www.theriojournal.com

Theriogenology 68 (2007) 93–99

Preliminary assessment of reproductive technologies in

wood bison (Bison bison athabascae): Implications

for preserving genetic diversity

J. Thundathil a,*, D. Whiteside a,b, B. Shea c, D. Ludbrook d, B. Elkin a,e, J. Nishi f

a Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada T2N 4N1b Calgary Zoo Animal Health Centre, Calgary, AB, Canada

c Alta Embryo Group, Calgary, AB, Canadad Bear Creek Animal Clinic, Grande Prairie, AB, Canada

e Wildlife Division, Government of Northwest Territories, Environment & Natural Resources, Yellowknife, NT, Canadaf Government of Northwest Territories, Environment & Natural Resources, Fort Smith, NT, Canada

Received 2 August 2006; accepted 5 April 2007

Abstract

Since the high prevalence of bovine tuberculosis and brucellosis in free-ranging wood bison in the Canadian north poses a threat

to nearby healthy bison populations, commercial bison and cattle ranches, and potentially to humans, there is considerable impetus

to salvage the genetics of infected bison and maintain a disease-free herd. In that regard, there is a great need to develop appropriate

reproductive technologies. Therefore, the objective of this study was to develop protocols to produce and cryopreserve wood bison

embryos (based on protocols used for cattle). Cumulus oocyte complexes (COC) aspirated from ovaries recovered after slaughter

were matured in vitro, and fertilized with either frozen-thawed semen or chilled epididymal spermatozoa. Although both sources of

spermatozoa resulted in acceptable rates of fertilization (64.4%, n = 45; 89.2%, n = 28, respectively) and cleavage (75.0%, n = 40;

92.5%, n = 40), production of morulae (7.5%, n = 40; 25.0%, n = 40) and blastocysts (7.5%, n = 40; 10.0%, n = 40) was low.

Morulae- and blastocyst-stage embryos were frozen-stored by vitrification. To our knowledge, this is the first report regarding the in

vitro production and cryopreservation of bison embryos for genetic recovery of diseased wood bison. These techniques have

substantial potential for conserving and managing the genetic diversity of wild bison, and may also have important management

implications for genetic salvage of diseased bison populations in North America.

# 2007 Elsevier Inc. All rights reserved.

Keywords: Wood bison; Spermatozoa; Embryo; Cryopreservation; Genetic diversity

1. Introduction

Conservation of genetic diversity is a keystone issue in

management of rare, threatened, and endangered wildlife

* Corresponding author at: Faculty of Veterinary Medicine, G380,

3330 Hospital Dr., NW University of Calgary, Calgary, AB, Canada

T2N 4N1. Tel.: +1 403 220 8244; fax: +1 403 210 3939.

E-mail address: jthundat@ucalgary.ca (J. Thundathil).

0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2007.04.020

[1–3]. Wood bison (Bison bison athabascae), a

subspecies of the American bison [4] and listed under

Appendix II of the Convention on International Trade in

Endangered Species of Wild Flora and Fauna (CITES),

are considered an endangered subspecies under the

United States Endangered Species Act. In Canada, wood

bison are classified as threatened [5]. One of the most

important factors complicating the preservation of wood

bison in northern Canada is the high prevalence of bovine

tuberculosis (Mycobacterium bovis) and brucellosis in

J. Thundathil et al. / Theriogenology 68 (2007) 93–9994

bison in and around Wood Buffalo National Park [5,6].

Furthermore, these diseases in free-ranging wood bison

also present a risk of disease transmission to nearby

healthy bison populations in the Northwest Territories

and northwestern Alberta, commercial bison and cattle

ranches in northern Alberta, and potentially to humans

who may hunt and consume infected animals [7,8]. As

recovery of wood bison is intrinsically tied to manage-

ment of the diseased populations, salvage efforts are

essential for conserving representative genetic material

[9,10], if diseased populations are to be removed from the

metapopulation [11].

Assisted reproductive technologies are critical tools

in genetic conservation. In addition to preserving the

genetics and extending the reproductive potential of

individual animals, assisted reproductive technologies

are extremely valuable for conservation of diseased

wildlife populations. For example, although control

measures for infectious zoonotic pathogens (e.g.

Brucella abortus) often require isolation and euthanasia

of the infected host, assisted reproductive technologies

may be the only practical method to preserve and utilize

germplasm from infected wildlife [12,13]. General

information regarding reproductive physiology and

estrous synchronization of bison has been published

[14–16]. Bison calves have been produced by super-

ovulation of infected bison donor cows, followed by AI

and embryo transfer without transmitting B. abortus to

recipient cows or calves [17], suggesting that assisted

reproductive technologies are an effective means of

salvaging the genetics of diseased bison. However, there

are no published data regarding in vitro production and

cryopreservation of bison embryos for genetic salvage.

The objective of this study was to evaluate the potential

of assisted reproductive technologies for the production

and cryopreservation of wood bison embryos, with the

ultimate objective of maintaining a disease-free wood

bison herd and preservation of their genetic diversity.

2. Materials and methods

Unless otherwise stated, all chemicals used were

purchased from Sigma–Aldrich, Oakville, ON, Canada.

Confirmation of tuberculosis in the Hook Lake Wood

Bison Recovery Project [18] resulted in depopulation of

the entire conservation herd in Spring 2006. A total of

30 bison cows (aged 4–10 years) were slaughtered at the

Agriculture and Agri-Food Canada Research Centre,

Lacombe, AB, Canada. Of these cows, 27 were

pregnant (estimated stage of gestation: 1–6 months).

Ovaries were collected approximately 30 min after

death and transported to the laboratory in a vacuum

flask. Sterile physiological saline (35 8C) was used both

as a transport medium and for washing the ovaries after

arrival. Thirty-eight ovaries had visible antral follicles;

a 5 mL syringe and 21.5 gauge needle were used to

aspirate antral follicles (2–6 mm in diameter). The

follicular fluid was examined under a stereomicroscope

(�600); cumulus oocyte complexes (COC) were

selected for in vitro maturation (IVM) if they had at

least two layers of a compact cumulus cell mass.

2.1. IVM

The selected COC were matured in vitro as described

below. Initially, they were washed (three times) in

Tissue Culture Medium 199 (with HEPES) supple-

mented with 50 mg/mL gentamicin and 3 mg/mL of

fatty acid free BSA. Maturation medium was prepared

by supplementing Tissue Culture Medium-199 with

25 mM sodium pyruvate, 25 mg/mL gentamicin,

0.5 mg/mL FSH, 5 mg/mL LH, 2 mg/mL estradiol-

17ß, and 10% fetal calf serum (Hyclone, Logan, UT,

USA). Maturation medium was transferred to the wells

(500 mL/well) of a multiwell dish, covered with

embryo-tested mineral oil, and equilibrated for 4 h

(in an atmosphere of 5% CO2 in air with 95% humidity

at 39 8C). Twenty-five COC were transferred to each

well and left in the incubator for 24 h to mature.

2.2. Evaluation of the maturation status of oocytes

After 24 h, a group of COC from each replicate were

examined to assess expansion of cumulus cells; these

cells were removed (denuded) by incubating the COC

with 1% hyaluronidase in phosphate-buffered saline

(PBS) for 2 min and vortexing for 1 min. After

denuding, the oocytes were examined and those with

the first polar body expelled were considered mature.

2.3. Preparation of spermatozoa for IVF

We used frozen-thawed semen (from two bison

bulls) and chilled epididymal spermatozoa (from two

other bulls) for IVF. Fresh ejaculates of bison semen

were cryopreserved using a standard protocol for bull

semen. A single ejaculate was collected (manual and

electro-stimulation) from each bull, extended in a Tris-

glycerol-egg yolk extender at 37 8C (sperm concentra-

tion �100–150 � 106/mL), cooled to 4 8C (minimum

of 4 h), and loaded into 0.5 mL straws (at 4 8C). The

straws were held horizontally �10 cm above liquid

nitrogen (in a styrofoam cooler) for 15 min and then

plunged into liquid nitrogen.

J. Thundathil et al. / Theriogenology 68 (2007) 93–99 95

Epididymal spermatozoa were recovered from two

adult bulls within 30 min after death. The epididymides

were dissected, transferred to physiological saline

(36 8C), and minced with scissors, releasing the sperm

into either physiological saline or Plasmalyte pH 7.4

(Baxter Corporation, Toronto, ON, Canada) held at

36 8C. The resultant sperm suspension was strained

through sterile gauze into sterile 15-mL conical tubes,

centrifuged at 1200 � g for 3 min, and the supernatant

was removed. The sperm pellet was extended with a

Tris-glycerol-egg yolk extender (20 � 106/mL) and

kept at 36 8C for 20 min, and preserved at 4 8C for 24 h

before IVF.

2.4. IVF

A stock medium of FERT-TALP [19], supplemented

with 6 mg/mL fatty acid-free BSA, 50 mg/mL genta-

micin, and 25 mM sodium pyruvate, 20 mM penicilla-

mine, 10 mM hipotaurine, 1 mM epinephrine, and 5 mg/

mL heparin, was used for fertilization. Fertilization

medium was transferred to the wells of a multiwell dish

(500 mL/well), covered with embryo-tested mineral oil,

and equilibrated in an atmosphere of 5% CO2 in air with

95% humidity at 39 8C for 4 h.

One hour prior to IVF, frozen semen samples were

thawed for 1 min in a water bath (35 8C). An aliquot

(0.5 mL) of either frozen-thawed semen or chilled

epididymal spermatozoa was transferred to a 1.5 mL

microcentrifugation tube, centrifuged at 500 � g for

5 min, the supernatant removed, and 0.5 mL of

fertilization medium layered over the sperm pellet.

The centrifugation tube was placed (with lid open) in

the incubator (5% CO2 in air with 95% humidity at

39 8C) for 1 h. Thereafter, the upper two-thirds of the

FERT-TALP medium (essentially all sperm in this

portion of the tube were motile) was removed and used

for IVF. A sperm suspension was added to each well,

which contained 10–15 oocytes (sperm concentration

1 � 106sperm/mL).

2.5. Evaluation of fertilization status

Following co-incubation with spermatozoa, a batch

of oocytes from each replicate was processed and

examined for fertilization status (formation of pronu-

clei). After 20 h of co-incubation with spermatozoa, the

COC were placed in the refrigerator (4 8C) and

subsequently denuded (as described for the evaluation

of maturation status of oocytes). Oocytes were placed

between two polylysine-coated glass slides with a small

drop of TCM-199 (with HEPES) and gently flattened,

fixed in a solution of glacial acetic acid and absolute

ethyl alcohol (1:3 ratio) for 24 h, and stained by placing

small droplets of aceto-orcein (1% orcein in glacial

acetic acid) at the corners of the cover glass. Oocytes

were examined microscopically (�400) and were

considered fertilized if they had two pronuclei.

2.6. Embryo development

After 24 h of co-incubation with spermatozoa, the

COC were removed from the fertilization medium and

cumulus cells were denuded by vortexing for 1 min.

Presumptive zygotes were cultured (20 in each 500 mL

drop) in synthetic oviduct fluid (SOF), comprised of

107.7 mM NaCl, 7.16 mM KCl, 1.19 mM KH2PO4,

25.06 mM NaHCO3, 0.3 mM sodium pyruvate, 2.5 mM

sodium lactate 60% syrup, 1 mM glutamine, 3 mg/mL

BSA, 250 mg/mL gentamicin, 1X BME essential amino

acids, and 1X MEM non-essential amino acids. The

SOF medium was transferred to the wells of a multiwell

dish (volume 500 mL [20]), covered with embryo-tested

mineral oil, and equilibrated at 39 8C and 5% CO2 in a

humidified atmosphere (95%) for at least 4 h. After co-

incubation with spermatozoa for 18–24 h, cumulus cells

were denuded by vortexing for 1 min and the

presumptive zygotes were transferred to SOF medium

and cultured at 39 8C and 5% CO2 in a humidified

atmosphere (95%) for 8 days (fertilization = Day 0).

Embryo development was assessed daily on Days 2–8.

2.7. Vitrification of embryos

On Day 8, a vitrification procedure originally

developed for bovine embryos [21] was modified and

used for the cryopreservation of morulae- and

blastocyst-stage embryos. Briefly, a metal straw holding

rack (7 cm high) was placed in a styrofoam box that was

filled with liquid nitrogen (surface �2.5 cm below the

straw-holding surface of the rack) and allowed to cool

for at least 10 min. A buffer solution (TL-HEPES;

Cambrex, Baltimore, MD, USA) was used as the

holding medium for embryos. A 3.5 M ethylene glycol

solution was prepared by mixing 1 mL of ethylene

glycol with 4 mL of holding medium. The vitrification

solution contained 7 M ethylene glycol, 0.5 M galac-

tose, and 18% Ficoll. Holding medium, 3.5 M solution

of ethylene glycol in holding medium, and vitrification

solution were transferred (500 mL aliquots) to the wells

of a multiwell dish. For loading straws, an embryo was

transferred from holding medium to 3.5 M ethylene

glycol and allowed to equilibrate for 2 min; the embryo

was then transferred to the vitrification solution and

J. Thundathil et al. / Theriogenology 68 (2007) 93–9996

loaded in a plastic straw (0.25 mL) within 30 s. The

straw was quickly plugged, left on the straw holding

rack for 5 min, and then plunged into liquid nitrogen.

2.8. Statistical analysis

Although the objective was to evaluate the potential

of assisted reproductive technologies for the production

and cryopreservation of wood bison embryos, the

frozen-thawed semen resulted in a poor cleavage rate in

Replicate 1. In Replicate 2, we used frozen-thawed

semen from a different bull and epididymal sperma-

tozoa that had been recovered (immediately after death)

from two other bulls and chilled for 24 h. Therefore,

Fig. 1. (A) Bison ovary with antral follicles; (B) cumulus oocyte comp

surrounded by several layers of cumulus cells and the ooplasm was darker

cumulus cell mass; (D) cumulus dissociated oocytes prepared for evaluation

Day 3 of culture (Day 0 = day of fertilization); and (F) morulae and blasto

only data from Replicate 2 were included in a Chi-

square analysis for determining the effects of the source

of spermatozoa (frozen-thawed vs. chilled epididymal

spermatozoa) on embryo development. Unfortunately,

the lack of access to additional ovaries prevented further

replication.

3. Results

3.1. Oocyte collection and in vitro maturation

A total of 307 COC were collected from 38 ovaries

(Fig. 1A and B). These COC were similar to those from

cattle, except that nearly all appeared dark, had cumulus

lexes recovered from abbatoir-derived bison ovaries. Oocytes were

than bovine oocytes; (C) cumulus oocyte complexes with expanded

of the maturation status of oocytes; (E) embryos (four to eight cell) on

cysts on Day 8 of embryo culture.

J. Thundathil et al. / Theriogenology 68 (2007) 93–99 97

Table 1

In vitro development of IVP bison embryos according to semen source

Replicate No. of oocytes cultured Bull Semen Cleavage on Day 2a (%) Morulae on Day 8 (%) Blastocyst on Day 8 (%)

1 80 A FT 28.7 12.5 5

2 40 B FT 75 a 7.5 a 7.5

2 20 C CE 90 b 20 b 10

2 20 D CE 95 b 30 b 10

In Replicate 2, within a stage of embryo development, values with different letters (a and b) differ (P < 0.05). Morulae and blastocyst productions

were expressed as percentages of oocytes cultured. FT, frozen-thawed; CE, chilled epididymal.a Day 0 = day of fertilization.

cell expansion, heterogeneity of cytoplasm, and

presence of vacuoles; these are all indicative of poor

quality in bovine COC [21,22]. Unfortunately, the

limited number of COC precluded rigorous selection

and only grossly aberrant COC [22,23] were discarded.

An acceptable proportion of COC had matured (80.0

and 86.6% of 20 and 30 COC in Replicates 1 and 2,

respectively; Fig. 1C and D).

3.2. IVP

Evaluation of a group of COC from Replicates 1 and 2

demonstrated that the fertilization rate was lower for

COC fertilized with frozen-thawed semen compared to

chilled epididymal spermatozoa (64.4%; n = 45 and

89.2%; n = 28). For Replicate 1, although cleavage rate

was very low (28.7%), 43% of the cleaved embryos

developed to the morulae stage and 50% of the morulae

developed to blastocysts. Although fertilization of

oocytes in Replicate 2 with a different bull resulted in

an acceptable cleavage rate, the cleavage rate for frozen-

thawed spermatozoa still appeared to be lower than that

of chilled epididymal spermatozoa obtained from two

other bulls. However, the percentage of blastocysts

produced was not different between treatment groups

(Table 1). Preimplantation IVP bison embryos (morulae

and blastocysts) are shown (Fig. 1 E and F, respectively).

3.3. Vitrification of embryos

A total of 27 embryos (21 morulae and 6 blastocysts)

derived during these studies were cryopreserved.

4. Discussion

Availability of a broad range of reproductive

technologies are important for conserving the genetic

diversity of American bison and maintaining disease-

free status by eradication of zoonotic diseases from

captive and free-ranging bison herds. Although assisted

reproductive technologies, e.g. AI, IVP, superovulation,

and embryo transfer, are commonly used for the

propagation of valuable germplasm in livestock species,

no information is available regarding IVP and

cryopreservation of embryos using spermatozoa recov-

ered from the epididymides after slaughter or from

frozen-thawed semen. Our results clearly demonstrated

the potential of these reproductive technologies for

salvaging the genetics of threatened wood bison. To our

knowledge, this is the first report on in vitro production

of bison embryos using sperm and oocytes recovered at

the time of slaughter, and their subsequent cryopre-

servation for preserving the genetics of a diseased wood

bison herd. These technologies may also be applicable

to plains bison.

This preliminary study demonstrated the potential of

chilled epididymal spermatozoa for IVP of embryos.

Chilled epididymal sperm yielded better fertilization

rates, increased cleavage rates, and percentage morulae

production than frozen-thawed semen; these differences

were attributed to the deleterious effects of freezing and

thawing. Similarly, in red deer (Cervus elaphus), there

was a similar trend for higher blastocyst formation with

the use of epididymal spermatozoa [24]. However,

further replication is necessary, due to the small number

of bulls used and the lack of a direct comparison of

frozen-thawed semen and chilled epididymal sperm

from the same bull. Furthermore, these differences were

not evident in the rate of blastocyst production.

Most of the recovered oocytes were dark, with

cumulus cell expansion, heterogeneity of cytoplasm, and

presence of vacuoles; all of these were a sign of poor

quality of oocytes in cattle [22,23]. Although most

oocytes had these characteristics, rates of IVM and

fertilization were acceptable. However, the rate of

blastocyst formation was low, due to the developmental

arrest of embryos at the 8- to 16-cell stage and the

morulae stage. These may have been due to their

morphological appearance; in cattle, oocytes with these

characteristics have poor developmental competence just

prior to blastocyst formation [25–27]. As this develop-

mental stage corresponds to the transition of the

J. Thundathil et al. / Theriogenology 68 (2007) 93–9998

dependence of embryo from maternally derived tran-

scripts to embryo-specific gene products in bovine

embryos, developmental arrest at this stage of embryo

development may be attributed to the poor quality of

oocytes [25] or to inadequate physical and chemical

composition of the embryo culture medium for bison

embryos. Furthermore, most of the bison cows used were

pregnant. Although a significant effect of a corpus luteum

of the reproductive cycle on the oocyte quality and in

vitro development of embryos has not been demonstrated

[27], the effect of pregnancy on the developmental

competence of oocytes remains unknown.

Cryopreservation of epididymal spermatozoa from a

variety of wildlife species has been successfully

accomplished, including blesbok, African buffalo,

springbok, and black wildebeest [28]. Cryopreserved

epididymal spermatozoa have been successfully utilized

for AI of Iberian deer (Cervus elphus hispanicus) [29]

and Spanish ibex [30]. Moreover, frozen-thawed

epididymal spermatozoa have been successfully used

for the in vitro production of morulae-stage embryos

in the Burchell’s zebra, (Equus burchellii) and the

Hartmann’s mountain zebra (Equus zebra hartmannae)

[31]. Since cryopreservation of epididymal spermatozoa

has the potential for long-term storage of valuable bison

genetics, it is important to optimize these procedures.

A vitrification procedure originally developed for

bovine embryos [21] was used in the present study.

Although optimal post-thaw viability has been reported

for bovine embryos, the post-thaw viability (ability to

expand and hatch following thawing and culturing) of

bison embryos cryopreserved by this method still needs

to be evaluated. Since these embryos are very valuable

genetic material, we did not evaluate post-thaw embryo

survival. In the future, these embryos will be transferred

to recipient bison. Furthermore, we propose to recover

ovaries and epididymides of plains bison after slaughter

and use them to optimize techniques for in vitro

production and cryopreservation of bison embryos.

In summary, although the methods used require

further refinement to optimize results, we showed that

IVP and cryopreservation of embryos using gametes

recovered after slaughter have substantial potential for

conserving and managing genetic diversity of wild

bison, and may also have important management

implications for genetic salvage of diseased bison

populations in North America.

Acknowledgements

We acknowledge financial support from the Depart-

ment of Environment & Natural Resources, Government

of Northwest Territories, Fort Smith, NT, Canada, and we

thank Alta Embryo Group, Calgary, AB for technical

assistance.

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