BIOLOGY OF REPRODUCTION 44, 1177-1182 (1991)

1177

In Vitro Fertilization of Goat Oocyte&

A. I. YOUNIS, K. A. ZUELKE, K. M. HARPER, M. A. L. OLIVEIRA,3 and B. G. BRACKETI’2

Department of Physiology and Pharmacology, College of Veterinaiy Medicine

The University of Georgia, Athens, Georgia 30602

ABSTRACT

Experiments were carried out to achieve fertilization (lyE) and initial embryonic development of goat oocytes in vitro. Oo-cyte/cumulus complexes were recovered from large follicles (>7 mm) of hormonally treated does and from 1-6-mm follicles

of ovaries from hormonally superstimulated and nontreated goats. Three different sperm treatment/IVF media were used: definedmedium (Brackets and Oliphant, Biol Reprod 1975; 12:260-274 111) with modifications (mDM); TALP (Bavister and Yanagimachi,

Biol Reprod 1977; 16:228-237 [2]), as modified by Parrish et al. (Theriogenology 1986; 25:591-600 [3]), i.e. modified TALP

(mTALP); and HEPES-buffered M199 with modifications (mH-M199). Immature oocytes (from 1-6 mm, small antral follicles)

were cultured for in vitro maturation (WM) in M199 buffered with bicarbonate and with modifications including supplementation

with 20% (v/v) goat serum (mB-M199) with either (a) 100 �g L}l/ml, (b) 5 �g FSH/ml, or (c) no added gonadotropin control.

Insemination of (in vivo or in vitro) matured oocytes was performed with swim-up separated and heparin-treated freshly ejac-

ulated sperm; additionally, caffeine was included in the mDM treatment. Use of mDM yielded better results than mTALP or mH-

M199 (p < .05). Results with oocytes after IVM were significantly better than those obtained with oocytes matured in vivo

(68.4% vs. 45.5%, p <0.05). Presence of LH or FSH during oocyte maturation improved both the WM and WE results over

those of the control (p < 0.05). The highest proportion of fertilized oocytes (fertilization rate) was achieved by combining the

use of mDM for sperm and WE with IVM in the presence of LH. LH provided the highest proportion of inseminated oocytes that

cleaved, 39.5% vs. 23.3 when WM was with FSH (p < 0.05). For fertilization, mDM afforded the best results (p < 0.05) whether

oocytes were matured in vivo or in vitro. Three pregnancies were initiated after oviductal transfer of 2. and 4-cell stage embryos

resulting from WM in the presence of LH, WE, and culture.

INTRODUCTION

In vitro fertilization (IVF) with development to live young

has been reported in several large domestic species in-

cluding cattle [4, 5) sheep [6,7], pigs [6], and goats [8]. Mor-

phological description of sperm-egg union [9] and ultra-

structural studies of goat zygotes obtained in vivo have

revealed many morphological features common to other

mammalian species [10].

Requirements for in vitro capacitation of mammalian

spermatozoa have been investigated extensively [11, 12]. The

defined medium (DM) for rabbit IVF [1] was modified and

used for treatment of goat spermatozoa [13-15]. Kusunoki

et al. [15] have shown that the acrosome reaction can be

induced by incubating washed ejaculated goat spermatozoa

in DM prepared without BSA. The motility of goat sper-

matozoa can be reversibly inhibited by elevation of CO2

tension [14]. Bou and Hanada [13], using zona-free hamster

eggs in vitro, demonstrated the capacitation of ejaculated

goat sperm following treatment with calcium ionophore

Accepted February 11, 1991.

Received October 15, 1990.

‘Supported by Veterinary Medical Experiment Station (91-222), and Program in

Biological Resources and Biotechnology, The University of Georgia.

2Correspondence: FAX: 404-542-5743.

3Current address: MAL. Oliveira, Universidade Federal Rural de Pernambuco,

Departamento de Medicina veterinaria, Rua D. Manoel de Medeiros S/N, Dois Ir-

maos, 50000 Recife PR, Brasilia

A23187 and caffeine. A lamb and a kid resulting from IVF

of in vivo-matured oocytes with A23187-capacitated sperm

were reported in Japan by Hanada [8].

Oocytes recovered surgically from follicles of eCG-treated

pregnant goats have been shown to be competent for fer-

tilization and development [16]. Xenogenous fertilization of

goat follicular or ovulated oocytes in the rabbit [17] or sheep

[18] oviduct has resulted in embryos (2- to 8-cell stages)

that developed into blastocysts and live offspring.

Objectives of the present studies are to develop LYF

technology for utility in goat breeding and to provide zy-

gotes for utility in production of transgenic animals capable

of expressing valuable molecular species. Progress in de-

velopment of a repeatable IVF procedure for goat oocytes

matured in vivo and in vitro, and the developmental po-

tential of resulting IVF embryos, reported earlier in prelim-

inary form [19], is reported here.

Animal Treatments

MATERIALS AND METHODS

Experiments were carried out during the fall of 1989.

Twenty-three doe goats (Angora, Alpine, Nubian, and Tog-

genburg) maintained on pasture isolated from males were

used as oocyte donors. Nine additional goats were used as

recipients for embryos resulting from IVF. Some of the oo-

cyte donors were hormonally superstimulated by admin-

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1178 YOUNIS ET AL.

istration of a total of 20 mg of FSH (F.S.H.-P, Schering Corp.,

Kenilworth, NJ) in six divided (evening and morning) i.m.

doses over 3 days: 5.0, 5.0; 2.5, 2.5; 2.5, 2.5 mg, respectively.

Concurrent with the fourth FSH injection, the donors re-

ceived a single i.m. injection of 125 �tg prostaglandin F2�

(PGF2a, Lutalyse, Upjohn Co., Kalamazoo, MI). Each oocyte

donor was anesthetized with i.m. injections of xylazine (0.2

mg/kg body weight, Rompun, Bay Vet Division, Miles Lab-

oratories, Inc., Shawnee, KS) and ketamine hydrochloride

(2-3 mg/kg, Vetalar, Parke-Davis Division, Warner-Lambert

Co., Morris Plain, NJ) and prepared for aseptic surgery.

Ovariectomy was performed 24 h after the last FSH injec-

tion, 48 h after PGFZa. Laparotomy and post-operative care

were as described below for embryo transfer.

Oocyte Treatment

In a controlled environmental room at 38#{176}C,the ovaries

were placed in a sterile petri dish containing 10 ml HEPES-

buffered medium 199, with Earle’s salts and L-glutamine (H-M199; Sigma Chemical Co., cat. no. 2520, St. Louis, MO)

modified by addition of sodium pyruvate (112 p.g/ml) and

gentamicin sulfate (50 �g/ml), mH-M199 without BSA or

serum. Under a stereomicroscope (16x), all large follicles

greater than 7 mm in diameter, were aspirated using a 20-

gauge needle attached to a 1-cc syringe. Oocytes with sur-

rounding cumulus cells were washed (4 times) in drops of

mH-M 199 without BSA or serum and evaluated for matu-

ration. Matured oocytes, i.e. with expanded cumulus cells

and/or visible first polar bodies, were randomly transfered

to droplets of medium for in vitro insemination, which was

initiated within 3 h of ovariectomy. Oocytes with signs of

degeneration were not used. Immature oocytes were sim-

ilarly obtained following dissection of small antral follicles

(1-6 mm in diameter) in mH-M199 without BSA or serum.

The oocyte/cumulus complexes selected as previously de-

scribed in the cow [22] were randomly assigned to treat-

ment groups and cultured for 24-25 h in bicarbonate-buff-

ered M199 (b-M199) with Earle’s salts and L-glutamine (Sigma,

cat. no. M5017) to which additions of sodium pyruvate (50

�g/ml), glucose (5.5 mg/mI), gentamicin sulfate (50 pg/

ml), and goat serum (heat-inactivated and mycoplasma-free;

GIBCO, Grand Island, NY) to 20% (v/v) were made. This

medium (mB-M199) was further supplemented with 100 �g

LH (NIADDK-0LH-26)/ml [21] or 5 p�g FSH (NIADDK-0FSH-

17)/mI [20] or nothing (no added hormone control). Con-

tamination of the NIADDK-oLH-26 with other anterior

pituitary hormones was negligible; determined by RIA,

contamination as percent by weight was <0.1% GH, <0.5%

TSH, <0.5% FSH, <0.1% PRL, and <0.5% vasopressin on

a molar basis (Parlow, A.F., Technical report no. 157, Pi-

tuitary Hormones and Antisera Center, 1000 W. Carson St.,

Torrance, CA 90509). The NM and all subsequent incuba-

tions of ova were maintained in droplets of medium under

silicon oil (Aldrich Chemical Co., Inc., Milwaukee, WI) and

a humidified 5% C02, 5% 02, and 90% N2 atmosphere at

38#{176}C.

Sperm Treatment and IVF

Fresh semen was obtained by electroejaculation or via

an artificial vagina from a purebred Nubian buck of proven

fertility. Three approaches identified by media tested for

sperm preparation were mDM, mTALP, and mH-M199. More

specifically, the three media were as follows: (1) defined

medium DM [1), prepared with 6.0 mg BSA/ml (rather than

3.0 mg/mI) and with gentamicin sulfate (50 �.tg/ml) and no

penicillin added, designated here as mDM; (2) Tyrode’s

medium modified to include albumin, lactate, and pyru-

vate, TAIP [2) prepared with HEPES (2.3 mg/ml) and with

other additions described by Parrish et al. [H-TALP, 3], re-

ferred to as mTALP here; (3) H-M199 supplemented as

described above and with 6.0 mg BSA/ml, designated mH-

M199. Each of these media contained BSA that was essen-

tially fatty acid-free (Sigma, cat. no. A-751 1). To each of the

above sperm-containing media, heparin (sodium salt, GradeII from porcine intestinal mucosa; Sigma, cat. no. H7005)

was added for 5 mm (10 p.g/ml) prior to transfer of sperm

to oocytes (insemination). Caffeine (388 �tg/ml final con-

centration) was also included in the mDM sperm treatment.

Routinely, a combination of two fresh ejaculates, from the

same buck, were diluted with 1.0 ml sperm medium to be

tested (mDM, mTALP, or mH-M199) just after collection and

initial gross evaluation of the semen (volume, color, gen-

eral appearance) and prior to examination for motility, con-

centration, and morphology. Sperm and IVF procedures were

similar to those used for cattle [21, 22]. Briefly, in each ex-

periment 0.2 ml of sperm suspension was overlaid with 1

ml sperm medium in each of six small test tubes (12 X 55-

mm Falcon, Becton-Dickinson Labware, Lincoln Park, NJ)

held at a 45#{176}angle at 38#{176}Cfor 1 h. Then, the top 0.8 ml

from each tube (swim-up fraction) was removed and pooled

into a sterile centrifuge tube (15 ml) and centrifuged at 350x g for 10 mm. After the supernatant was discarded, the

sperm pellet was resuspended with 4 ml fresh medium and

centrifuged as before. The resulting sperm pellet was trans-

ferred into 100 �tl of heparin (10 p�g/ml)-containing sperm

medium in a microcentrifuge tube. At this point, 388 p.g/

ml caffeine was also included in the mDM treatments.

Motility, concentration and morphology were assessed. Si-

multaneously with sperm incubation, oocytes were evalu-

ated (40 x) and those with properly expanded cumuli weretransferred into droplets. Aliquots of sperm suspensions (6-

10 �.d) to give a final concentration of 106 sperm/mi were

added to the droplets, each 50-pA droplet containing 5 oo-

cytes. Oocytes were incubated with sperm for 24 h.

In Vitro Culture and Embryo Transfer

Ova were removed from sperm-containing media for co-

culture on a monolayer of cumulus cells in mH-M199 pre-

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IN VITRO FERTILIZATION OF GOAT OOCYTES 1179

pared with 10% goat serum (v/v) and no BSA. The pro-

cedure for preparation of the cumulus cell monolayer was

similar to that used for cattle [22]. This involved culture of

cumulus cells isolated during oocyte retrieval and inde-

pendently cultured until their use as monolayers at the end

of the insemination interval. Ova were examined for pro-

gressive development in culture at successive 24-h inter-

vals.

Animals were taken off feed and water 24 h prior to

administration of drugs for anesthesia. For embryo transfer

the reproductive tract was exposed via a lateral flank inci-

sion in does previously treated with acepromazine (0.2 mg/

kg, Bay Vet Division, Miles Laboratories), xylazmne (0.13 mg/

kg), and ketamine (2-3 mg/kg). Embryos in 2- to 4-cell

stages were surgically transferred via a tomcat catheter (4.5

cm, 3.5 French, open-ended catheter; Sherwood Medicals,

St. Louis, MO) into recipients’ oviducts ipsilateral to cor-

pora lutea. Five embryos in less than 50 p.l medium (mH-M199 with 10% goat serum) were deposited approximately

2 cm into the oviductal ampulla of each of nine recipient

does given PGF2a 120 h earlier. Closure was routine and

recovery from anesthesia was complete within 3 h. Goats

were maintained in the surgical area until standing, then

moved to a stall where food and water were provided dur-

ing a 24-h interval before return to pasture. Antibiotics were

used as indicated and additional postoperative care includ-

ing suture removal was routine.

Statistical Analyses

Data included the numbers of large follicles (>7 mm in

diameter) per doe (both ovaries) and the numbers of ma-

tured and immature oocytes recovered per doe; data are

reported as mean ± SEM per treated donor and per un-

treated donor; proportions of oocytes completing matura-

tion (maturation rate); each mature ovum exhibiting an ex-

panded cumulus oophorous, Metaphase II chromosomal

plate, and/or a first polar body; proportions of inseminated

oocytes (ova) that were fertilized (fertilization rate), as-

sessed at 48 h postinsemination; fertilized ova that had two

pronuclei + sperm tail or that were cleaved; and cleavagerate, indicating proportion of inseminated ova that cleaved

to at least the 2-cell stage within 48 h after insemination.

Representative ova (5-10) from each group were mounted

on slides, fixed in acetic alcohol for 24 h, and stained with

1% aceto-orcein stain. Nomarski optics facilitated identifi-

cation of Metaphase II chromosomal configurations, first

polar bodies, pronuclei and sperm tail remnants. Degen-

erated oocytes were found with heterogeneous-appearingcytoplasm, fragmentation, absence of a normal surrounding

complement of cumulus cells, etc.Proportions were compared by Chi-square analysis and

differences of p <0.05 were considered significant.

RESULTS

Qocyte Recovery and Hormonal Responses

The numbers of visible large follicles and the mean

numbers of oocytes isolated from treated and untreated do-

nors are shown in Table 1. The mean number of large fol-licles (at least 7 mm in diameter) seen (both ovaries) after

hormonal superstimulation was 33.7 ± 14.9 (range of 9-

65), which was 15-fold higher than for untreated donors

(2.2 ± 1.0). Total oocytes aspirated from ovaries of treated

does averaged 29.5 ± 10.6; of these, a mean of 3.9 (13.2%)

were degenerated and, hence, eliminated from further study.

Total oocytes aspirated from ovaries of untreated does av-

eraged 17.2 ± 4.4; on average, only 0.8 (4.7%) were dis-

carded due to their degenerated appearance at time of re-

covery. Numbers of oocytes aspirated from large follicles

judged to be in vivo-matured by their expanded cumulus

cells and/or first polar bodies are reported (Table 1). The

discrepancy between numbers of matured oocytes (2.8) and

large follicles (2.2) aspirated in the untreated group might

be explained by recovery of mature oocytes from medium-sized (5-6 mm diameter) follicles or aspiration of adjacent

follicles. Fixation and staining of representative ova en-

abled confirmation of nuclear maturation as evidenced by

Metaphase II chromosome plates and/or first polar bodies.

The mean number of in vivo-matured oocytes was 13.8 ±

6.0 per treated donor, 5-fold higher than for nontreated do-

nors (2.8 ± 1.8). Immature oocytes recovered from follicles<7 mm diameter of hormonally treated and untreated doeswere characterized by the presence of compact and unex-

panded cumuli and homogeneously dark ooplasm. Thesewere cultured and matured in vitro in experiments re-ported below. The mean numbers of immature oocytes se-

TABLE 1. Comparison of large ovarian follicles and oocytes recovered from hormonally

superstimulated and untreated doe goats.

Hormonallytreated

No. ofdoes

Num bers per doe (mean ± SEMI

Large follicles±7 mm diameter

In vivo-maturedoocytes

Immature oocytes

from follicles<7 mm diameter

Yes

No1211

33.7 ± 14.922b ± 1.0

13.8� ± 6.0

28d ± 1.811.8 ± 3.413.6 ± 3.2

“Hormonal treatment (3-day protocol described in text) consisted of 20 mg FSH and 125 �g PGF2�#{149}�da > b, c > d (p < 0.05).

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1180 YOUNIS El AL.

TABLE 2. Results of mDM, mTALP, and mH-M199 sperm and insemination treatments and use of in vivo-matured oocytes for goat IVF.”

Treatment

Number of ova

Inseminated Fertilized (%)t Cleaved (%)� Unfertilized (%) Degenerated (%)

mDM 55 25 (45.5)” 8 (1451d 20 (36.4) 10 (18.2)”mTALP 52 10 (19�2)b 0 (0.0)” 12 (23.1) 30 (57.7)9mH-M199 50 18 (36.0)c 4 (8.0)’ 13 (26.0) 19 (38.0)’

“Mature oocytes obtained from follicles >7 mm diameter from hormonally treated donors.

tPronuclear and/or cleaved.tTwo- and 4-cell stage reached within 48 h after insemination.

> b (p < 0.025); a, c and b, c (n.s.d.); d > e (p < 0.025); d, f and e, f (n.s.d.); g < h (p < 0.025); h, i and g, i

(n.s.d.).

lected for culture from ovaries of treated and untreated do-

nors were not significantly different (11.8 for treated donors

and 13.6 for untreated donors). Proportions of oocytes that

evidenced degeneration, assessed at 48 h after recovery, were

higher (p < 0.05) from treated donors than from untreated

donors, i.e. 49 of 157 (31.2%) vs. 17 of 88 (19.3%), re-

spectively.

IVF of Oocytes from Hormonally Treated Donors

Data summarized in Tables 2 and 3 reveal differences in

fertilization and cleavage rates according to different con-

ditions for sperm and insemination. The approach identi-

fied with use of mDM was significantly better than those

identified with mTALP and mH-M199 (p < 0.05) as re-

flected by cleavage rates for both in vivo- (Table 2) and in

vitro- (Table 3) matured oocytes. Cleavage represented

continued viability in contrast to fertilization, which in-

cluded the pronuclear stage. At 48 h postinsemination, the

pronuclear stage characterized development severely de-

layed at best and most likely, arrested. The highest fertil-

ization rate was obtained when mDM was used as sperm

and fertilization medium for oocytes matured in vitro withLH; this rate was significantly greater than for oocytes ma-

tured in vivo (56.7% vs. 45.5%,p < 0.05). Only a few oo-

cytes were fertilized when sperm and ova were treated ex-

actly according to conditions used routinely for successful

cow IVF (mTALP). The use of mTALP was associated withhigh rates of degeneration and death of gametes. Heparin

and caffeine in the mOM treatment resulted in effective goat

sperm preparation for IVF. Proportions of goat spermato-

zoa that were vigorously motile (after swim-up) ranged from

90-99%. Motility was well-maintained for up to 3 h in all

three media used. After capacitation treatments, i.e. at the

time of insemination, 60-75% of spermatozoa exhibited

progressive motility. Morphological observations consis-

tently indicated more than 90% normal spermatozoa in se-

men and less than 1% abnormal forms in suspensions pre-

pared for insemination.

IVF of Qocytes from Untreated Donors

Experiments were carried out to investigate the effects

of LH (100 p.g/ml) or FSH (5 p.g/ml) during in vitro mat-

uration on fertilization and development of goat oocytes

obtained from ovaries of untreated does. Data summarized

in Table 4 show that the addition of LH or FSH enhanced

cumulus expansion and maturation of oocytes cultured for

24-25 h over the “no-added-hormone” control (p < 0.05).

The fertilization rate was similar for LH and FSH treatments.

However, the highest proportion of cleavage development

was seen after the LH treatment for NM, i.e. 39.5% vs. 23.3%

and 11.1% after NM with FSH and no-added-hormones, re-

spectively (� < 0.05). Only two ova (unfertilized) were de-

generated in these experiments and both were matured with

FSH.

Development of eight of 22 early embryos to the 8- to

16-cell stage was observed after in vitro coculture with cu-mulus cells; six of the 8- to 16-cell embryos developed from

oocytes matured with LH and, two from oocytes matured

TABLE 3. Results of mDM, mTALP, and mH-M 199 sperm and insemination treatments and use of in vitro-matured oocytes for goat IVF.

Treatment

Number of ova

Inseminated Fertilized (%)t Cleaved (%)� Unfertilized (%) Degnerated (%)

mDM 30 17 (56.7)” 10 (33#{149}3)C 10 (33.3) 3 (10.0)”

mTALP 30 8 (267)b3 (100)d 12 (40.0) 10 (33.3)’

mH-M199 28 10 (35.7)” 3 (,07)d 14 (50.0) 4 (14.3)”

“Oocytes from follicles 1-6 mm diameter from hormonally treated donors were matured in presence of 100 �g LH/ml of mB-M199.

tPronuclear and/or cleaved.*Two- and 4-cell stage reached within 48 h after insemination.

> b, c > d, e < f (p < 0.025).

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TABLE 4. Effects of in vitro maturation of goat oocytes with LH (100

�g/ml) or FSH (5 �g/ml) on IVF and cleavage.”

Number of ova

“Sperm preparation and IVF were in mDM.

tPronuclear and/or cleaved.

lTwo- to 4-cell stage reached within 48 h after insemination.

“9a>b,c>d,e>f>g(p<0.05).

IN VITRO FERTILIZATION OF GOAT OOCY�ES 1181

Maturation

treatment

Oocytes

cultured

Matured

(%)Fertilizedt

(%)Cleaved�

(%)

LH 38 38 (100.0)” 26 (68.4)’ 15 (39.5)”

FSH 33 30 (90.0)” 18 (60.7)’ 7 (23.3)’

mB-M199 15 9 (506)b 4 (444)d 1(11.1)”

with FSH (Table 4). Tubal transfer of five 2- to 4-cell stage

embryos developing after maturation in vivo with LH and

IVF in mDM to each of nine recipients resulted in three

pregnancies confirmed by ultrasonography. Although three

of nine recipients sustained embryonic development initi-

ated by IVF, resorption occurred in two recipients at ap-

proximately 2 mo, and the other pregnancy resulted in

abortion of a 3.5-mo fetus.

DISCUSSION

This report is the first to document pregnancy initiation

by embryo transfer after in vitro maturation and fertiliza-

tion of goat oocytes. Abortion of one and resorption of the

other two pregnancies suggested transfer of five embryos

to each recipient doe to be excessive and inappropriate to

allow complete gestational development. The initial report

of term development in this species involved use of in vivo-

matured oocytes [8]. Our goal was to establish an IVF sys-

tem whereby goat oocytes matured in vivo or in vitro could

be fertilized and cultured to stages suitable for gene injec-

tion and embryo transfer. 1W technology requires meth-

odologies for sperm preparation, oocyte maturation, fertil-

ization, and for fulfilling developmental potential in

conjunction with embryo transfer to appropriate recipients.

Heparmn added in all sperm treatments appeared to be ef-

fective for in vitro capacitation of goat spermatozoa. The

medium seemed to be more determinant than the pres-

ence of heparin in relation to resulting proportions of 00-

cytes undergoing fertilization and cleavage. Best results were

obtained when mDM was used as sperm and fertilization

medium. Heparin apparently binds to spermatozoa and plays

a role in sperm uptake of calcium [12,23]. Heparin has been

shown to capacitate fresh or frozen/thawed bull sperm in

a dose-dependent manner that leads to fertilization rates

above 70% [11]. Niwa and Ohgada [24] reported that caf-

feine acts synergistically with heparmn to enhance efficacy

of sperm for bovine IVF. Addition of caffeine (2 mM) to

heparin in mTALP treatment of goat sperm for IVF did not

enhance results (Younis and Brackett, unpublished data).

For oocyte maturation, two approaches were taken to

rescue ovarian oocytes from their normal fate of degen-

eration and to induce development of others. The first was

superstimulation of does with exogenous hormones to en-

hance folliculogenesis and preovulatory maturation; this was

followed by isolation and insemination of in vivo-matured

oocytes. Second, oocytes were treated to accomplish in vivo

maturation. The numbers of antral follicles developing in

ovaries of hormonally treated goats were greater than for

controls and also greater than reported for the sheep [25]

or for the cow [26, 27]. Effects of exogenous hormones in

stimulation of goat follicles have been investigated [27, 28].

The two most commonly used gonadotropin preparations

for superovulation are eCG and FSH (of porcine origin).

Comparison of effectiveness of these hormones in goats re-

vealed FSH to be superior [29,30,31]. Ovulation and IVF

rates were improved by FSH, although abnormal endocrine

patterns of estradiol, progesterone, and LH were observed

in the treated goats [30].

In the present work, an initial high dose [5.0 mg, BID.)

followed by a constant dose of 2.5 mg FSH twice daily for

2 additional days (20 mg total) led to an average of 33.7

large follicles per animal. This is higher than the average

number of corpora lutea (17.7 per cycle) reported by Nuti

et al. [31] to result from progesterone pretreatment fol-

lowed by a constant dose of 2.5 mg FSH twice daily for 3

days (15 mg total). Including those retrieved from small

follicles requiring in vitro maturation, many more oocyteswere recovered for IVF than were accounted for by grossly

visible (surface) follicles. Oocytes from hormonally treated

donors resulted in lower fertilization and cleavage devel-

opment than those obtained from untreated donors. This

probably reflected improper follicular and oocyte matura-

tion induced by exogenous FSH treatments. This conclu-

sion is further supported by the higher proportion of de-

generation seen among oocytes from hormonally treated

donors. Several experiments have shown that most prep-

arations used for superovulating sheep and cattle adversely

affect a high proportion of the oocytes and lead to abnor-

mal maturation including abnormal protein synthesis [32],

a high proportion of degenerated oocytes [33, 34], prema-

turely activated oocytes [34], and oocytes unable to mature

beyond the diakinetic stage [34]. Callensen et al. [33] have

reported that certain superovulatory treatments have been

associated with disturbed normal oocyte and follicular de-

velopment leading to embryos of inferior quality in the cow.

The ability of a mature oocyte or cumulus-oocyte complex

to fertilize and develop normally into live offspring re-

quires appropriate dynamics within its microenvironment

during the preovulatory, maturational interval.

Current efforts to rescue ovarian oocytes by isolation as

immature oocytes have demonstrated that 90-100% of the

cultured oocytes could be matured to Metaphase II in vitro

in the presence of gonadotropmns and serum. Not unex-

pectedly in light of our recent work in cattle [19,20,25],

best results of fertilization were obtained with oocytes that

were matured in medium containing a high concentration

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1182 YOUNIS ET AL.

of LH. The 68.4% fertilization rate afforded by present con-

ditions was higher than that (57.7%) observed by Song and

Iritani [10] who cultured oocytes with compact cumulus cells

for 25 h and inseminated them with epididymal sperma-

tozoa preincubated for 6 h in a modified KRB medium. Also,

the present fertilization rate was higher than that (49.4%)

described by Hanada [8] and apparently without the high

incidence of polyspermy, 33.3% of fertilized oocytes. Mor-

phological aspects of goat fertilization have been published

[10,36]. Recent progress has been made in culture of IVF

goat embryos up to the 16-cell stage in the presence of

oviductal cells [37]. Attempts to achieve xenogenous fertil-

ization of goat follicular or ovulated oocytes in the rabbit

[16] or sheep [17] oviduct have had limited success; a low

proportion of embryos (2- to 8-cell stage) have been shown

to develop into blastocysts or live offspring.

IVF in many species has evolved into a useful research

tool. Animal biotechnology programs hold promise for

achieving, in a single generation, improvements in com-

mercial livestock. Application of IVF technology in the goat

promises more rapid success in gene transfer experimen-

tation than is possible in cattle and other livestock with longer

generational intervals. An aim of experiments reported here

was to obtain a large number of synchronized zygotes suit-

able for microinjection of novel genes. Other applications

of IVF technology in goats will include improvements in

production by extending the use of valuable semen, eval-

uation of sperm and/or oocyte quality, and in many facets

of experimental embryology.

ACKNOWLEDGMENTS

The authors thank Amanda Latimer for her support and encouragement; Dr. Roy

Silcox, Levent Kestintepe, and Katharine Mackie for assistance in various aspects of

this project; and Joanne Foster for secretarial assistance. The gift of purified pituitary

hormone from NIDDK of Maryland School of Medicine, is gratefully acknowledged.

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