible that some insemina-ions were performed in the uterine horn contralateral tohe side with the POF. The observed low pregnancyate under high sperm doses might therefore be reflec-ive of a reduced ability of frozen-thawed beluga sper-atozoa to undergo uterine migration and that it is

ikely that some inseminations were performed in theterine horn contralateral to the side with the POF. Thismpediment could be overcome in future AI proceduresy performing bilateral, low dose, deep IU insemina-ions.

The progesterone profiles during the first 30 d post-nsemination from three conceptive cycles are the firsteported from beluga with known conception dates. Inhese profiles, there was a clear divergence of serumrogesterone after Day 14 post-AI; this was due to theemise of the CL in non-pregnant animals. In otherpecies, the demise of the CL, as reflected by a declinen circulating progesterone concentrations, typically oc-urred during a 2 to 3 d window if a conceptus is notresent [41]. If this relationship holds true for theeluga, maternal recognition of pregnancy probablyccurs, by a yet to be determined mechanism, betweenays 14 and 21 post-ovulation.Through the application of previous research in

perm biology and advances in the endoscopic insem-nation technique, this study demonstrated the first usef cryopreserved beluga semen for AI. Such findingsncourage the development of gamete resource banksnd validate the use of the ovulation induction protocolreviously developed for this species. With climatehange in the arctic moving at increasing rates, futureffects on beluga populations cannot be predicted [42];owever, application of the information gained by theresent research may eventually prove to be an impor-ant part of the overall effort to maintain healthy, sus-
ainable populations of this species.

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cknowledgements

We thank the animal care laboratory, animal trainingnd animal care staff at SeaWorld San AntonioSWSA), SeaWorld San Diego (SWSD), Kamogawaea World (KSW), and Mystic Aquarium and Instituteor Exploration (MA-IFE) for their support. For tech-ical assistance, we thank Michelle MorrisseauSWSD). For assistance with ultrasonographic exams,e thank former MA-IFE veterinary intern Dr. Mich-

lle Davis. For semen collection and processing, wehank Sherry Dickerson and Chris White (SWSA), and

ild Arctic staff, in particular Mitzi Synnott andicole Grovhoug (SWSD). We thank Dr. Hideki Koi

Kameda Medical Center) for serum E2 analysis. Wehank Brad Andrews (SeaWorld Parks and Entertain-ent Corporation, SEA) and Kazutoshi Arai (KSW) for

heir support of this project. This research was con-ucted under the NMFS permit number 116-1691, wasunded by SEA, KSW and MA-IFE, and is a SeaWorldechnical contribution number 2009-05-T.

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