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Theriogenology 69 (2008) 349–359

In vitro oocyte maturation, fertilization and culture after ovum

pick-up in an endangered gazelle (Gazella dama mhorr)

F. Berlinguer a, R. Gonzalez b, S. Succu a, A. del Olmo b, J.J. Garde c,G. Espeso d, M. Gomendio b, S. Ledda e, E.R.S. Roldan b,*

a Department of Animal Biology, Universita degli Studi di Sassari, 07100 Sassari, Italyb Reproductive Ecology and Biology Group, Museo Nacional de Ciencias Naturales (CSIC), c/Jose Gutierrez Abascal 2,

28006 Madrid, Spainc Instituto de Investigacion en Recursos Cinegeticos (CSIC-UCLM-JCCM), 02071 Albacete, Spain

d Estacion Experimental de Zonas Aridas (CSIC), 04001 Almerıa, Spaine Institute of General Pathology, Pathological Anatomy, and Veterinary Obstetrics – Surgery Clinic,

Universita degli Studi di Sassari, 07100 Sassari, Italy

Received 22 June 2007; received in revised form 1 October 2007; accepted 2 October 2007

Abstract

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of

biological material of great genetic value for the establishment of genetic resource banks of endangered species. Studies exist on

sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte

collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian

stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-

up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56

punctured follicles (62%) (N = 6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal

vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of

matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6–8 cell stage but none progressed to the

blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more

studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates

for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-

up from FSH-stimulated endangered gazelles.

# 2008 Elsevier Inc. All rights reserved.

Keywords: Oocyte; Sperm; Fertilization; Embryo; Gazelle

1. Introduction

Habitat preservation is a priority in biodiversity

conservation, but it is often difficult to avoid habitat

* Corresponding author. Tel.: +34 91 411 1328x1245;

fax: +34 91 564 5078.

E-mail address: roldane@mncn.csic.es (E.R.S. Roldan).

0093-691X/$ – see front matter # 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2007.10.001

destruction and, in some endangered species, other

causes such as over-hunting are responsible for the

declines in population size. Therefore, complementary

strategies for biodiversity conservation, such as captive

breeding, the establishment of genetic resource banks

(GRBs) and the development of assisted reproductive

techniques (ARTs) are needed. Genetic resource banks

preserve current levels of genetic variability, facilitate

genetic flow between natural and captive populations

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359350

[1–4], and could be regarded as a form of ‘‘life

insurance’’ in case wild populations continue their

decline.

The World Conservation Union has recognized the

potential role that captive breeding may play in

conservation [5] and has recommended that taxa with

low numbers of individuals in the wild should be

considered for captive breeding. However, reproductive

processes may be impaired in captivity as a consequence

of behavioral or genetic incompatibilities, inadequate

diet, disease, or due to the risks inherent to small

population size such as inbreeding depression [6].

Reproductive biotechnologies can improve the repro-

duction of wild animals in captivity, allowing for the

recovery and future use of reproductive biomaterials, and

facilitating the exchange of gametes both within and

between populations thus avoiding the negative effects of

inbreeding and overcoming behavioral incompatibilities

between individuals. Various ARTs have been applied

successfully in several species of wild mammals [7–12].

The Parque de Rescate de la Fauna Sahariana of the

Estacion Experimental de Zonas Aridas (EEZA), in

Almerıa, Spain, was created in 1971 with the objective of

avoiding the extinction of the large ungulates of the

Western Sahara. Mohor gazelle (Gazella dama mhorr

Bennet 1833) is a subspecies of dama gazelle for which a

breeding program has been established. The mhorr

subspecies occupied the western strip of the Sahara desert

and became extinct in the wild in 1968 due to excessive

hunting. The Red List of Threatened Species [13]

categorizes G. dama as ‘‘critically endangered’’ and the

species is included in Appendix I of CITES. The captive

population of Mohor gazelles was founded with two

males and nine females [14], and this conservation

program has been managed carefully to minimize

inbreeding effects. At present the captive population is

distributed among several institutions in Europe and

North America, all derived form the original population

bred in Almerıa [14]. At the end of 2003, the total captive

population of Mohor gazelle consisted of 277 indivi-

duals, with 183 individuals (66.5%) within the European

Breeding Program (EEP), and 92 individuals (33.5%) in

the North American Breeding Program (SSP) [14].

The success of captive breeding programs initiated

with a small founding population relies to a great extent

on avoiding the deleterious effects of inbreeding on

reproduction [15]. There is evidence showing the

deleterious effect of inbreeding on reproductive success

both in male and female Mohor gazelles and in other

related endangered North African gazelles [16].

Inbreeding adversely affects ejaculate quality in

gazelles with relatively high coefficients of inbreeding

[17,18]. Reproductive performance is negatively

affected as the coefficient of inbreeding increases in

Cuvier’s (G. cuvieri), Mohor and dorcas (G. dorcas

neglecta) gazelles [19]. At the EEZA, although efforts

have been aimed at minimizing the levels of inbreeding

in the captive populations, there are several limiting

factors: the population is small in size, effective

population size is even smaller because space restric-

tions do not allow all animals the opportunity to breed,

and some individuals die before they can reproduce. In

addition, moving animals between different populations

is risky and costly. These problems can be overcome by

cryopreserving gametes from most of the individuals so

that they can be used to increase their reproductive

opportunities even beyond death, and to facilitate gene

exchange between populations. Previous work has

focused on the management of natural breeding and on

the development of semen freezing protocols for the

three species to initiate a GRB [16,20,21]. No studies

have been carried out on the collection, maturation and

fertilization of oocytes in endangered gazelle.

The development of procedures in endangered

gazelles for in vitro maturation, fertilization and culture

(IVMFC) would allow the rescue of biomaterials from

females with genetic value for establishing GRBs.

Oocyte collection by laparotomy or laparoscopic pick-

up, followed by IVMFC, has been extensively studied in

domestic ruminants [22,23] and several studies have

been performed in non-domestic ruminant species

including red or sika deer [24,25], and mouflons

[26,27]. Several factors affect the success of follicle

growth stimulation [28,29], and ovum pick-up (OPU)

[30], including the possibility of repeated collections

[31]. In addition, conditions for in vitro maturation and

fertilization have been described [22,23].

The aims of the present study on G. dama mhorr

were to develop a protocol for (1) ovarian stimulation

for the recovery of immature oocytes, (2) in vitro oocyte

maturation, fertilization and embryo culture and (3) to

evaluate the ability of cryopreserved spermatozoa to

fertilize in vitro matured oocytes. We regard the

development of these techniques to be a first step

towards the rescue of female reproductive biomaterials

for the preservation of genetic diversity.

2. Materials and methods

2.1. Reagents

All chemicals used in the present study were

purchased from Sigma Chemical Co. (Madrid, Spain),

unless otherwise indicated.

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359 351

2.2. Animals

All animal procedures were performed in accordance

with the Spanish Animal Protection Regulation,

RD223/1988, which conforms to European Union

Regulation 86/609. Procedures also adhered to the

Guiding Principles for Biomedical Research Involving

Animals, as issued by the Council for the International

Organizations of Medical Sciences, and recommended

by the journal.

The study was carried out in December 2004, using

seven healthy mature (aged >2 years) female Mohor

gazelles from the breeding herd maintained and

managed by the Estacion de Zonas Aridas (EEZA) at

the Parque de Rescate de la Fauna Sahariana (PRFS) in

Almerıa, south-eastern Spain. The PRFS is located at

3685001000N and 282704800W and is 100 m above sea

level. The animals were kept in a group in the same

enclosure without the presence of males or young. The

enclosure was visually opened to the exterior and was

provided with shadow areas. Their diet consisted of

commercial pellets, fresh alfalfa hay and barley grain.

Water and mineral salts were available ad libitum.

2.3. Estrous synchronization and stimulation of

follicular growth

Estrous cycles in the gazelle were synchronized by

insertion of controlled progesterone internal drug

release devices (CIDR, type G, 330 mg progesterone;

InterAg Hamilton, New Zealand) for 15 days. CIDRs

were replaced on day 10, and removed during ovum

pick-up (OPU) on day 15. Follicular growth was

stimulated by the administration of a total of 5.28 mg of

ovine FSH (Ovagen, ICPbio, Auckland, New Zealand)

given in four equal doses administered at 8:00 and

18:00 h on days 13 and 14. One female was removed

from the experiment because of stress during handling;

data from this animal were excluded from analyses.

Blood samples were collected for plasma progester-

one and 17b-estradiol quantification on days 0 (before

insertion of CIDR) and 10 (before replacement of

CIDR), at each time of FSH administration (day 13,

8:00 h and 18:00 h; day 14, 8:00 h and 18:00 h) and

before OPU on day 15. Blood samples were centrifuged

at 3500 rpm for 15 min, and plasma was recovered and

frozen at �20 8C and stored at �80 8C until assayed.

Progesterone and 17b-estradiol concentrations were

determined in duplicate by radioimmunoassay at the

Laboratoire de Dosages Hormonaux, Institut Nationale

de la Recherche Agronomique (Tours, France).

2.4. Oocyte recovery

Cumulus-oocyte complexes (COCs) were recovered

using a semi-laparotomy technique. Female donors

were anesthesized with a combination of an intramus-

cular administration of xylazine (0.2 mg/kg body

weight; Rompun, Bayer, Barcelona, Spain) and

intravenous injection of ketamine chlorohydrate

(15 mg/kg body weight; Imalgene 1000, Merial, Lyon,

France) and then secured in a cradle in dorsal

recumbency. When necessary, surgical anesthesia was

maintained using halothane inhalation. The effects of

xylazine were reversed by intravenous administration of

yohimbine chlorohydrate (0.03 mg/kg body weight;

Sigma, Madrid, Spain).

Ovum pick-up was carried out following procedures

described previously [28]. Briefly, an endoscope was

inserted into the abdominal cavity about 10–15 cm

cranial to the udder, and 4–5 cm to one side of the

midline, while atraumatic grasping forceps were

inserted in a contralateral position through a 3–4 cm

long laparotomy incision. After endoscopic visualiza-

tion, the ovaries were fixed at their ligament origin and

exposed through the laparotomy incision. Follicles

present on the ovarian surface were counted and

aspirated with a 2-ml syringe fitted with a 25-gauge

needle carrying 0.5 ml of warm medium, placed in a

15 ml plastic tube (Falcon, Becton Dickinson, Lepont

De Claix, France) and kept in a water bath at 38 8C until

processed. The collection medium was Hepes-buffered

TCM-199 supplemented with antibiotics (50 mg/ml

streptomycin plus 50 IU/ml penicillin), polyvinyl-

alcohol (PVA, 0.1%, w/v) and heparin (15 IU/ml).

After each OPU session, the ovaries were washed with

warm physiologic solution.

2.5. In vitro maturation

The COCs derived from each female donor were

kept separate throughout the in vitro procedures.

Following oocyte collection, the content of each tube

was poured into a 60 mm Petri dish (Falcon #35-1007,

Becton Dickinson) and observed under a stereomicro-

scope (Nikon SMZ-1500). Oocytes that were degener-

ated and/or with expanded cumulus cells were removed.

After rinsing three to four times in Hepes-buffered

TCM-199 with PVA, heparin and antibiotics, the COCs

were placed in four-well culture dishes (Nunclon,

Nalgene, Nunc International, Roskilde, Denmark)

containing the in vitro maturation medium (TCM-199

plus 10% v/v heat-treated estrus sheep serum, 10 mg

ovine FSH/LH/ml, 1 mg estradiol/ml and 0.1 mg

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359352

glutamine/ml), covered with 250–300 ml of mineral oil

and cultured at 38.5 8C under 5% CO2 in air and

maximum humidity.

2.6. In vitro fertilization

After 24 h, oocytes were washed in Hepes-buffered

TCM-199 followed by gentle pipetting with the aid of

an automatic micropipette to dissociate surrounding

cumulus cells. Fertilization medium consisted of

synthetic oviduct fluid (SOF) [32] supplemented with

heat-inactivated 2% (v/v) estrus sheep serum, 1 mg

hypotaurine/ml and 10 mg heparin/ml. Oocytes were

inseminated with cryopreserved spermatozoa. To avoid

confounding effects due to male-to-male variations, the

same male was used to fertilize oocytes from different

females. In preliminary trials, semen samples from

various males were tested for cryosurvival.

Semen was collected by electroejaculation using a

sine-wave stimulator (P.T. Electronics, Boring, OR,

USA) and evaluated for volume, concentration,

motility, viability, membrane integrity, morphology,

and acrosome integrity, as described previously [21]. A

sperm motility index (SMI) was calculated as follows:

[sperm % motility + (quality of motility � 20) � 1/2].

Semen samples were centrifuged at 700 � g for 5 min

and resuspended in a Tes-Tris–glucose diluent with 5%

egg yolk and 6% glycerol [21]. Sperm suspensions

(adjusted to 400 � 106 spermatozoa/ml) were loaded

into 0.25 ml plastic straws, cooled to 5 8C for 1.5 h and

allowed to equilibrate for an additional 2 h. The straws

were frozen in nitrogen vapors for 10 min and then

plunged into liquid nitrogen. Straws were thawed for

30 s at 37 8C in a water bath and the content of each

straw was poured into a conical glass tube and assessed

for sperm motility and acrosome integrity. Spermatozoa

were selected by using a swim-up procedure. Briefly,

spermatozoa in the cryodiluent (0.25 ml) were placed

gently in the bottom of a round-bottom glass tube

(15 mm � 75 mm) which contained 1 ml SOF medium

(as used for IVF) and incubated at 38.5 8C under 5%

CO2 in air. After 30 min the upper layer of 0.3 ml was

removed (aspirating the sperm suspension at a point

located 3 mm below the surface), and sub-samples were

evaluated for sperm motility and acrosome integrity.

Aliquots of 50 ml of sperm suspension (from the upper

layer) were transferred to 1.5-ml tubes and further

incubated at 38.5 8C under 5% CO2 in air for 1 or 24 h.

Sub-samples were taken for assessment of motility and

acrosome integrity.

The final concentration of spermatozoa used during

IVF was 1 � 106 spermatozoa/ml. Gametes were co-

incubated in four-well culture dishes under mineral oil

for 24 h at 38.5 8C under 5% O2, 5% CO2 and 90% N2.

Sperm samples obtained at the end of gamete co-culture

were examined for motility and acrosome integrity.

2.7. In vitro culture

After 24 h of gamete co-culture, presumptive

zygotes were washed in culture medium to remove

spermatozoa and cell debris. They were transferred to

500 ml of SOF medium supplemented with 0.4% BSA

(w/v) and nonessential and essential amino acids at

oviductal concentrations [33] under mineral oil and

cultured in a humidified atmosphere at 38.5 8C under

5% O2, 5% CO2 and 90% N2. Cleaving embryos were

separated from 1-cell presumptive zygotes after 36 h of

culture. Embryo development was monitored for

several days, every 24 h, until it was arrested.

2.8. Evaluation of meiosis progression

The resumption and progression through meiosis

was evaluated retrospectively after in vitro fertilization.

Non cleaved oocytes and embryos were stained with

Hoechst 33342 and propidium iodide (10 mg/ml each)

and visualized in a microscope (Nikon E-400) equipped

with epifluorescence to determine maturation status

and/or fertilization by the presence of �2 pronuclei or

decondensed sperm heads. Fluorescence of Hoechst and

propidium iodide was observed simultaneously using an

Hg excitation beam and a filter set (UV-2A Nikon)

consisting of a UV330-380 excitation filter, a DM400

chromatic beam splitter and a 420 barrier filter.

3. Results

3.1. Oocyte harvesting, culture and fertilization

Of 56 follicles aspirated from six females (9.3 � 1.7

follicles per female), 35 COCs were recovered

(5.8 � 1.5 oocytes per female), resulting in a overall

recovery rate of 62% (mean � S.E.M., 58.6 � 9.2%

recovery per female) (Table 1).

A total of 29 cumulus-surrounded oocytes (Fig. 1)

were selected for in vitro maturation (83% of recovered

oocytes). When examined after culture, 3% (1/29) were

found at the germinal vesicle (GV) stage, 7% (2/29) at

metaphase I (MI), 21% (6/29) were degenerated or not

classified, and 69% (20/29) advanced to metaphase II

(MII) (Table 1, Figs. 1 and 2).

Semen collected from four males was evaluated pre-

and post-cryopreservation and one was selected for IVF

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359 353

Table 1

Summary of results of ovum pick-up and in vitro culture of immature oocytes from Gazella dama mhorr

Animal Recovery ratea (%) Maturation rateb (%) Fertilization ratec (%) Cleavage rated (%)

ND 778 3/5 (60) 2/3 (66.7) 2/2 (100) 2/2 (100)

ND 917 1/5 (20) 1/1 (100) 1/1 (100) 0/1 (0)

ND 1158 11/16 (68.8) 8/9 (88.9) 2/8 (25) 2/8 (25)

ND 1160 5/9 (55.6) 3/4 (75) 2/3 (66.7) 1/3 (33.3)

ND 1191 7/12 (58.3) 3/7 (42.9) 1/3 (33.3) 1/3 (33.3)

ND 1219 8/9 (88.9) 3/5 (60) 0/3 (0) 0/3 (0)

Total 35/56 (62.5) 20/29 (68.9) 8/20 (40) 6/20 (30)

Mean � S.E.M. (range) 58.6 � 9.2 (20–88.9) 72.2 � 8.4 (42.9–100) 54.2 � 16.9 (0–100) 31.9 � 14.9 (0–100)

Data are shown for each individual animal and as total and mean � S.E.M. (range) results of the experiment. Recovery rate is expressed as recovered

oocytes over the total of punctured follicles, maturation rate as matured oocytes over the number of COCs included in culture, and fertilization and

cleavage rates over the number of matured oocytes.a Recovery rate: number of COCs recovered/number of punctured follicles.b Maturation rate: number of matured oocytes/number of COCs included in culture.c Fertilization rate: number of oocytes fertilized/number of matured oocytes.d Cleavage rate: number of cleaving embryos/number of matured oocytes.

based on superior sperm survival during cryopreservation

and subsequent post-thaw incubation for 2 h at 37 8C.

Fresh semen characteristics for the male (ND 932)

designated to use for IVF were: volume, 4.95 ml; sperm

concentration, 600 � 106 spermatozoa/ml; motility,

Fig. 1. Oocytes recovered by ovum pick-up (OPU) and embryos obtained aft

Cumulus-oocyte complex (COC) recovered by semi-laparoscopic OPU with

vitro maturation; (B) COC included in maturation medium; (C and D) 4-cell a

BSA and amino acids under 5% CO2, 5% O2, 90%N2. Bar = 20 mm.

90%; morphologically normal spermatozoa, 86% and

intact acrosomes, 95%. Sperm characteristics immedi-

ately after thawing and after 2 h incubation were:

motility, 65% vs. 50% and intact acrosomes, 62% vs.

62%, respectively. Sperm characteristics after swim-up

er in vitro maturation, fertilization and culture from Mohor gazelle. (A)

expanded cumulus cells and degenerated oocyte, not selected for in

nd 8-cell embryos after in vitro culture in SOF supplemented with 0.4%

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359354

Fig. 2. Retrospective assessment of meiosis progression after 36 h in vitro fertilization in SOF. (A) Germinal vesicle; (B) metaphase I with

disorganized plate (C) metaphase I; (D) metaphase II, (E) fertilized oocyte with decondensing chromosomes and sperm head showing chromosome

decondensation (arrow). Oocytes were stained with Hoechst 33342 and propidium iodide and examined using fluorescence microscopy.

Bar = 20 mm.

and after co-incubation with COCs are shown in

Table 2. The proportion of motile spermatozoa was

improved after swim-up, although sperm concentration

was reduced from �400 � 106 to an average of

5.6 � 1.5 � 106 (range 1.6–8.9 � 106) spermatozoa/ml

before and after swim-up, respectively. The percentages

of motile and acrosome-intact spermatozoa were reduced

during co-incubation with COCs (Table 2).

Fertilization and cleavage rates of matured oocytes

were 40 and 30%, respectively (Table 1, Figs. 1 and 2).

Six oocytes cleaved after IVF, yielding two 2-cell

embryos, two 4-cell embryos and two 6- to 8-cell

embryos (Fig. 1C and D). None of the embryos

developed to the morula or blastocyst stage.

3.2. Progesterone and estradiol levels during

synchronization and FSH stimulation

Plasma levels of progesterone and 17b-estradiol

during CIDR and FSH treatment are shown in Fig. 3.

Progesterone concentrations increased from the day of

CIDR insertion (0.72 � 0.17 ng/ml) until the day of

replacement of the vaginal device (5.3 � 1.42 ng/ml).

Then, plasma concentrations of progesterone decreased

from the day of CIDR replacement until the beginning

of FSH treatment (0.88 � 0.17 ng/ml). Henceforth,

progesterone levels remained low, despite the presence

of CIDRs. Plasma concentrations of 17b-estradiol

showed a decreasing pattern, reaching its lowest level

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359 355

Table 2

Sperm motility and acrosome integrity after thawing, swim-up and incubation of Mohor gazelle spermatozoa

Sperm parameters Upon thawing After swim-up After 1 h incubation After 24 h incubation At end of IVF (24 h)

Motility (%) 53.1 � 1.9 77.5 � 3.4 64.2 � 5.7 5.1 � 3.5 0.0 � 0.0

Quality of motility (0–5) 1.7 � 0.1 2.7 � 0.1 2.4 � 0.1 0.1 � 0.1 0.0 � 0.0

SMI 43.4 � 1.6 66.3 � 2.6 56.5 � 3.4 3.9 � 2.3 0.0 � 0.0

NAR (%) 66.5 � 2.7 57.0 � 2.5 43.0 � 2.7 16.8 � 2.9 16.0 � 2.3

DAR (%) 30.0 � 2.6 30.3 � 2.6 35.5 � 2.3 38.8 � 4.4 40.2 � 5.2

MAR (%) 3.5 � 0.3 12.8 � 0.5 21.5 � 3.3 44.5 � 3.9 43.8 � 7.5

Spermatozoa (400 � 106 cells/ml) in 0.25 ml straws were thawed at 37 8C for 30 s, transferred to a 12 mm � 75 mm glass tube with 1 ml SOF

medium, and incubated for 30 min at 38.5 8C under 5% CO2 in air. The upper 0.3 ml was recovered and evaluated. Aliquots were incubated for 1 or

24 h under similar conditions or were used for co-culture with oocytes during 24 h. See Section 2 for details. Results are means � S.E.M. (N = 4).

SMI, sperm motility index; NAR, normal apical ridge (i.e., spermatozoa with intact acrosomes); DAR, damaged apical ridge; MAR, missing apical

ridge.

on the morning of day 14, with a slight increase

occurring afterward.

4. Discussion

This study represents, to the best of our knowledge,

the first attempt to generate gazelle embryos by in vitro

maturation of oocytes recovered by using ovum pick-up,

with subsequent in vitro fertilization and culture. Our

results indicate that gazelle embryos can be generated

following this approach and, furthermore, that cryopre-

served gazelle spermatozoa can be successfully used for

in vitro fertilization. Although the results reported here

are based on a limited number of individuals, a common

problem when dealing with endangered species, these

results are important for the future development of

genetic resource banks for this and related species.

Fig. 3. Plasma progesterone and 17b-estradiol levels during treatment

with CIDR and ovine FSH in Mohor gazelles. Day 0, insertion of

CIDR; day 10, replacement of CIDR; day 13 a.m., FSH injection

(8:00 h); day 13 p.m., FSH injection (18:00 h); day 14 a.m., FSH

injection (8:00 h); day 14 p.m., FSH injection (18:00 h); day 15, ovum

pick-up. Results are means � S.E.M.; N = 6 females.

In earlier studies, work on the preservation of gazelle

gametes has concentrated on the characterization of

semen traits [34,35] and in the development of semen

cryopreservation protocols in three endangered gazelle

species, including Mohor gazelle [16,20,21,36]. How-

ever, none of these studies have explored whether

cryopreserved gazelle spermatozoa are able to fertilize

in vitro. In the present study we have used a semen

preparation method (swim-up) for in vitro fertilization

and tested the fertilizing ability of frozen-thawed

spermatozoa. We found that after swim-up there was

an improvement in the proportion of motile sperma-

tozoa, the quality of motility and, hence, the sperm

motility index. On the other hand, after swim-up, sperm

concentration was low, indicating poor recovery of

spermatozoa during this procedure, and the percentage

of cells having an intact acrosome decreased. Incuba-

tion of spermatozoa for up to 24 h led to a reduction

of motility and acrosome integrity over time. The

percentage of spermatozoa with intact acrosomes was

about 57% at the start of incubation, 43% after 1 h of

incubation, and about 17% at the end of incubation. The

value of spermatozoa with intact acrosomes found at the

end of sperm incubation and at the end of the in vitro

fertilization period were very similar (16–17%).

The percentage of fertilization in vitro was rather low

(40%) and it may be due in part to a rapid decrease in

spermatozoa with intact acrosomes during incubation.

Nonetheless, the present results demonstrate that

frozen-thawed Mohor gazelle spermatozoa can function

to fertilize oocytes in vitro.

Several staining methods and sperm incubation

assays have been used for estimating sperm fertilizing

ability. However, in vitro fertilization assays may offer a

more accurate assessment of post-thaw sperm function

in vitro. A positive relation between the sperm motility

index observed after incubating spermatozoa for 3 or

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359356

6 h and fertilization has been found in the scimitar-

horned oryx [37]. A similar relationship has been

described between sperm longevity and in vitro

fertilization success in the cheetah [38] suggesting that

sperm longevity could be a better predictor of potential

fertility than a single assessment of motility after

thawing. These results should be taken in account for

future studies of semen evaluation in gazelles.

Administration of FSH enhances the number of

oocytes obtained by increasing the number of follicles

that, based on their size and morphological character-

istics, are adequate for aspiration [39]. The response of

female gazelles to treatment with ovine FSH revealed a

rather poor rate of follicular growth. In domestic

ruminants, variation in ovarian response to super-

ovulatory treatment relates to many factors, such as

ovarian status at the beginning of treatment, [40,41],

hormonal profile [42], interval between the last

exogenous gonadotropin stimulation and follicle aspira-

tion [43,44] and donor treatment [45,46]. Among

gazelles, failure to respond could be due either to stress

during animal handling or to insufficient FSH doses.

With regards to the former, improvements could

perhaps be obtained in the future by animal training,

with repeated captures and handling, that may

eventually lead to reduced stress. Another option to

minimize stress could be the use of long-acting

neuroleptics. Use of tranquilizers such as perfenazine

enanthate or haloperidol chlorohydrate in Spanish ibex

(Capra pyrenaica) females has allowed the collection of

transferable embryos after a superovulatory treatment

with ovine FSH [47]. Moreover, administration of the

long-acting neuroleptic flufenazine decanoate prior to

collection of oocytes from European mouflon (Ovis

orientalis musimon) resulted in a better response to the

hormonal treatment, increasing the number of punctu-

red follicles per donor [26]. The only animal that did not

receive neuroleptic treatment had been previously

submitted to regular handling and showed a good

response to superovulatory stimulation [26].

Poor follicular growth could also be due to

insufficient FSH doses. In sheep, successful stimulation

of follicular growth occurs with four doses of FSH (as

used in our study), allowing for a good collection of

healthy oocytes with subsequent fertilization and

development in culture. Constant doses of ovine FSH

seem to produce better results than decreasing ones

[28], although other studies have found no differences

between FSH administration regimes [48,49]. We found

that plasma concentrations of estradiol decreased

during exogenous FSH administration, supporting the

hypothesis that the amount of FSH was insufficient to

adequately stimulate follicular growth. In sheep, FSH

treatment leads to an increase in the number and size of

gonadotropin-responsive follicles and in a subsequent

increase in circulating levels of estradiol [50]. Thus,

estradiol is considered to be a reliable marker of

follicular function [51]. Decreases in follicular secre-

tion of estradiol are related to deficiencies in follicular

health and, moreover, a decrease in the developmental

competence of the oocyte [52]. Therefore, a possible

explanation for the limited ovarian response seen in our

study is that follicles were not responding adequately to

FSH, with stress being an additional factor leading to a

reduced response.

The proportion of oocytes recovered from punctured

follicles (62%) was similar to that obtained using

endoscopic recovery in sheep (55%) [53]. However, it

was lower than that obtained in sheep using laparotomy

(80%) [54] or in goats employing laparoscopy (90%)

[55]. In non-domestic ungulates, oocyte recovery rates

ranged from 36 to 46% in red deer (Cervus elaphus)

[24] and were 57% in sika deer (Cervus nippon) [25],

which are closer to the values obtained in our study.

More studies are needed to improve results of oocyte

harvesting and to explore the possibility of repeated

oocyte collection from the same females.

The size of the follicles found on the ovarian surface

of gazelles was small (2–3 mm). It is known that an

oocyte is unable to resume and progress through

meiosis until it reaches a given size. In sheep, a direct

relationship exists between follicular size and the ability

of oocytes to progress through meiosis [56]. Thus, the

capacity to advance to metaphase II increases with

follicle and oocyte sizes in lambs and adult sheep [56].

It is not clear whether this relationship exists in gazelles,

but it is possible that oocytes contained in small follicles

have a reduced maturation capability.

The overall maturation rate obtained at the end of the

in vitro incubation period was about 70%, with

considerable variation between females, and this result

is similar to data reported in other wild ruminants. In red

deer, a maturation rate of about 55% was found for

oocytes harvested by laparoscopy [24] and a range of

14–85% for oocytes recovered from slaughterhouse

ovaries [25]. In sika deer, 14–76% of oocytes aspirated

by laparoscopy matured in vitro [25]. In domestic sheep,

oocytes recovered by OPU showed a 80–90% matura-

tion rate when conditions similar to those used for

gazelles were employed [28]. Clearly, the fertilization

rate we obtained with gazelle oocytes in the present

study was rather low (40%) as compared to the �80%

fertilization rate achieved in domestic sheep using

similar methodology [28].

F. Berlinguer et al. / Theriogenology 69 (2008) 349–359 357

When gazelle presumptive zygotes were cultured in

vitro, we found that although there was cleavage up to

the 6- to 8-cell stage, none of the embryos progressed to

the morula or blastocyst stage, suggesting the existence

of a developmental block. In sheep, about 15–20% of

embryos develop to the blastocytst stage when a similar

methodology is used [28]. One possible explanation for

our results is that embryo culture conditions were not

adequate for gazelle embryos. In vitro embryo culture

procedures for wild ungulates are currently limited to

protocols developed for domestic animals. Using such

protocols, blastocysts have been obtained after oocyte

in vitro maturation, fertilization and culture in the axis

deer, (Axis axis) [57], red deer [25,58], sika deer [25],

and European mouflon [26]. Adjustments are therefore

needed for improvements in gazelle in vitro embryo

culture, but progress in this area will be slow due to the

scarcity of gametes and embryos from this endangered

species. Additional factors to consider are that

differences exist, even between closely related species,

in the requirements of embryos for in vitro development

[8]. An alternative to overcome the difficulties of in

vitro embryo culture could be an early transfer to

recipient females. In deer, in vitro matured and fertilized

oocytes were capable of establishing pregnancies when

embryos were transferred to recipient hinds at the 2- to

4-cell stage, with this transfer being more efficient than

that of 8-cell embryos [59]. In future gazelle work we

will explore the feasibility of early embryo transfer, i.e.,

oviductal transfer of 2- to 4-cell embryos.

Another likely explanation for poor embryo develop-

ment in culture relates to inadequate oocyte quality and

limited developmental competence. Studies on ewes

treated with progestagen/gonadotropin for in vitro

embryo production have indicated that oocyte quality

(i.e., the ability to develop into a viable embryo after in

vitro maturation, fertilization and culture) seems to

depend on both the reproductive status of the female and

the hormonal treatment employed [28,29]. Since,

gazelles showed a limited response to FSH treatment,

as revealed by poor follicular growth, it is possible that

oocytes had a poor degree of cytoplasmic maturation and

this had an important bearing on post-fertilization deve-

lopment. The fundamental processes involved in the

acquisition of oocyte developmental competence remain

unknown, but there is increasing evidence that such

competence is acquired while still in the follicle [43].

Oocyte pre-maturation and final maturation occurring

during follicular growth are considered crucial steps in

determining the outcome of embryo production [60].

Taken together, our results of in vitro maturation,

fertilization and culture in the Mohor gazelle, achieved

using protocols developed originally for sheep, demon-

strate, for the first time, the production of embryos in

vitro from this endangered species. Although more

studies are needed to improve in vitro conditions, these

results reveal the feasibility of achieving in vitro

fertilization, with frozen-thawed semen, of in vitro

matured oocytes collected from FSH-stimulated endan-

gered gazelles. Aspects that need improvement include

oocyte harvesting conditions, stimulation for the

development of healthier oocytes, and in vitro culture

of embryos to overcome a developmental block that

prevents progression to the blastocysts stage. These

improvements would allow the generation of a

sufficient number of oocytes and embryos for cryo-

banking or transfer.

Acknowledgements

The authors are grateful to Eulalia Moreno for

permission to study the gazelles, Jesus Benzal for

laboratory support, and the staff of the Parque de Rescate

de la Fauna Sahariana, EEZA, CSIC (Juan Belzunces,

Alfonso Lopez, Oscar Salinas, Luis Pozo, Gema Morales,

Leire Correa) for their help in handling the animals. This

study was supported by the Spanish Ministry of

Education and Science (REN 2003-01587, CGL2006-

13340/BOS) and Acciones Integradas (HI20030336).

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