BIOLOGY

REPRODUCTION

OF

43,

In Vitro

733-744

Fertilization

A. M. DONOGHUE,2’3 P. WOLF,7 National

and Embryo Development In Vitro in the Tiger (Panthera tigris)1

PETRINI,7

Park,3

L. G.

Henry Minnesota

T. GROSS,6

Institution,

Washington,

National Cancer Institute, Medical Center,5 Minneapolis,

Doorly

Zoo,6

Zoological

Omaha,

Gardens,7

Nebraska

Apple

and In Vivo

L. ARMSTRONG,6 R L. TILSON,7 and D. E. WILDT3

SIMMONS,6

Smithsonian

of Viral Carcinogenesis,4 Veterans Administration

SEAL,5 D.

U. S.

L. A. JOHNSTON,3’4 K

Zoological

Laboratory

(1990)

District

of Columbia

Frederick, Minnesota

Maryland 55417

20008 21701

68107

Valley,

Minnesota

55417

ABSTRACT A study

was

conducted

developed

in the

5)

i.m.

LU eCG

I) were tigers licles oocyte

468

fofficles

2 500

and samples

volume

was

30

were

fertilized.

±

in Trial

embryos

ability

81.4

from

2 females

LI, and

3 live-born in Ham’s

sperm

96

contaminated

oocytes

(n

of 4 of the cubs

72

follicular

to live-born

original

were for

=

delivered

(95.9%)

20).

advanced after

in vitro. embryo

‘This tutes

12,

1990.

2, 1990.

work

was

supported,

of Health

(HD

23853),

and the Friends

Center

‘Correspondence:

Zoological

Park,

in part, NOAHS

of the Ann

Smithsonian

by grants

National

M. Donoghue,

Institution,

to DEW,

Opportunities

(New Zoo

from

the

National

in Animal

Health

of Animal 20008.

Health,

Insti-

Sciences)

(FONZ).

Department Washington,

DC

7 male

the

overall

and

the

sperm

mature

were

oocytes

were

two-cell

resulting

I and

excluded

107

sixteen-cell 14 (30.4%) embryos

days

No two-

after stage,

embryo and

to early are

in Trial

capable

mean

48 and (±

from

(10.5%)

as

in Trial

IL, 3

SEM)

percentage

ejaculate

of structurally

co-cultured from

number

collected

tigers,

with 227

embryo

cleavage

embryos

LI. A pregnancy

The

of advancing

or

cleavage

to four-cell transfer.

10 (63.4%)

parthenogenetic

15 (26.8%)

blastocysts.

all (fol-

Fofficle

inseminated,

stage.

2 females

treatment,

were

as mature,

=

Of the

were

occurred 56 cleaved

were

morulae.

data

demonstrate

to morulae

Of and

require extensive habitat (-25 000-50 000 km2) it is necessary to propagate tigers in individual captive breeding programs [4]. In this context, attempts are being made to manage most of the world’s captive tigers under guidelines that provide breeding recommendations for sustaining maximum genetic diversity within a given subspecies [3, 4]. Reproductive biotechniques including in vitro fertilization (IVF) and embryo transfer could play an important role in managing tigers, especially when desirable animal pairs are separated geographically by long distances or in selected cases of sexual incompatibility or infertility [5]. There is a growing database on the reproductive physiology of the tiger. In North America, the Siberian tiger (P.t. altaica) is seasonally polyestrous; females demonstrate a 5day estrus every 25.0 ± 1.3 days between January and early June [6]. In contrast, electroejaculation data suggest that Siberian and Bengal (Pt. tigris) tigers produce sperm throughout the year [7-9]. Unlike the majority of nondomestic feud species, which produce more than 60% pleiomorphic sperm/ejaculate [8, 10-12], tiger ejaculates

INTRODUCTION

June April

from

therefore,

hCG

(n in Trial

development

oocytes

qualified

motile

358

after

previously

or 5000 treated

follicles/female).

X 106,

106

were

of follicular

of 456

occasion; 20.6

5)

=

transfer.

The tiger is one of 36 threatened or endangered felid species [1]. Since 8 subspecies of tigers were recognized formally in 1969 by the International Union for Conservation of Nature and Natural Resources, 3 (Panthera tigris vigata, Pt. sondaica, Pt. balica) have become extinct, and 1 (P.t. amoyensis) is critically endangered (only about 50 animals remain) [2]. The primary causes for loss of free-living tigers are extensive habitat fragmentation and a declining prey base [3]. About 250 individuals are required to sustain adequate genetic diversity within a tiger subspecies [3]. Because a natural, free-ranging population of this size would

Accepted Received

section and

The

6-52

collected

to the

in Trial

at the

to morulae,

oocytes

oflpring

were

hours basis

system

(n

of which

good-to-excellent-quality

donors

by Caesarian

h, 14 (25.0%)

and,

cleaved

Eighty-six

oocyte

X

in air. Of the

in culture

187

±

0.05

CO2

the

(82.9%)

on each 105.9

processing,

(5

A total

378 were

was

of 5%

oocytes,

h, 20 (43.5%)

to fertilize

and

swim-up

On

these,

recovered

2 500

to twenty-six

of year.

I, 8 electroejaculates

(38CC)

became

FlO

Of

culture

either

6 females

(range,

or time

sperm/ejaculate

After

fertilized

oviducts

in vitro

in culture

-

1990),

Twenty-four

dose

were

with

transabdominaily.

oocytes/female). sperm

fertilization/embryo

treated

gonadotropins

by eCG

Trial

atmosphere

control

for

3.4

Motile

2.0%

h later.

aspirated

0.05)

of motile

±

195

the

cultured

of tiger

blastocysts

3 males.

84

were

II (January

to exogenous

> ±

i.m.

were

During

number

remaining into

cultured

the

from

was

Oocytes

in 1 female

28.5

tigers

In Trial

[U hCG

(p

of an in vitro

tiger

10 female

h later.

responded

as degenerate. the

84

oocytes

unaffected mean,

ml,

to the

1989),

2 000 and

all females

collected 0.7

surgically

46

and

in noninseminated

transferred

the

i.m.

in a humidified

Of the

observed

embryos

Lm.

(6.6%)

oocytes

calculations.

LU hCG

recovery;

were 7.5

tiger

2 000

were

sperm/ejaculate

fewer

1 (July

in diameter), (97.4%

semen

In Trial

to laparoscopy, rate

adaptability

the

cat.

LU eCG

recovery

immature,

normal

with

subjected

2 mm

and

was

and

given were

to evaluate

domestic

National

733

734

DONOGHUE

contain low proportions of morphologically abnormal spermatozoa (range, 21.8-37.5%) [7-9, 13].There have been a few attempts to stimulate ovarian activity in the tiger with exogenous gonadotropins. Serial injections of commercially available FSH-P have been only partially effective [14, 15], and the need to give repeated injections of FSH-P is a disadvantage when studying stress-susceptible wildlife species. Vaginal artificial insemination (Al) has not resulted in pregnancies [15], perhaps because exogenous gonadotropins alter the endogenous endocrine milieu, thereby compromising cervical sperm transport [7]. One attractive alternative to Al is IVF. The resulting embryo can be seen and characterized and, if pregnancy does not occur after transfer, at least the general timing of the reproductive failure is known. Particularly important is that IVF does not require identification of estrus or prediction of ovulation, two characteristics often difficult to determine in nondomestic species [5]. In our laboratory, IVF has been used to produce viable-appearing embryos of domestic cats (Fells catus) [16-18], leopard cats (F. bengalensis) [19],and pumas (F. concolor) [12]. In each species, eCG stimulates ovarian follicle development consistently, although the number of resulting oocytes varies between and within species [12, 16, 19]. In domestic cats, the sperm/oOcyte coculture system routinely results in 65-80% of all mature oocytes cleaving in vitro [17, 18, 20]. In contrast, fewer ovarian follicles are available for aspiration in leopard cats, and oocyte degeneration rate 24 h after insemination is 5-fold higher than in domestic cats [19]. In pumas, eCG is also effective for stimulating follicular growth, but a high proportion of the aspirated oocytes (31%) are immature [12]. Overall cleavage rate in vitro is low (25%), which may be related, in part, to the remarkably high proportion of pleiomorphic spermatozoa (>90%) ejaculated by male pumas [12]. This study was conducted as part of a long-term effort to explore the conservation of fertilization mechanisms among the Felidae family [5]. To extend our comparative observations further, we examined the utility of IVF in the tiger, the largest of all felid species. Our specific objectives were 1) to examine the effectiveness of eCG dose and consecutive treatments at different times of the year on ovarian follicle development and oocyte quality; 2) to assess the IVF ability of laparoscopically aspirated follicular oocytes exposed to homologous, processed spermatozoa; and 3) to determine the in vitro and in vivo developmental competence of these embryos.

MATERIALS

AND

METHODS

Animals The study was conducted over a 12-day period in July 1989 (Trial I) and a 10-day period in January 1990 (Trial II) at both the Minnesota Zoological Gardens (Apple Valley, MN) and the Henry Doorly Zoo (Omaha, NE). A total of 11

ET

AL.

adult (5-11 yr of age) female tigers (5 Pt. altaica; 6 P.t. tigris) were used as oocyte donors. Ten females were used in Trial I and 6 in Trial II. Six of the tigers in Trial II also served as oocyte donors in Trial I, so these females provided additional data on the impact of two consecutive gonadotropin treatments on ovarian responsiveness and 00cyte recovery and quality. All tigers in both trials had normal reproductive histories (i.e., evidence of reproductive cyclicity), and 5 were monoor multiparous. As part of an existing management protocol, 2 tigers (#2 and #8) had previously received implants of a progestagen (medroxyprogesterone acetate)-containing contraceptive [21]; these silastic implants were removed 6 wk before the onset of Trial I. Seven adult (5-13 yr of age) males (2 P.t. altaica; 5 Pt. tigris) served as sperm donors. All males were either proven fertile on basis of live cubs sired or had produced high-quality electroejaculates consistently in previous studies [9, 13]. Each tiger was housed separately with free access to an outdoor area (5.5 X 6.8 rn) during daylight hours; during the night, each tiger was confined indoors in a 2.6 X 2.6-rn enclosure. Each animal was fed a carnivore diet (Nebraska Brand Feline Diet, North Platte, NE) daily, and water was available ad libiturn. Induction of Ovarian and Oocyte Recovery

Activity,

Laparoscopy

Female tigers were administered gonadotropins without regard to a specific stage of the reproductive cycle. In Trial I,either 2 500 (n = 5) or 5000 (n = 5) IU eCG (Equitech Incorporated, Atlanta, GA) were administered i.rn. Findings from this trial indicated no effect of eCG dose on follicle or oocyte number or on oocyte quality or fertilization ability. Therefore, all tigers in Trial II were treated with a single 2 500-lU dose of eCG i.m. Females at the Minnesota Zoological Gardens received eGG via blow-dart delivery. The tigers at the Henry Doorly Zoo were placed in a restraint cage, and the eCG was injected by syringe. In both trials, the same injection approaches were used respectively at each institution to give each tiger 2 000 IU hCG i.m. (Sigma Chemical Company, St. Louis, MO) 84 h after eCG. Oocytes were recovered 24-26 h after hCG treatment by use of a standard laparoscopic procedure for tigers [15] that was modified to allow transabdominal oocyte aspiration [12, 16]. In brief,a surgical plane of anesthesia was induced with xylazine (Rompun#{174}, Mobay Corp., Shawnee, KS; 0.5 mg/kg body weight, i.m.), diazepam (Valium#{174}, Hoffman LaRoche, Nutley, NJ; 0.1 mg/kg, i.m.), and ketamine hydrochloride (Vetalar#{174},Parke-Davis, Detroit, MI; 5.0 mg/kg, i.m.). To sustain this plane of anesthesia, each female was intubated and maintained on halothane gas/oxygen or given supplemental iv. injections of ketamine HC1 (1.5 mg/kg). Each animal was placed in a supine, head-down position on a surgical table and surgically prepared, and a 10-mmdiam. 180#{176} laparoscope (Richard Wolf Medical Instruments Corporation, Rosemont, IL) was inserted into the abdomi-

IN VITRO

FERTILIZATION

cavity through a 2-cm-long skin incision made near the umbilicus. Ovaries were evaluated for number of ovarian follicles(clear structures with a well-defined border measuring 2 mm or more in diam.), corpora hemorrhagica (CH), or corpora lutea (CL) according to our previous criteria [15]. Follicle diameter was measured using a 2-mm-diam. Verres needle probe inserted transabdominally [16]. The Verres needle was also used to position and secure the ovary for oocyte aspiration using a procedure described previously for the domestic cat [16], leopard cat [19], and puma [12]. A 22-g, 4-cm-long needle attached to size 100 polyethylene tubing (inner diam., 0.86 mm; Clay Adams, Parsippany, NJ) was rinsed with 2-3 ml of Ham’s FlO medium (Irvine Scientific, Santa Ana, CA) containing 10% fetal calf serum (Irvine Scientific) and 40 units heparin/mi of medium. A siliconized collection tube (Terumo Medical nal

IN

THE

TIGER

735

Corporation, Elkton, MD) was attached to the free end of the polyethylene tubing, and the aspiration system was driven by a vacuum pump (Gast Manufacturing Corporation, Benton Harbor, MI). Distinct follicles 2 mm in diam. were perforated with the needle while gentle negative pressure (100 mm Hg) was applied with the vacuum pump. After aspiration of follicles from one ovary, the collection tube and aspiration needle were replaced, and the procedure was repeated for the contralateral ovary. Collection tubes from each animal were emptied into separate plastic culture dishes that were examined by stereomicroscopy. Each oocyte-cumulus cell complex was evaluated for maturational status and classified as 1) mature if corona radiata and cumulus oophorus cells were loosened and expanded (Fig. la); 2) immature if the oocyte had a tightly compacted corona radiata; or 3) degenerate if the oocyte appeared ab-

w

b

0

a d FIG.

1.

(a) Mature

tiger

oocyte

with

expanded

corona

radiata

and cumulus

cell mass.

tiger embryos after 30 Ii of culture. Ic) Sixteen-cell and morula stage, in vitro-fertilized blastocyst, in vitro-fertilized tiger embryo after 96 h of culture. Magnification for a-d:

(b) Two-

tiger x300.

and four-cell

embryos

after

stage,

in vitro-fertilized

72 h of culture.

Id) Early

736

DONOGHUE

normal, pale, and/or lacked an apparent corona radiata. Oocytes were washed 3 times in Ham’s FlO under oil and placed in fresh medium (without heparin). Because neither the Minnesota Zoological Gardens nor Henry portable

Doorly Zoo incubation

custom

culture

cm-thick

had tissue-culture system was

chamber

plastic

plate was constructed CA). Oocytes and/or in the chamber, a plastic hose chamber, the ervoir.

After

(5

containing

X 12

a simplified and tested.

X 22 cm)

2 air-ports

and

by C.B.S. Scientific embryos in culture

the

chamber

chamber removed

was and the Melrose

made

of 1.2face-

for

2 mm,

time

via the res-

both

chamber was placed Park, IL) maintained

was regassed each for oocyte/embryo

A

Company (Del Mar, dishes were placed

gassed

air-

inside at 38#{176}C.

the culture examination

AL.

for subjective estimates of sperm percent motility [8, 10]. A 10-1.il aliquot of semen was used to calculate sperm concentration by a standard hemocytometer method [10]. Sperm morphology was evaluated in detail after fixing a 25-)il ali-

a removable

and one of the air-ports was attached to a tank of 5% GO2 in air. To humidify CO2 gas was bubbled through a water

ports were occluded, a drying oven (I.abLine, The were

facilities, developed

ET

dishes and/or

quot in 1% glutaraldehyde and examining sperm/male at 400X [8, 10, 22]. Semen was processed for IVF by means described

swim-up

procedure

of highly motile, Electroejaculates 10-ml il

conical

individual

of a previously

recovering

a population

structurally normal spermatozoa [16, 23]. were centrifuged (300 x g, 8 mm) in a tube,

of fresh

for

300

the

Ham’s

FlO

supernatant

was

was

gently

layered

removed,

and

onto

the

The sperm were allowed to migrate from the the overlying medium during a 1-h interval at supernatant layer was recovered by aspiration, tility was evaluated, and the solution was diluted an WE concentration

of 0.5

X

motile

106

100

pellet.

pellet into 22#{176}C. The sperm moto provide

sperm/mi

for fer-

tilization.

washing. Insemination Endocrine

Activity

in Female

Tigers

Mature

To establish baseline hormonal jected taken

to eCG/hCG treatment, from each of the females

ration immediately induced. The blood the

serum

was

after was

and

validated

for

blood sample day of oocyte

by

tiger

centrifugation thawed by RIM

of variation

was aspi-

[6]. The

and

estradiol-17

oocytes

X

10-mm

then

cytes

or fewer

placed

into

assay

has

of variation of 20%. of 10 pg/ml and a

of 17%.

were

plastic

males

frozen

Sperm/Oocyte

CoCulture

inseminated

with

of diluted motile sperm (0.05 X 106) affin oil (Fisher ScientificCompany,

was

and analyzed for espreviously described

a sensitivity of 2 pg/mI and a coefficient The progesterone assay has a sensitivity coefficient

in tigers sub-

a surgical plane of anesthesia allowed to clot for 1-3 h, and

separated

the

norms

a 10-mi on the

(-80#{176}C). Samples were later tradiol-173 and progesterone

and

were

determine

culture

dish.

maintained per

the the

5%

and

CO2-in-air

rate

Oocytes

culture

or more representative tion) were cultured

To

obtain

and spermatozoa

collected from were obtained donors

in Trial

II, thus

providing

istics casion

Semen for

Evaluation 1W,

and

8 electroejaculates

I were

used

as a source

consecutive

data

on

of sperm

dardized

female. In Trial II, each ejaculate males was split and used to inseminate The procedure

electroejaculation [8, 10] using

gitudinal electrodes and a total of eighty 3-7-volt

characterOn one octwice within females. For I, individual oocytes from

from the oocytes

technique a rectal

was

probe

an AC, 60-Hz sine-wave electrical stimuli were

a ‘-20 mm interval. Total ejaculate volume was and each semen sample was examined immediately

chamber control

Eighteen-twenty removed from hyaluronidase

(10

dishes

oowere

at 38#{176}C. To oocytes

(one

each collecin medium

hours after the incubator and solution (Type 1-

Company) for 3 mm loosely attached sperm.

Oocytes were returned to the incubator in 100-pA drops fresh, equilibrated Ham’s FlO under oil and examined h after

of 30

insemination.

in Trial

ejaculate

24 h to inseminate oocytes from 4 different the remaining 6 groups of oocytes in Trial electroejaculates were used to inseminate only

2 females.

were

the 7 males in Trial I, and 3 electroejaculates from 3 males in Trial II. Three of the 7 sperm

and individual male fertilizing capacity. in Trial I, Male #1 was electroejaculated

a single dividual

Processing

drops

mature oocyctes from under standard conditions

containing no spermatozoa. insemination, oocytes were washed 3 times in a 0.2%

par-

Lawn, NJ) in a 35 from individual fe-

fertilization

of parthenogenesis,

aliquot

lightweight

sperm

the

S, from bovine testes; Sigma Chemical to remove residual cumulus cells and

Electroejaculation

Fair

in separate

drop),

a 100-pA

under

3 infrom a stan-

with

3 lon-

ejaculator; given over

recorded, (25 X)

Assessment

of Fertilization

Our fertilization polar bodies, two cell

stage

structures spermic.

relied on the presence or cleavage to at least

[12, 16, 19]. Oocytes within Oocytes

#33342 (H342; a DNA-specific counterstained cal Company) ml, 15 mm, terference

criteria pronuclei, the failing

more

than

two

were considered were treated with

nuclear polyHoechst

bisbenzamide; Sigma Chemical Company), fluorescent stain [16]. Oocytes were with 0.1% Trypan blue dye (Sigma Chemi-

for 1-2 23#{176}C), and contrast

with

cytoplasm to cleave

of two the two-

and

for germinal vesicles, spermic fertilization.

mm, then

incubated examined

fluorescence pronuclei,

in H342 (0.09 using differential optics

polar

(250X bodies,

and and

mg/ in400X) poly-

IN VITRO

Embryo

Transfer/Culture

Embryos

cleaving

to the

two-cell

assigned a quality grade [24,25]. or excellent quality were those

stage

IN THE TIGER

FERTILIZATION

or greater

ysis [27] was the proportion

were

In brief, embryos of good that were perfectly sym-

immature,

metrical (or only slightly asymmetrical), spherical, and uniformly dark. Fair- or poor-quality embryos were those that were degenerating partially or severely, were pale, or contained lyzed A portion

blastomeres. of the good/excellent-quality

embryos

were

transferred surgically to the oviducts of five of the original oocyte donors. A sixth recipient was treated with the eCG/ hCG regimen described above but not subjected to oocyte aspiration. All recipients were multiparous. At 48 h after initialoocyte recovery, each recipient was anesthetized and surgically prepared (as described above). A mid-ventral abdominal incision were exposed

was made, and the ovaries bilaterally. Each ovary was

and oviducts examined for

of 2 pA of physiological saline in 50 polyethylene the tip of which was inserted into the fimbriated oviduct ipsilateral to the ovary containing the most,

tubing, end of normal-

appearing CL. The embryos were deposited by gentle injection, and the tubing was re-examined under the stereomicroscope to ensure that the embryos were delivered Four of six

recipients

however, from

received

on which

the

Wilton,

described

had

MA)

[26].

Each

-30

and

pregnancy

hormone

transfer,

and

tigers the

non-

a silastic progesterone improgesterone (Steraloids,

recipient -60

was

days

anesthetized

later

and

ultrasonography probe). Ultrasound

abdominal wall along to the flank regions. diagnosis,

(one

one

the

(as

subjected

to

(Equisonics, scans were

both teat lines In the event

original

implant

and of a

was

degenerate,

effect of eCG dose on classified as mature,

or fertilized.

RESULTS Follicles

were

observed

in 10 of 10 tigers

in Trial

I (range,

6-52/female) and in 6 of 6 tigers in Trial II (range, 6-52/ female) (Table 1). Female #5 in Trial I and females #4 and #5 in Trial II (both of which produced the fewest follicles) had CH on the ovaries at the time of laparoscopy (25 h post-hCG) and thus had begun ovulation before all follicles could be aspirated. Luteal tissue of unknown origin was

observed

on

and

#6,

Trial

I). These

surface,

and

their

were

advanced

ovarian gested

that

the

they

culating of these

ovaries

of 2 additional

CL were

slightly

females raised

yellow-orange

(#3

above

coloration

in age

and

not

the sug-

formed

as

re-

2) failed to affect the ability follicle numbers comparable

progesterone, Table 2 females to produce

to those produced the luteal status

by the of females

other tigers #3 and #6

(Table 1). Likewise, had no detrimental

impact on the proportion of mature oocytes recovered (Table 1). On the day of oocyte collection, there was a close relationship

treatment;

two

aspirated

radiography or and a 3.5-rn-Hz

carried out on the extending laterally positive

been

each received 35 mg crystalline

above)

abdominal Model-LS300S

further

of embryo

oocytes

aspirated female) plant containing Inc.,

day

no

used to compare the of oocytes eventually

a result of the recent gonadotropmn injections. Regardless, the presence of CL (and the simultaneous elevation in cir-

of CL and preovulatory follicles.Two- to four-cell (12-16/female) were aspirated in a total volume

number embryos

737

between

steroid hormone with no detectable

ovarian

morphology

concentrations luteal tissue

licles produced 34.5 pg/ml and

estradiol-1713 progesterone

concentrations levels of 3.7

The 5 tigers with luteal tissue (#3, [Trial II]) produced estradiol-173 rable to those of progesterone was

no

of year treated

effect

in tigers with levels ranging (p

> 0.05)

and

#5,

greater ng/ml or

than less.

#6 [Trial I] and #4, #5 concentrations compa-

no CL, even in the presence from 3.0-40.0 ng/ml. There

of eCG

on ovarian response with 2 500 IU eCG

circulating

(Tables 1 and 2). Females and only preovulatory fol-

dose,

subspecies,

to treatment. produced

or time

In Trial I, females 29.6 ± 6.2 follicles

placed immediately with a 50-mg progesterone implant to sustain the pregnancy. Some of the original embryos also were placed in culture to determine the ability of tiger 1W-produced embryos

compared to 27.0 ± 6.2 follicles produced by females treated with 5000 IU. For both trials, the mean number of follicles in Pt. altaica (30.5 ± 3.8) was no different from that ob-

to advance bryos were

eCG in July (Trial ± 4.0) comparable

to later developmental transferred into fresh

after insemination and examined at 72 and 96 h after insemination.

stages Ham’s for

in vitro. These FlO medium

stage

em30 h

of development

Statistics Mean mean.

values Differences

are

presented in follicle

as number

±

standard and

oocytes

error

of the collected

between eCG treatment groups, tiger subspecies, or time of year were measured by Student’s t-test [27]. The same statistical approach was used to compare ejaculate characteristics between the Pt. altaica and Pt. tigris subspecies within trials and over time. In Trial I, Chi-square (X2) anal-

served

in Pt.

tigris

(28.2

± 5.5).

Overall,

tigers

I) produced numbers to those produced

January (Trial II, 29.8 ± 6.6). For both trials, a total of 456 468 aspirated follicles (97.4%),

were collected from a mean of 28.5 ±

Of the

classified as mature, as degenerate. There

48 (10.5%) as immature, and 30 (6.6%) was no effect (p > 0.05) of eCG dose

I), tiger efficiency

gers

receiving

378

with

3.4 oocytes/female.

(Trial covery

total,

oocytes providing

treated

of follicles (28.9 by tigers treated in

subspecies, or time or maturation/quality exogenous

(82.9%)

oocytes

of year on oocyte status. Of the

gonadotropmns

(#1, #2, #3) demonstrated a remarkable follicle and oocyte number/quality status ment periods (Table 1).

were

re5 ti-

twice, consistency between

3 in treat-

738

DONOGHUE

TABLE 1. Ovarian activity, oocyte (Trial I) and/or January (Trial II). Animal number

Trial

recovery,

and

ET AL.

oocyte

quality

in female

tigers

treated

Number

Ovarian activity on day of aspiration

Collected

Mature

with

eCG/hCG

in July

of oocytes

Immature

Degenerate

I II

1’

40 follicles

36

28

0

1b

48 follicles

48

45

2

I II

2”

28 follicles

28

24

2

2

2b

28 follicles

28

22

4

2

I II

3 3

32 follicles, 3 CL 28 follicles

32 28

22 26

8 2

2 0

I II

4 4

52 follicles 6 follicles, 7 CH

52 6

47 6

4 0

1 0

I II

5”' 5

6 follicles, 17 follicles,

6 CH 7 CH

6 15

6 14

0 0

0

I

6

32 follicles, 6 CL

32

22

8

2

I

7

16 follicles

15

10

2

3

I

8”’

22 follicles

20

16

3

1

9”

39 follicles

39

35

0

4

10

22 follicles

22

13

9

0

11b

52 follicles

49

42

4

3

8

1

eCG.

‘Received

5000

lU

bReceiveci

2500

lU eCG.

‘P.t. “Pt.

altaica female. tigris female. Served as a nonaspirated,

There

was

no

difference

(p

>

embryo

0.05)

recipient

over

time

in Trial

or

II.

be-

tween subspecies group in any of the ejaculate traits measured. Semen characteristics by individual sperm donor for each trial are provided in Table 3. Overall mean ejaculate characteristics

were

as follows: TABLE oocyte

January

Trial

II

II

ejaculate

volume,

7.5

± 0.7

ml; sperm concentration, cent motility, 85.4 ± 106;

and

ejaculate,

± 20.6

x

81.4

± 2.0%.

included

cells

with

2. IVF results and circulating estradiol-17 and aspiration) in female tigers treated with eCG/hCG (Trial II). Animal number

17.3 2.0%;

percent

morphologically The

either

most

progesterone (on day of in July (Trial I) and/or

oocytes 1%)

Estradiol-17p pg/mI

Progesterone ng/ml

1’ l

27/28 22/41

(96.4%)

103.0

3.7

(53.7%)

109.0

0.5

21c

12/22 10/22

(54.5%)

95.3

(45.5%)

94.9

1.8 0.7

I

3

13/20

(65.0%)

58.6

II

3

23/25

(92.0%)

105.0

I

4

28/43

(65.1%)

162.0

II

4

4/6

(66.7%)

40.5

I II

5 5

5/6 10/13

(83.3%) (76.9%)

126.0

6

9/20

(45.0%)

79.6

7

8/9

(88.9%)

34.5

0.7

8

11/15

(73.3%)

145.0

0.5

(21.2%)

140.0

0.5

9”

II

7/33

96.4

28.0 0.4 0.4 19.0 18.3 3.0 40.0

10

11/12

(91.7%)

85.0

0.2

11

27/41

(65.8%)

87.2

0.4

‘Received 5000 IU eCG. bReceiv(( 2 500 IU eCG. ‘Pt altaica female. “p.t. tigris female.

sperm

normal

prevalent

a proximal

No. mature fertilized

2c

± 3.9 X 106/ml; motile sperm/ejaculate,

per105.9

sperm/

pleiomorphisms droplet

(5.0%),

bent

IN VITRO

TABLE

3.

Semen

characteristics

and

IVF results

in individual

FERTILIZATION

tigers

IN THE

electroejaculated

in July

739

TIGER

(Trial

I) and/or

January

(Trial

II). No.

Animal number

Trial

Ejaculate volume

Spermatozoa/mI (ml)

eiaculate

Sperm motility (%)

(X1O#{176})

Motile sperm/ ejaculate (x106)

Morphologically normal sperm

oocytes

fertilized/no. (%)

inseminated

(%Y

17 8 33

85 80 80

173 83

73.0 70.5

35/37 21/42

(94.6)

1b

12.0 13.0 7.0

184

71.5

32/63

(50.8)

I II

2’ 2’

6.5 5.0

10 16

95 90

62 72

83.0

5/6

(83.3)

84.5

33/39

(84.6)

I II

3’ 3’

7.0 6.0

7 8

80 90

39 43

88.5

13/20 31/47

(65.0)

87.0

I

4b

7.0

48

75

252

84.0

11/15

(73.3)

I

5’

7.0

16

80

90

84.0

28/43

(65.1)

I

6’

7.0

21

95

140

88.0

7/33

(21.2)

I

7’

5.0

6

90

81.5

11/12

(97.7)

I I II

1” 1.b

‘Collected on subsequent bpt altaica male. ‘P.t. tigris male. dAJI oocytes were

27

inseminated

Culture interval 30

h

72 h 96 h

4.

(66.0)

days.

with

the same

number

of motile

sperm.

wise, there was no effect (p> 0.05) of subspecies (Pt. altaica, 14.1 ± 3.0 versus Pt. tigris, 14.2 ± 3.6) or time of year (July, 14.5 ± 2.7 versus January 16.0 ± 3.4) on number of fertilized oocytes/female. The 2 tigers that had contraceptive implants removed 6 wk before the onset of Trial I (#2 and #8) produced numbers of follicles, mature oocytes, and fertilization rates similar to those of nonimplanted counterparts (Tables 1 and 2). Furthermore, the incidence of IVF was comparable between females treated twice with gonadotropmns at different times of the year. By 30 h after insemination, 178 of the 187 (95.2%) cleaving embryos were at the two- or four-cell stage of development (Table 4). The remaining 9 embryos contained 8 blastomeres. Of this total, 45 were used for a parallel embryo cryopreservation study (Donoghue, Johnston, and Wildt, unpublished). Of the remaining 142 cleaved embryos, 12, 12, 16, and 16 two- or four-cell embryos were transferred surgically into oocyte donors #1, #2, #7, and #8 in Trial I; and 15 two- to four-cell embryos were transferred to each oocyte donor #3 and the recipient not subjected to oocyte aspiration (#8), respectively. Pregnancies were not evident on either diagnostic day according to radiography or ultrasound in any nonprogesterone-supplemented recipient in Trial I or in the oocyte-aspirated/progesterone-supplemented recipient in Trial II. However, 61 days from the day of embryo transfer, 2 distinct fetuses and heart-beat activity were observed in the progesterone-supplemented recipi-

midpiece (5.0%), or abnormal acrosome (2.7%). The number of motile sperm/ejaculate in the one male (#1) electroejaculated on consecutive days was reduced by half on the second day of collection, primarily because of a reduction in sperm/mi of ejaculate (Table 3). Of the 358 mature oocytes inseminated, 227 (63.4%) were fertilized on the basis of the criteria described in Materials and Methods. In Trial II at the Minnesota Zoological Gardens, the gamete cocultures for 2 oocyte donors were inexplicably contaminated by 30 h after insemination. These oocytes, which were Hoechst-stained to confirm fertilization, were excluded from subsequent cleavage rate calculations. Of the remaining 195 uncontaminated fertilized oocytes, 187 (96.0%) cleaved to at least the two-cell stage of development (Fig. Ib). All other oocytes failing to cleave (n = 8) contained two pronuclei after Hoechst-staining. Of the cleaved embryos, 165 met the excellent (11.9%) or good (76.1%) quality-rating criteria. Parthogenetic cleavage was not observed in any of the 20 control oocytes. Among individual sperm donors and over both trials, the IVF rate varied more than 3-fold (Table 3). Because all insemination solutions contained similar numbers of structurally normal, motile spermatozoa, the fertilization differences among males could not be attributed to a specific ejaculate characteristic(s). In Trial I, the mean IVF rate was unaffected (p > 0.05) by eCG dose (2 500 LU, 63.5% versus 5000 IU, 57.7%). Like-

TABLE

(50.0)

Culture

rate

and development

56 46

tiger

embryos. Stage

No. embryos 187

of in vitro-fertilized

2-cell 73

5 1

(39.0%)

(8.9%) (2.2%)

8-cell

4-cell 105

(56.1%)

13 (23.2%) 1 (2.2%)

9

(4.8%)

9 (16.1%)

4

(8.7%)

of development 16-cell

Morula

-

-

14 (25.0%)

15 (26.8%)

-

20 (43.5%)

14 (30.4%)

6 (13.0%)

Early

blastocyst -

740

em

DONOGHUE

(#8)

not

subjected

to oocyte

collection

(Fig.

2).

ET AL.

The

ficult

progesterone implant was removed from this female 105 days after embryo transfer. Three live cubs were delivered by Caesarian section 48 h after implant removal. One cub died

within

tions. Totals

1 h of birth of 56 and

because

46 embryos

of respiratory were

complica-

allowed

to culture

or impossible

to implement.

One

alternative

strategy

is to rely on common animal models for defining mechanisms and developing concepts and techniques for later application to rarer species [5, 28]. This approach is effective given that reproductive mechanisms are conserved among taxonomically always appear

for

related species. to be the case

for

Interestingly, this certain reproductive

does

not char-

72 and 96 h, respectively. At 72 h, 29 of 56 (5 1.8%) embryos either contained 16 blastomeres (n = 14) or were at the

acteristics within the Felidae family. For example, males from most felid species vary markedly in ejaculate characteristics

morula (43.5%)

(n = 15; Fig. ic). By 96 h, 20 morulae, and 14 (30.4%) were

[8, 10, 11,29], pituitary-gonadal ships, and adrenal reactivity

4; Fig.

31]. Likewise, among felid

stage of development of the embryos were

early-stage

blastocysts

(Table

ld).

eCG/hCG produced

DISCUSSION

ever, These results vitro fertilization, spring

after

LW and

representing idae. sons.

are the embryo

However, First, the

the

first to demonstrate successful in culture, and production of off-

embryo

largest

transfer

of the

great

in a Panthera cats

in the

this study is significant for eventual utility of reproductive

ogy in the emerging field of conservation biology on a solid database of fundamental biological This knowledge, usually obtained from basic

Fel-

other reabiotechnol-

critical to making sound decisions about how to conduct the actual artificial breeding attempt. Because by definition, endangered species are available in few numbers, a basic research approach for detailing species biology often is dif-

Ultrasonograph of tiger fetus into a hormone-synchronized

differences to a given

the

present

study

cross-species

utility

is unique

because

of a given

IVF

it

demonstrates

protocol

,--,‘

were similar domestic cat

between

to recent data using the same [18, 20]. Secondly, the positive

suits of the conventional mobile; that

present

study were conditions. is, all the technology

obtained under The NP system was transferred

laboratories,

We

experience

contamination

cyte cultures in a clinical

during laboratory

Trial II, no normally

doubt used

tine

veterinary

laboratory did

diagnostics

(including

fecal

*:

63 days after in vitro fertilization recipient. V = embryonic vesicle;

and 61 days F = fetus.

after

surgical

sysre-

less than was made between in two

oo-

related to working for conducting rou-

i

..-

FIG. 2. of embryos

considerable responsiveness

hormone challenge, both in number of follicles and resulting oocyte quality [12, 16, 19]. How-

in the tiger tem in the

will depend information. research, is

-

we have observed species in ovarian

two species (cat and tiger) separated considerably on the evolutionary tree [32]. The mean number of oocytes recovered, oocyte quality, and NP and embryo culture rates

species family

two

the

hormonal inter-relationto specific stressors [7, 8, 29-

transfer

parasites).

This

IN VITRO

experience standards

reinforces of quality

the control

mandate [33].

for

maintaining

ovarian activity in farm P in tigers is interesting follicular activity puma, and Indian no evidence

that

follicular oocyte rates)

livestock because

efficiently desert the

ineffectiveness preparation

in the cheetah, cat [14, 15, 28,34,35].

dosages

recruitment,

demonstrated a rather injections of FSH-P used for inducing

The this

of eCG

number

tested

quality (based on subsequent were similar in the two treatment

trial, there was substantial between individuals, which tions made feud species formity in

the

recovered, collection

overall

within occasions.

variation

among

of eCG

and

hCG

[19],

these

apparent

data

adverse

are

at a 6-month

also

effects

interval

the

3 females

known

demonstrate of treating

with

eCG

and

there

appeared

to be

no

relationship

sequent

ability

of these

oocytes

good-to-excellent-quality less

of the

recovered and

embryos.

magnitude oocytes

forming

aspiration

The

ability

had

cleaved

ovarian luteal progesterone cyte

of the

tissue at the had to collect

an

and time

context,

form

regardto eCG,

chance

Likewise,

ex-

impact

presence

numbers

oocyte

on

all

of fertilizing

the

of

high circulating concentrations of follicular recruitment and

a negligible large

and

response

equivalent

embryos.

the

of follicles and the sub-

cleave,

In this

ovarian

because

between

to fertilize,

no

in other speon the longfurther study.

tent of ovarian responsiveness (i.e., the number developing or oocytes recovered) in the tiger

of fertilizable

of oo-

integrity. oocytes

from tiger ovaries under estrogenic or progestogenic influence suggests that embryo production in vitro in this species was largely independent of reproductive status. Most (>82%) of the 456 tiger oocytes collected during both trials were classified as mature, indicating that the hormonal maturation vations

regimen

used

in vivo.

was These

in eCG/hCG-challenged

adequate data

are

for

provoking

similar

leopard

to recent cats

in which

sensitivities

uniform

of tigers

response

of year of the trials: ing the nonbreeding

categorized

as imma-

of pumas were

oocyte obser-=12%

to eCG may

females season

also

revealed immature

that and

[121. This weight basis

were challenged with exogenous midbreeding season [12]. Normal production optimal

oocytes

pumas. 63.4%,

far

20%

cleavage

and

than

for

in the

pumas by

covered. tion/culture

may the

have

high

experiences

with

oocytes was cats [18,20] in pumas

onthe

several

posand used

been

suboptimal,

proportion

of

are

[12].

tiger over

an assertion immature

sup-

oocytes

re-

Second, there were differences in the fertilizamedium used for the two species (tiger, Ham’s

for

mKRB

that the quality proved gamete gers

compared

cies

surveyed

IVF of domestic

Ringer medium

to the to date

were

dominant toplasmic semen used pleiomorphic

bicarbonate on the IVF of

but recent to using

cat oocytes

[18].

of the spermatozoa contributed interaction and embryo formation pumas. [8,9,

classified

abnormal droplet

as structurally

features or a bent

produces

81.4%

normal,

FlO

It is likely to the imin the tifetid

consistently

were cells midpiece.

spe-

produces Of the 11 of all sperand

the

pre-

with a residual cyIn contrast, puma

has contained and one rare ejaculates

evidence Ham’s

nondomestic

tiger

spermatozoa/ejaculate. in this study,

for IVF attempts sperm forms,

coryi)

Of the

11], the

the fewest pleiomorphic electroejaculates examined

concolor

of inseminated

There

puma and tiger oocytes is unknown, indicates that there is little advantage

matozoa

interfered eCG/hCG

differences in fertilization the gonadotropin regimen

PlO versus puma, modified Krebs [mKRB]). The significance of culture

over

the and

have

achieved

[12].

duronset

during activity

(96.0%) of fertilized a considerable increase

pumas

time

after rate

previous

results

sible reasons for the species embryo cleavage rates. First, ported

may

cleavage our

proportion cleavage, rate

more

ovarian acthe pumas

fertilization rate for tiger to recent results in domestic

higher

The to the

gonadotropins cyclic ovarian maturation

exceeded

The overall comparable about

NP

the

hCG.

when natural In contrast,

animals

oocyte

vivo

administration. Nevertheless, tiger

in these

in

or

be related

were given gonadotropins (July) or just before the

of the breeding season (January) tivity was assumed to be minimal.

and

occasions

However,

were

before insemination eCG dose on a per

to species-specific

25%

were

oocytes

in vitro maturation even though the

Likewise, a high cytes underwent

an immune

on two

hCG.

mean

[36] and rhein leopard

that tigers

required occurred

with

recovered adminis-

to induce in rabbits observations

follicular

ture [19]. In contrast, a similar study 43 of 140 oocytes (30.7%) recovered

hormone

response observa-

in the

oocytes Multiple

of the previous observations of refractoriness cies, the impact of repeated eCG injections term responsiveness of the tiger ovary warrants There

each

individual tigers on Of the 5 tigers treated

counted and respectively.

response and ovarian refractoriness sus monkeys [37]. Similar to recent cats

and

fertilization/cleavage groups. Within

variability in ovarian was consistent with

number of ovarian follicles was less than 15 and 18%, trations

results;

recovered,

in the domestic cat [16] and other nondomestic [12, 19]. However, there was surprising uniovarian responsiveness, including number of

mature oocytes two consecutive twice,

leopard, There was

affected

of oocytes

741

and the interval from eCG to hCG administration was comparable to that used in the tigers in the present study. These variations in in vivo oocyte maturation may be attributable

of FSHstimulates

lion,

TIGER

of aspirated

high

Single injections of eCG and hCG were at least as effective as or more effective than a similar hormone treatment given to domestic cats [16-18, 20], leopard cats [19], or pumas [12]. In previous studies, the tiger striking lack of response to repeated [15], a common exogenous gonadotropin

IN THE

FERTILIZATION

in

more than 90% subspecies (Fells which

more

than

50% of the spermatozoa are afflicted with severely deformed acrosomes. Therefore, it seems reasonable to speculate that the marked LW/cleavage differences between these two species may be related to different degrees of teratospermia. More detailed studies from our laboratory recently

742

DONOGHUE

have

focused

on the

in vitro

ability of spermatozoa normal spermatozoa) spermatozoa) domestic troejaculates cessed so tained

comparable

numbers

sperm and

ing spermatozoa interpreted this spermic

males

of motile,

from the to mean

domestic rates

cat

normospermic animals. that the spermatozoa

inherently

compromised

influencing

the

these results puma support successful

in culture

to the

morula

stage

a similar mestic beyond

rate.

Preliminary

cat embryos, the morula

assertion

that

tiger stage

there

preimplantation

embryos in vitro,

is a late

embryos

to all tisper-

ual

of certain

like

feud

species

[18].

do-

in which

[39]. This observation bryonic development lier stage of growth:

appears unique, in other species mouse, two-cells

fertilizable tiger also

thus

of

is possible follicular

enous Studies

at least

the one in a tiger

that

overly

content

may

hormone patterns are in progress

enous progesterone cies and to explore cedure on subsequent emphasize at the

the

need

cat

[35]

culture

medium

transfers embryos

resulting

NP

from

have

born offspring. In this study, the transfer bryos to a progesterone-supplemented in a single viable pregnancy. The reasons failures are unknown and are, in part, poor understanding of the mechanisms tation/pregnancy in felids in general mestic licles

on

transfer, ular one

cat). However, the ovaries presumably

growth after concentrations

we observed of all recipients reflecting

and

Indian

resulted

of being

fertilized

desert in live-

by our implanthe do-

many unovulated folon the day of embryo

a secondary

wave

of follic-

oocyte aspiration. Circulating progesteron the day of transfer (2.5-4.5 ng/ml)

the

perturbed

treaten-

early

and

of females,

blastocysts.

biologically competent young. Taken together,

teams.

that

in vitro

and

in vitro

culture

of structurally Tiger embryos

importantly,

as demonstrated these results

nondomestic may not

This

species, always be

approach

an

may

NP

adapted stimfollicles capable

in vivo.

The

and cleaving gonadotropic

these

in

conditions,

normal allowed

readily to the late morula a partial developmental Most

em-

of developing oocytes that were

developing

optimal

directed

cat is readily to an eCG/hCG

sperto cul-

stage block

embryos

but to were

by the birth of live support the contention an extensive mandatory

be

existing expertise and may more efficiently. This especially

considering the life propagation

endog-

studies

ating embryos in vitro given that an adequate available in a suitable animal model. Additionally, ings suggest that, for some species, it may be apply reproductive biotechnology using mobile research

it

preimplantation

in felids. has demonstrated

or the naturally high incidence matozoa in electroejaculates.

exploiting sources

oc-

of individnormal

numbers of mature oocytes being fertilized vitro may have resulted from the successful

that, for certain search approach

establish-

the formation of CL. the utility of exog-

fundamental

large numbers of mature

of 1W tiger emrecipient resulted for the pregnancy complicated regulating (including

more

survival and implantation In summary, this study

becoming cat [16]

hyper-

women is rate after producing

aspiration

ulus by producing and a high percentage

[18].

of domestic

have

controlling

ture in vitro developed appeared to experience

in the

thorough

by disrupting both to assess

for

mechanisms

overcome the morula-to-blastocyst vitro is regulated partially by the Previous

explaining

for the domestic tigers responded

block in of pro-

ovarian

Because this pregnancy oocytes were not collected,

procedure developed to the tiger. Female

In this

know

the gonadotropin altered endogenous

for

the

We

profiles alin gonad-

implants for facilitating NP pregnanthe impact of the oocyte collection proluteal function. Overall, our findings

stimulation

tein

follicles.

Likewise,

partially

pregnancy. in which

four-cells [41]; pig, four-cells [42]; sheep, eightto sixteencells [431; cow, eightto sixteen-cells [44]. Studies in progress suggest that the ability of domestic cat embryos to type

[46].

bryo

since blocks to emoccur at a much ear[40]; hamster, two- to

developmental presence and

cats

readily recent

the domestic cat is similar to the rabbit, a species embryos also arrest in vitro at the morula stage

context,

mature, for the

ment curred

number of solution. 1W readily was

domestic

recipients,

also

block

accessory

that abnormal endocrine rate and embryo recovery

docrine patterns in a fashion detrimental to the embryo. If hyperestrogenism indeed occurred, then this condition may have been countered in the progesterone-supplemented

of homolo-

do not develop supporting our

of many

observation

from a previous study ter embryo transport

copulating

oocytes, may have

to fer-

that,

developmental

concurrent

estrous,

levels were above norno doubt related to the

many ment

impossible because normal

indicates

natural

with NP protocols in cause of low pregnancy Although effective for

of domestic cat em[18]. In both species, to advance in vitro at

evidence

for

stimulation associated thought to be a common embryo transfer [47,48].

of development

in agreement with recent observations bryos produced by the same approach Ham’s FlO medium allowed embryos

range

otropin-treated

in ability

interaction

temporal

normal

in the context of previous the idea that a major factor

in vitro

in the

tigers [45]. However, estradiol-1713 mal (38.8-135.0 pg/ml), a finding

oocytes

We have of terato-

gous gametes from felid species may be the pleiomorphic spermatozoa in the insemination The observation that embryos resulting from advance

procon-

males resulted in compared to us-

tilize in vitro. In the present study, it was evaluate the impact of teratospermia on 1W gers produced high numbers of structurally matozoa. However, 1W studies in the

were

swim-up oocytes

structurally

immature

from teratospermic zona penetration

are

zona-penetrating

cat populations were used to inseminate

Coculturing

with normal lower binding

and

recovered from normospermic (>60% and teratospermic (

In vitro fertilization and embryo development in vitro and in vivo in the tiger (Panthera tigris).

A study was conducted to evaluate the adaptability to the tiger of an in vitro fertilization/embryo culture system previously developed in the domesti...
2MB Sizes 0 Downloads 0 Views