ELSEVIER

EFFECT OF TESTOSTERONE AND DIBUTYRYL C-AMP ON THE MEIOTIC COMPETENCE IN PIG OOCYTES OF VARIOUS SIZE CATEGORIES

J. P&r,’ 0. Teplh,’ J. Rozinek* and F. Jilek2

’ Research Institute of Animal Production Department of Reproductive Biology

104 00 PraFa 10 - Uhiineves, Czech Republic University of Agriculture

Department of Veterinary Sciences 165 21 Praha 6 - Suchdol, Czech Republic’

Received for publication: October 20, 19% Accepted: October 9, 1995

ABSTRACT

Spontaneous meiosis resumption was investigated in small pig oocytes of various size categories. Two parameters were studied: 1) the possibility for inhibiting spontaneous meiosis resumption in small oocytes of various size categories and 2) the level of meiotic competence of small oocytes in which meiosis resumption had been temporarily blocked. It was observed that 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) combined with 0.5 FM testosterone were able to prevent spontaneous maturation in pig oocytes of various size categories cultured in vitro for up to 10 d. Moreover, a culture of these oocytes with dbcAMP and testosterone has a beneficial effect on the integrity of the oocyte-granulosa cell complex. When oocytes were allowed to mature after long-term inhibition of maturation, the percentage of oocytes able to resume maturation was significantly increased. However, the portion of oocytes which completed maturation, reaching the metaphase II (M II) stage, did not increase. It was found that dbcAMP, in combination with testosterone, also efficiently inhibited spontaneous maturation in fully grown pig oocytes. When the inhibitory effect of dbcAMP and testosterone was reversed in fully grown oocytes, maturation was significantly accelerated.

Key words: pig oocyte, meiotic competence, dbcAMP, testosterone

INTRODUCTION

There is a large pool of small follicles with small growing oocytes in which meiotic competence is not fully developed in the ovaries of domestic mammalian species. These oocytes do not mature when cultured in conventional culture systems. They remain arrested at the prophase of the first meiotic division, or they resume meiosis but do not complete meiotic division by extrusion of the first polar body (11, 12, 13). The use of such oocytes for in vitro fertilization and other biotechniques is

’ Acknowledgments We thank Dr. Martin Vitek for statistical advice and Mrs. Lucy Westcott for editorial assistance with this manuscript. The study was supported by grant 5082 from NAZV MZe of the Czech Republic.

Theriogenology 46:97-106. 1996 Q 1696 by Elsevier Science Inc.

0093-691 X/96/$15.00 PII SOO93-691X(96)00145-3

Theriogenology

substantially limited in contrast with fully “j

rown oocytes from larger follicles that mature spontaneously during in vitro culture (15, ).

It seems essential for in vitro oocyte growth to prevent spontaneous meiosis resumption during culture until the oocytes reach full meiotic competence (19). The maintenance of the functional integrity of an oocyte with somatic follicular cells also seems to be very important (19). If the arrest of germinal vesicle breakdown could be maintained in vitro in growing oocytes, these oocytes could acquire full meiotic competence and the competence for further development. After maturation and fertilisation they could develop into viable embryos.

In rodent studies, dbcAMP or hypoxanthine were used for the inhibition of precocious meiosis resumption during the in vitro culture of small oocytes. More recently, Eppig (8) succeeded in inhibiting maturation in mice using in vitro co-culture of growing oocytes with granulosa cells.

In large domestic mammalian species both dbcAMP and hypoxanthine are themselves only able to partially block or delay oocyte maturation (18, 16). However, it was demonstrated that dibutytyl cyclic adenosine monophosphate (dbcAMP) in combination with testosterone inhibits meiotic maturation in fully grown pig oocytes (16).

The aim of the present study was to investigate 1) the level of meiotic competence in pig oocytes of different sizes, 2) the possibility of inhibiting spontaneous meiotic maturation in pig oocytes of various size categories using a combination of dibutyryl cyclic adenosine monophosphate (dbcAMP) and testosterone, 3) the effect of prolonged inhibition of meiosis resumption on oocyte growth and the integrity of oocyte-somatic cells complex, 4) the reversibility of prolonged inhibition of meiosis resumption in oocytes of different sizes and 5) nuclear maturation after reversal of the inhibition of meiosis resumption in fully grown pig oocytes.

MATERIALS AND METHODS

Oocyte Collection and Culture

Porcine ovaries were obtained from a local slaughterhouse from gilts at an unknown phase of the oestrous cycle. The ovaries were transported to the laboratory in a saline solution at 370C within 45 min.

Fully grown oocytes were aspirated from 2- to 5mm follicles using a syringe fitted with a 20-gauge needle. Only oocytes with compact cumuli were chosen for further study.

Small oocytes of different size categories were obtained from thin strips (lo- to 15mm length, l-mm width) dissected from the surface of the ovaries using a surgical blade. The stnps of ovarian tissue were placed in 3-cm Petri dishesa containing Tyrode lactate (TALP) medium (2). Oocytes were liberated from their follicles by opening the follicular wall using the tip of a 25gauge needle. The internal diameter of the oocytes (without zona pellucida) was measured with an ocular micrometer mounted on

a Nunc, Roskilde, Denmark

Theriogenology 99

a microscope. Only oocytes surrounded with several layers of cumulus cells were chosen for further experiments. The oocytes were handled in a temperate box at 37oC, and all manipulations were completed within 1.5 h.

Before culture the oocytes were washed three times in a culture medium and then cultured in 100 ml droplets of culture medium covered with paraffin oil, 37OC in a mixture of air with 5% C02.

Oocytes were cultured in modified Earle’s salts medium 199 (E199) (14): El99 mediumb supplemented with 0.039 ml 7.5% solution of sodium bicarbonate per mililiter of medium, calcium lactateC (0.6 mglml medium), sodium pyruvate’ (0.2 mglml) gentamycin” (0.025 mglml), HEPES’ (1.5 mg/ml) and 10% bovine serumd.

At the end of the culture, the oocytes were mounted on slides, fixed with acetic alcohol (1:3, v/v) for at least 24 h and stained with 1% orcein. The oocytes were then examined under a phase-contrast microscope.

Experiment 1

The level of meiotic competence during in vitro culture was investigated in small pig oocytes of different size categories and in fully grown pig oocytes during their culture in vitro. After liberation from the follicle the oocytes were measured using an ocular micrometer and were assigned to groups according to their internal diameter (i.e., oocyte diameter without the zona pellucida). Oocytes with an internal diameter of 70, 80, 90, 100 and 110 pm, and fully grown oocytes (internal diameter approximately 120 pm) were used in this experiment. Oocytes whose diameter did not fit into the above mentioned size categories were discarded from the experiment. The oocytes were cultured in culture medium for 48 h after which they were fixed and the stage of their nuclear maturation was evaluated.

Experiment 2

The combined effect of dbcAMP” with testosterone” on nuclear maturation was investigated in small oocytes of 3 size categories (80, 90 and 100 pm). Combinations of dbcAMP (mM) with testosterone (PM) were chosen as follows: 1 with 0.5, 0.5 with 0.5, 0.1 with 0.5, 1 with 0.25, and 1 with 0.1 respectively. Oocytes cultured In the dbcAMP testosterone-free medium served as the controls. At the end of a 48. h culture, the oocytes were fixed and their nuclear maturation was evaluated.

Experiment 3

Possibilities for long-term inhibition of nuclear maturation in small pig ooc different sizes using dbcAMP and testosterone were tested in this experiment. r?

es of ocytes

of 3 size categories (80, 90 and 100 pm) were cultured in a medium supplemented with 1 mM dbcAMP and 0.5 t.tM testosterone for intervals of 1 to 10 d. Control oocytes were cultured for the same intervals in a medium without dbcAMP and testostemne. The internal diameter of the oocytes was measured every day during culture, and the integrity of complexes of oocytes with their surrounding cumulus cells was

b USOL,Prague, Czech Republic ’ SIGMA, St. Louis, USA d ZD HustopeCe, Czech Republic ’ SERVA, Heidelberg, Germany

Theriogenology

morphologically assessed. At the end of the culture the oocytes were fixed and their nuclear maturation was evaluated.

Experiment 4

This experiment was designed to test possibilities for a reversal of long-term inhibition of maturation in small oocytes of different size. Oocytes of 3 size categories (80, 90 and 100 pm) were first cultured in a medium supplemented with 1 mM dbcAMP and 0.5 PM testosterone for various interval (0, 2, 4, 7 or 10 d), then they were washed 3 times and cultured for 48 h in a dbcAMP testosterone-free medium. At the end of the culture, the oocytes were fixed and their nuclear maturation was evaluated.

Experiment 5

This experiment was designed to investigate the time sequence of nuclear maturation in fully grown pig oocytes after reversal of the inhibitory effect of dbcAMP and testosterone. Fully grown oocytes were cultured for various lengths of time (0, 1 or 2 d) in a medium supplemented with 1 mM dbcAMP and 0.5 PM testosterone. They were then washed 3 times and cultured for various periods (0, 3, 6, 8, 12, 16, 18 or 24 h) in a dbcAMP testosterone-free medium. At the end of culture, the oocytes were fixed and their nuclear maturation was evaluated.

Statistical Analysis

Each experiment was carried out 4 times. Data are expressed as the mean & SEM) percentage. Frequencies were subjected to arcsin transformation and were checked by analysis of variance followed by Duncan’s test. For all comparisons a P value of 0.05 was considered to be significant.

RESULTS

Experiment 1

In this experiment we tested competence for spontaneous maturation in small pig oocytes during their 48 h of in vitro culture. The results are shown in Figure 1. Small oocytes of different size categories and fully grown oocytes (internal diameter 120 pm) were cultured in this experiment and their competence to resume meiosis was shown to be dependent on the oocyte’s internal diameter.

Three size cate based on the results o B

ories of small pig oocytes were chosen for further experiments Experiment 1. The first group involved oocytes with an internal

diameter (without zona pellucida) of 80 pm. These represented oocytes at the early stage of acquisition of meiotic competence, since approximately only 10% of them underwent GVBD after 48 h of culture and their maturation did not proceed beyond the metaphase I stage. The second experimental group was represented by oocytes with an internal diameter of 90 pm. In this size category a significantly higher portion (22%) of the oocytes was able to resume meiosis during the 48-h culture, but none of them completed meiotic maturation to reach metaphase II stage. The third group was comprised of oocytes with an internal diameter of 100 pm, of which a limited portion exhibited fully developed meiotic competence. Thirty-six percent of these oocytes resumed meiosis after 48 h of in vitro culture, and 7% of them were observed to complete meiotic maturation by extrusion of the first polar body.

Theriogenology

P e 60

r C e 60

: 40 a 9 e 20

0

Internal diameter of oocyte (pm)

n = 60 75 115 I20 60 95

101

*The percentage of GVBD B the percentage of M II. I

Figure I. Meiotic maturation in pig oocytes of different internal diameters cultured for 48 hours in vitro.

Experiment 2

The effect of various concentrations of dbcAMP and testosterone on the spontaneous resumption of meiosis in growing pig oocytes from three chosen size categories (internal oocyte diameters of 80, 90 and 100 pm) was investigated. The results are shown in Table 1. The addition of 1 mM dbcAMP and 0.5 PM testosterone to the culture medium completely inhibited spontaneous maturation in all 3 size categories of growing oocytes during 48 h of in vitro culture. Decreasing the concentration of dbcAMP down to 0.1 mM allowed for meiotic resumption during the 48-h in vitro culture in a limited portion of oocytes with internal diameters of 90 and 100 pm. Oocytes with an internal diameter of 80 pm remained at the stage of germinal vesicle (GV) despite a decrease in dbcAMP concentration after 48-h of in vitro culture. A similar situation was observed after the culture of small oocytes in a medium with lower concentrations of testosterone. Decreasing the concentration of testosterone down to 0.25 pM allowed for meiosis resumption in a portion of the oocytes with an internal diameter of 90 and 100 pm cultured for 48 h in vitro, but oocytes with an internal diameter of 80 pm remained at the GV stage.

Experiment 3

The possibility of long-term inhibition of maturation in small oocytes in a medium containing 1mM dbcAMP and 0.5 pM testosterone was tested in this experiment. Oocytes of three size categories (internal diameter 80, 90 and 100 pm) were cultured in vitro for up to 10 d. At the end of culture the oocytes of all 3 size categories were

102 Theriogenology

Table 1. The effect of dbcAMP and testosterone on germinal vesicle breakdown in small pig oocytes. Oocytes were cultured for 48 h in medium with different levels of dbcAMP and testosterone (T).

Culture medium

1 mM dbcAMP + 0.5 pM T

0.5 mM dbcAMP + 0.5 pM T

0.1 mM dbcAMP + 0.5 pM T

1 mM dbc AMP + 0.25 pM T

1 mM dbcAMP + 0.1 pM T

OdbcAMP+OT

80

% of GVBD

(n)

o+oa

(120) o+oa

(116) o+oa

(96) 0-oa

(115) o+oa

(66) 9.5+2.lb

Oocyte diameter (pm)

90 100

% of GVBD % of GVBD

(n) (n)

o+oa o+oa

(120) (120) 10.0 + 1.4b 13.8 + 1.3b

(122) (120) 22.5 + 2.1’ 30.2 + 1.7’

(103) 4.OYzab 8.0 + 1 .gb

(102)

II.52 1.4b (117)

20.0 + 1.6d

(103) (82) 21.5t2.6’ 36.0 + )2.9C

a - d significant differences (PcO.05) in maturation rate between the oocytes of different size categories are indicated by different superscripts

observed at the GV stage, with no increase in the portion of oocytes with signs of degeneration. The integrity of the complex of oocytes with follicular cells in dbcAMP testosterone-free medium was broken quite quickly, and oocytes cultured in this medium were denuded within 2 d. When small oocytes are cultured in medium with 1 mM dbcAMP and 0.5 pM testosterone, the integrity of the complex was maintained up to 5 d (Figures 2 and 3). However, we did not observe significant growth in oocytes cultured during this experiment.

Experiment 4

Possibilities of reversal of the longterm inhibition of maturation in small pig oocytes was investigated in this experiment. Oocytes of the 3 size categories (internal diameter 80, 90 and 100 pm) were precultured in a medium with 1 mM dbcAMP and 0.5 PM testosterone for intervals of up to 10 d and subsequently cultured in a dbcAMP testosterone-free medium for 48 h. The results are shown in Table 2.

In oocytes with an internal diameter of 80 pm a maximal GVBD percentage (23%) was observed when the oocytes were allowed to mature for 48 h after a 3day preculture in a medium with dbcAMP and testosterone. None of these oocytes were able to complete maturation and reach the metaphase II stage. A 3day preculture significantly increased GVBD percentage in oocytes with an internal diameter of 80 pm compared with that of oocytes allowed to mature without preculture (GVBD 8%). A further extension of the preculture period with dbcAMP and testosterone increased the portion of oocytes degenerating during their subsequent 48 h culture in dbcAMP testosterone-free medium.

Theriogenology 103

Figure 2. The growing pig oocyte with an internal diameter of 90 wrn cultured for 3 days in the medium without dbc AMP and testosterone. The oocyte became denuded, and only a few follicular cells remained attached to the zona pellucida.

Figure 3. The growing pig oocyte with an internal diameter without zona pellucida of 90 pm cultured for 3 days in the medium with 1 mM dbc AMP and 0,5 PM testosterone. The integrity of complex of oocyte with follicular cells is preserved.

104 Theriogenology

Table 2. The reversal of the dbcAMP (ImM) and testosterone (0.5 PM) inhibitory effect in small pig oocytes. The oocytes were precultured with inhibitor for various periods and were then cultured for 48 h in medium without dbcAMP and testosterone.

No of days of preculture

GVBD

Percentage 01

GV Degenerated n oocytes

a) Small pig oocytes with internal diameter of 80 pm

0 7.8 f I .2a 88.0+2.P 6.2 + 2.0a 2 17.5+2.1b 79.0 + 1.7ab

72.0 + 2.5b

3.8 + 2.1~

3 22.8 + 3.ib 7.2 + 3.2a

4 a.8 f 1 .3a 84.5 + 2.0a 6.8 2 2.9a

7 2.3 + 1.7’ 83.2 + 2.ga 14.0 + 3.6b

10 3.4 2 1.3c 74.0 + 3.2b 22.0 + 4.4c

b) Small pig oocytes with internal diameter of 90 pm

110 105

113

120

115

aa

0 20.0 + 2.2a 73.2 + 3.3a

63.0 + 3.2bc

6.8 + 2.ga

2 20.5 + 2.3a 6.5 + 2.aa

3 23.5 + 2.7a 65.8 + 2.2b lo.8 + 3.4a

4 32.0 + 2.2b 59.8 + 2.2c a.2 + 2.5a

7 47.2 + 2.5’ 42.0 + 2.1d 10.2 + 3.5a

10 22.5 + 2.1a 46.0 + 4.5d 32.0 + 4.5b

c) Small pig oocytes with internal diameter of 100 pm

96

a2

73

115

120

131

0 36.5 f: l.ga 59.5 + 1 .oa 5.5 + 1 .gab a2

2 39.2 f: 1 .oa 58.0 + 1 .aa 2.a+i.oa 63

3 42.2 + 1 .2a 50.0 + 1 .6a 7.8 + 2.4ab 94 4 64.5 + 1 .7b 26.0 + l.ab 9.2 I 1.5bc 115

7 37.0 + 2.5a 43.0 + 2.3’ 17.5 + 3.6’ 76

10 23.0 + 2.1’ 40.0 + 4.5c 38.5 + 3.gd 103

e-d Significant differences (PcO.05) in oocyte nuclear status within each size category of oocyte are indicated by different superscripts.

In oocytes with an internal diameter of 90 urn a maximal GVBD percentage (47%) was observed when they were allowed to mature after a 7-d preculture period in medium with dbcAMP and testosterone, which significantly increased the percentage of GVBD above the level observed in oocytes of the same size category that were allowed to mature without preculture (GVBD 20%). None of the oocytes maturing after preculture reached the metaphase II stage, and the prolongation of preculture time with dbcAMP

Theriogenology 105

and testosterone up to 10 d significantly increased the portion of degenerated oocytes during their subsequent 48-h culture in dbcAMP testosterone-free medium.

Oocytes with an internal diameter of 100 urn exhibited the highest GVBD percentage (65%) following 48 h of culture in the dbcAMP testosterone-free medium and a 4 day preculture in dbcAMP plus testosterone supplemented medium. In this experimental group the percentage of oocytes resuming maturation was significantly higher (GVBD 65 vs 36O/6) than in control oocytes cultured for 48 h without preculture in dbcAMP plus testosterone. The percentage of oocytes reaching the metaphase II stage (9%) did not differ significantly when compared with that of the control oocytes (M II 7%). Further prolongation of the preculture period only caused an increase in the percentage of oocytes degenerating during 48 h culture following preculture with dbcAMP and testosterone.

Experiment 5

In this experiment the reversal of the inhibitory effect of dbcAMP and testosterone was investigated in fully grown pig oocytes (an internal diameter of 120 pm) that have full meiotic competence. Results are shown in Table 3.

Table 3. The time sequence of GVBD in fully grown pig oocytes after their preculture with dbcAMP (1 mM) and testosterone (0.5 PM) for 0, 24 or 48 h and subse- quent culture in dbcAMP-testosterone-free medium (24 h). Note the increased maturation rate in oocytes precultured with dbcAMP and testosterone.

Hours after inhibitory reversal

0

Preculture periods (hours) 0 24 48

% of GVBD % of GVBD % of GVBD (n) (n) (n)

o+oa 16.9 f. 1.9b 20.7+2.1b

(131) 3 otoa

(153) 6 2.2 0.6a +

(120) 8 3.8 l.Oa 2

(120) 12 II.45 1.8a

(102) 16 42522.6

(120) 18 91.2 3.8ab +

(120) 24 95.2 3.7a +

(120) 15.8 of: 2.0b

(80) 18.5 of. 2.4b

(107) 44.0 + 2.0b

(96) 75.8 + 2.2b

(114)

80.5 t 3.1”

(99) 89.4 + 3.ga

(80) 19.2 + 2.5b

(80) 56.0 2 3.0’

(80) 98.0 2 1.7’

(118) 100.0 + oc

(120)

99.2 + 3.4b

(80) 95.0 + 2.6a

0 Significant differences (P c O.Ol] of GVBD percentages between but not within the groups with different preculture periods are indicated by different superscripts.

Theriogenology

Control oo Y

es were cultured in a dbcAMP and testosterone-free medium. In these oocytes GV D occurred approximately after 16 h of in vitro culture. Fully grown oocytes, precultured for 24 h with dbcAMP and testosterone, exhibited 17% of GVBD at the end of the culture period. During subsequent culture of these oocytes in the dbcAMP testosterone-free medium, meiosis resumption was significantly accelerated. The GVBD percentage had increased significantly after 8 h of culture. Further acceleration of GVBD was observed after prolonging the preculture period to 48 h. Twenty-one percent of the oocytes were observed to undergo GVBD at the end of 48 h of preculture in medium with dbcAMP and testosterone. During subsequent culture in a dbcAMP testosterone-free medium, the GVBD percentage had increased significantly after a 6-h culture.

DISCUSSION

In the present study, meiotic competence was investigated in small pig oocytes, and similar to the findings of previous studies (12,19), we have shown that competence for meiosis resumption is acquired gradually during oocyte growth.

The inhibitory effect of dbcAMP and testosterone on spontaneous meiosis resumption was observed in small pig oocytes; moreover, these oocytes became denuded after a si

LA nificantly longer time interval than the small pig oocytes cultured in

medium without d MP and testosterone and after the reversal of the long-term inhibition of maturation they exibited higher meiotic competence. Carroll et al. (4) also observed that the addition of dbcAMP to the culture of mouse isolated primary follicles in collagen gels significantly improving the ability of the oocytes to resume meiosis. According to these authors, the effects of dbcAMP are caused by the induction of proliferation in granulosa cells. On the other hand, Chesnel et al. (5) showed that the beneficial effect of dbcAMP on the acquisition of meiotic competence is not mediated through the follicular somatic cells since dbcAMP improved maturation even in denuded growing mouse oocytes. Nevertheless, it seems that the positive effect of dbcAMP and testosterone on the integrity of the complex of oocyte with granulosa cells is the significant cause for increased meiotic competence in small pig oocytes in our study. The reasons for the different preculture periods needed for the maximum effect on the ability to resume meiosis in oocytes of different size categories after reversal of the inhibitory effect are not clear.

The inhibitory effect of dbcAMP and testosterone is complete only when both compounds are present in sufficient concentrations. Neither dbcAMP nor testosterone alone inhibited GVBD. This is contrary to that demonstrated in mouse oocytes, in which both dbcAMP or testosterone alone can inhibit GVBD, and the effects of both inhibitors are distinct and distinguishable (6).

In our study, significant oocyte growth was not observed, although the integrity of oocytes with surrounding granulosa cells remained intact for a extended time interval during culture with dbcAMP plus testosterone. This is in contrast to the increased ability of these oocytes to resume meiosis. It seems, however, that increased meiotic competence in vitro is not necessarily closely connected with oocyte growth. Canipari et al.(3) described the acquisition of meiotic competence in growing mouse oocytes which did not reach their full size. Sato et al. (17) demonstrated improved meiotic competence in not fully grown bovine oocytes by the addition of dbcAMP to the culture medium without any effect on oocyte growth. Bachvarova et al. (1) observed a positive

Theriogenology 107

effect of granulosa cell in denuded oocytes after reestablishment of their gap junctional connection with the granulosa cell monolayer.

The present study also confirmed earlier observations (16) that dbcAMP combined with testosterone inhibits spontaneous GVBD in vitro in fully grown pig oocytes. The GVs in arrested oocytes remained intact according to criteria published by Motlik and Fulka (12). However, the time sequence of GVBD after washing out inhibitors is characterised by signrficant acceleration. GVBD was completed within 12 and 8 hours after the preculture of oocytes in a medium with dbcAMP and testosterone for 24 and 48 h, respectively. This is a significantly shorter time than observed in pig oocytes maturing in vivo (12) or in vitro (12, present study). This acceleration could not be due to the delayed onset of GVBD occurring under the influence of dbcAMP and testosterone, because GVBD was not observed and GVs remained intact after 48 h culture with Inhibitors. It therefore appears possible that the inhibitory effect of dbcAMP and testosterone did not prevent all events involved in GVBD, and mimics only a part of the inhibitory influence that is exerted on the oocyte by the somatic components of the follicle. This processes occurin

8 in small oocytes can be also responsible for their

increased meiotic competence. imilar GVBD acceleration in fully grown pig oocytes was observed after the inhibition of spontaneous meiosis resumption by cycloheximide (11) or by the inhibitory factor from the cumulus cells (14).

Based on our data, we can conclude that dbcAMP plus testosterone inhibits spontaneous meiotic maturation in growing pig oocytes during their long-term culture in vitro. Such oocytes exhibited increased ability to resume meiosis but their ability to complete meiotic division by the extrusion of the polar body and to reach the metaphase II stage remained very limited. These data should be taken into account when attemps will be made to develop culture systems supporting in vitro growth in pig oocytes.

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