Theriogenology 41:969-980, 1994

MEIOTIC COMPETENCE OF PREPUBEKTAL GOAT OOCYTES

A. Martino, T. Mogas, M.J. Palomo and M.T. Paramio’

Departament de Patologia i de Producci6 Animals Facultat de Veterindria, Universitat Autbnoma de Barcelona

08 193 Bellaterra, Barcelona, Spain

Received for publication: April 6, 1993 Accepted: November 15, 2993

ABSTRACT

The object of this work was to evaluate in vitro maturation of follicular oocytes from the ovaries of prepubertal goats obtained from the slaughterhouse. To obtain the oocytes, follicles were dissected and classified according to their diameters. In the first experiment, oocytes were matured in vitro with granulosa cells. No significant differences were detected in the percentages of maturation between adult and prepubertal goat oocytes recovered from follicles of 2.5 to 6.0 mm in diameter (81.82 vs 72.47%, respectively). The percentage of maturation increased to 88.0% in prepubertal goat oocytes from 3.0 to 6.0-mm follicles. In the second experiment, the percentage of maturation of prepubertal goat oocytes was greater after 27 than after 24 h. In the third experiment, the maturational capacity of prepubertal goat oocytes according to follicular diameter was evaluated. The percentages of maturation after 27 h of culture with no granulosa cells were 24.14, 56.60 and 74.78%, respectively, for follicles 1.0 to 1.9 mm, 2.0 to 2.9 mm, and 3.0 to 6.0 mm in diameter. As the follicular diameter increased, growth of the oocyte as well as a greater number of oocytes with more cumulus cell layers were observed. A correlation between the diameter of the oocyte and its competence to complete in vitro maturation was also observed. Oocytes with more cumulus cell layers showed only a slight superiority in their capacity for maturation in large-size follicles (3.0 to 6.0 mm), but the difference was not significant. In conclusion, oocytes from prepubertal goats complete their growth and reach meiotic competence in follicles larger than 3.0 mm. With these oocytes it is possible to obtain in vitro maturation results similar to those from adult goats.

Key words: goat oocyte, in vitro maturation, meiotic competence

INTRODUCTION

Over the last decade considerable advances have been made in the production of embryos from in vitro fertilization (IVF) in numerous domestic species. In the case of the goat, the first birth using the IVF procedure on ovulated oocytes was achieved by Hanada in 1985 (15). In the last few years, many studies on in vitro maturation (IVM) and IVF of goat oocytes have appeared (36,3,10), especially because of the interest generated in this species by the

Acknowledgments This work was supported by CICYT (Project No. Gan 89-241). ’ Correspondence and reprint requests.

Copyright 0 1994 Buttetworth-Heinemann

970 Theriogenology

production of transgenic animals which produce substances of pharmacological interest in the mammary gland (12). Recently, the first case of a kid born from an oocyte matured and fertilized in vitro was reported (7).

In most of the studies in goats, oocytes are recovered surgically or after the slaughter of animals that have been treated with hormones; these methods are costly and greatly limit the number of oocytes available. The use of ovaries from slaughterhouses to obtain oocytes for IVM and IVF, developed mainly using bovine oocytes, provides an abundant and cheap source of oocytes, although their quality is variable (6). In Spain, goat ovaries can be reliably obtained from slaughterhouses, but the animals are killed when they are very young, at approximately 2 mo of age, before reaching puberty. The use of prepubertal animals is of interest because of the possibility of reducing the generational interval, a factor which is generally unchangeable and which is one of the main limiting factors in genetic improvement and in biotechnological studies (1,2). In the bovine, prepubertal calves treated hormonally have been used as oocyte donors for IVM (8) and for the production of viable embryos by IVM-IVF (1,17). Recently, interest has been shown in the use of prepubertal calf ovaries taken from the slaughterhouse for the in vitro production of embryos, although the success rate is usually lower than for those taken from adult animals (20,28,34).

The influence of follicular diameter on the meiotic competence of the oocyte has been demonstrated in the pig and the cow (24,25). In the goat, important differences between oocytes collected from follicles of 1 to 2 mm and follicles of 2 to 6 mm have been observed (IO), although it has not been possible to accurately determine the follicular size at which the oocyte reaches meiotic competence. In other studies differences exist in the follicular size from which oocytes were collected (1 to 6 mm or 3 to 6 mm; 36,3). Also, different in vitro maturation times, from 24 to 30 h, have been used, but best maturation rates have been reported in studies using 24 to 25 or 27 h (36,3,10).

The objectives of this work were to evaluate the IVM of prepubertal goat oocytes compared with those from adults (Experiment 1); to compare IVM after 24 or 27 h (Experiment 2); and to determine the follicular diameter at which meiotic competence of the oocyte is reached as well as to study the number of oocytes obtained from slaughterhouse ovaries in relation to the selection criteria of the oocytes (Experiment 3).

MATERIALS AND METHODS

Experiment 1

To collect the oocytes from adult animals, 6 multiparous Murciana goats were used. They were stimulated with 7 mg of porcine FSH (Ovaset FSH-LH, Sanofi, France) distributed in 3 doses (3,2,2 mg) separated by 12 h, beginning at Days 11-13 of the estrous cycle. The goats were slaughtered 24 h after the last injection of FSH (10). The ovaries were transported to the laboratory in isothermic containers in less than 5 min.

The ovaries of the prepubertal goats came from approximately 2 mo-old females, and were obtained from a commercial slaughterhouse close to the laboratory. They were transported in PBS + penicillin-streptomycin at an average temperature of 33.5 k 3.2”C (30 to 37°C). The

Theriogenology 971

average transport time was 52 f 12 min (20 to 75 min).

The ovaries were washed 3 times in PBS + penicillin-streptomycin. Follicles were completely dissected from ovarian tissue, and their diameters were carefully measured under a stereoscopic microscope with a calibrated eyepiece. For adult goats, follicles with a diameter between 2.5 and 6.0 mm were selected. Prepubertal goat ovaries were randomly separated into 2 groups. In the first group 2.5 to 6.0~mm follicles were selected, while in the second group 3.0 to 6.0-mm follicles were used. The cumulus-oocyte complexes were released in 199 medium (Flow, Irvine, Scotland) with NaHCO, 4mM, penicillin, streptomycin and buffered with 20 r&l Hepes (pH 7.35). Oocytes which had 3 or more complete layers of cumuli cells and an homogeneous cytoplasm were selected and washed 3 times in the medium described above. Granulosa cells were obtained from follicles with the fewest signs of atresia and were washed in the same medium. The whole process took place at a laboratory temperature of 35°C. The average time elapsed from slaughter to the beginning of culture was 4 h 53 min * 1 h 19 min (in an interval of between 2 h 35 min minimum and 8 h 45 min maximum).

The oocytes were cultured in 2 ml of the medium described previously enriched with 10% inactivated bovine fetal serum, 1 ,ug/ml 17S-estradiol, 10 pg/ml FSH and 10 &ml LH, together with 1x106/ml granulosa cells. The culture was carried out at 38.5”C for 27 h (10). At the end of culture, the oocytes were separated from the cumulus cells with a fine pipette in PBS with 150 IU/ml Hyaluronidase (Serva, Heidelberg, FRG), and were fixed for 3 to 4 d in acetic acid 90%-ethanol (l/3 v/v) at 4°C. Afterwards they were stained with 1% lacmoid and the nuclear state was observed under a phase contrast microscope.

Experiment 2

Only ovaries from prepubertal females were used; they were collected as described in Experiment 1. After dissecting and opening the follicles of 3.0 to 6.0 mm in diameter, as described in Experiment 1, the oocytes with 1 to 2 complete layers of cumulus cells were selected. Some of the oocytes were randomly selected, separated from the cumulus cells and fixed before culture, as described above. The oocytes were cultured for 24 or 27 h in the same maturation medium as in Experiment 1, in the absence of granulosa cells, and were processed in the same way.

Experiment 3

Ovaries from prepubertal females were used. They were obtained using the same procedures as for the previous experiments. The follicles were dissected and measured as described previously. Follicles were divided into 3 groups according to their diameter: 1.0 to 1.9 mm, 2.0 to 2.9 mm and 3.0 to 6.0 mm. In a second series of replicates the follicles were classified into 2 additional groups: 2.0 to 2.4 mm and 2.5 to 2.9 mm. Within each of the 5 groups the oocytes were processed as in Experiment 1, but they were separated into 2 classes according to the number of layers in the cumulus cells: CUM 1 (3 or more complete layers) and CUM 2 (1-2 complete layers). The same maturation medium was used as in Experiment 2. The oocytes were cultured in the absence of granulosa cells. At the end of culture the oocytes were processed as described above. Before fixing, the diameters of some of the oocytes were measured with the aid of an inverted microscope with a graduated eyepiece (x 300).

972 Theriogenology

Statistical Analysis

The percentages or the frequencies were compared 2 x 2 with a x2 test. The average diameters of the oocytes were compared 2 x 2 with a Student’s t-test. Multiple regression analysis was applied between the average diameters of the oocytes and the percentages of Metaphase I and Metaphase II.

RESULTS

Experiment 1

No significant differences were observed in the percentage of Metaphase II between the oocytes from adult and prepubertal goats for follicles of 2.5 to 6.0 mm diameter (81.82 vs 72.47%, Table 1). However, in the oocytes from prepubertal goats collected from slightly larger diameter follicles (3.0 to 6.0 mm), the percentage of Metaphase II improved significantly, reaching 88.0%.

Table 1. Effect of age and follicular size on the IVM of goat oocytes

% Meiotic stage (No. of oocytes) Source of oocytes

No. of oocytes GV GVBD META I ANA- META UNC

TEL0 I II

Adult Follicles

2.5 to 6.0 mm

66

(k

3.03 10.61 81.82ab 4.55

(2) (7) (54) (3)

Prepubertal Follicles

2.5 to 6.0 mm

178 1.69 1.12 21.91 72.47a 2.81

(3) (2) (39) (i) (129) (5)

Prepubertal Follicles

3.0 to 6.0 mm

125 8.80 88.0b 3.20

(k (& (11) (i) (110) (4)

GV = Germinal Vesicle, GVBD = Germinal Vesicle Breakdown, META I = Metaphase I, ANA-TEL0 I = Anaphase-Telophase I, META II = Metaphase II, UNC = Unclassified. a,b Values with different superscripts differ significantly (PcO.05).

Experiment 2

Most of the oocytes (85.42%, Table 2) evaluated before culture were in the germinal vesicle stage. In the rest of the oocytes it was not possible to determine the nuclear stage with exactitude because most of them had abnormal nuclear configurations.

Theriogenology 973

A decrease in the proportion of oocytes arrested at Metaphase I and an increase in Metaphase II were observed when passing from 24 to 27 h of culture (Table 2). The percentage of oocytes which resumed meiosis was higher than 98% in both cases.

Table 2. Effect of the duration of the culture on the nuclear stage of prepubertal goat oocytes

Duration of the culture

No. of oocytes GV

% Meiotic stage (No. of oocytes)

GVBD META ANA- META II UNC I TEL01

Oh 48 85.42 0a 14.58

(41) (0) (7)

24 h 73 (k (0”) 42.47b 1.37 53.42b 2.74

(31) (1) (39) (2)

27 h 56 1.79 (k 16.07’ 1.79 73.21’ 7.14

(1) (9) (1) (41) (4)

GV = Germinal Vesicle, GVBD = Germinal Vesicle Breakdown, META I = Metaphase I, ANA- TEL0 I = Anaphase-Telophase I, META II = Metaphase II, LJNC = Unclassified. qbgcValues in the same column with different superscripts differ significantly (PcO.05).

Experiment 3

Table 3 shows that the capacity of the oocytes for completion of nuclear maturation is acquired progressively with an increase in follicular diameter. In follicles of between 1.0 and 1.9 mm, nearly all the oocytes (>99 %) were capable of resuming meiosis, although most (71.12%) were arrested in Metaphase I, and only 24.14 % reached Metaphase II. In follicles of 2.0 to 2.9 mm diameter, there was a great improvement and more than half of the oocytes were capable of reaching Metaphase II. Most follicles (74.78%) larger than 3 mm were completely competent. The great difference between follicles of less than 2 mm and follicles greater than 2 mm suggested the subdivision of the follicles in the 2.0 to 2.9 mm group into 2 further groups. Few differences in the percentages of Metaphase II were observed between oocytes from follicles of 2.0 to 2.4 mm and 2.5 to 2.9 mm (52.70 and 62.64%, respectively). Significant differences remained between follicles of 1.0 to 1.9 mm and 2.0 to 2.4 mm.

For all groups of follicles, most of the oocytes which did not reach Metaphase II were arrested in Metaphase I. The percentage of oocytes at the germinal vesicle stage was always less than 2 %. Only one oocyte was observed in Anaphase-Telophase I.

It was observed that the diameter of the oocytes increased with an increase in the size of the follicle (Figure 1). As occurred with the percentage of maturation, there was a big change between follicles of 1.0 to 1.9 mm and 2.0 to 2.9 mm, which was maintained for follicles between 1.0 and 1.9 mm and 2.0 and 2.4 mm in diameter. There was a positive

974 Theriogenology

correlation between the diameter of the oocyte and the percentage of Metaphase II (x=0.98, P=O.O03), and a negative correlation between the diameter of the oocyte and the percentage of Metaphase I (r=O.98, P=O.O03).

Table 3. Meiotic competence of prepubertal goat oocytes according to follicular diameter

Follicular No. of diameter oocytes

(mm) GV

% Meiotic stage (No. of oocytes)

GVBD META I ANA- META II UNC TEL0 I

1.0 to 1.9 232 0.86 2.59 71.12 24.14 a 1.64

(2) (6) (165) (56) (3)

2.0 to 2.9 212 0.94 0.47 40.57 56.60 b 1.42

(2) (1) (86) (i) (120) (3)

3.0 to 6.0 230 (i) (i) 25.22 (& 74.78 ’

(58) (172) (k

2.0 to 2.4 241 (i) (i) 46.47 0.41 52.70 b 0.41

(112) (1) (127) (1)

2.5 to 2.9 175 1.14 35.43 62.29 b 1.14

(2) (62) (109) (2)

GV = Germinal Vesicle, GVBD = Germinal Vesicle Breakdown, META I = Metaphase I, ANA-TEL0 I = Anaphase-Telophase I, META II = Metaphase II, UNC = Unclassified. a,b~c Values with different superscripts differ significantly (PcO.05).

The percentage of oocytes with 3 or more layers of cumulus cells (CUMl) with respect to the total number of oocytes selected for culture (CUM 1 + CUM 2) improved on changing from follicles of 1.0 to 1.9 mm to follicles of 2.0 to 2.9 mm, and from the latter to those of 3.0 to 6.0 mm (Table 4). A difference was observed between the relation between the number of oocytes cultured (CUM 1 + CUM 2) and the number of follicles, on comparing small (60.75%) and medium (72.85%) or large follicles (73.67%). The rest of the uncultured oocytes corresponded to oocytes with partially or totally expanded cumulus, denuded oocytes, or oocytes with signs of cytoplasmatic degeneration. All these data would indicate that the quality of the oocytes increases with an increase in follicular diameter. The number of follicles of 3.0 to 6.0 mm was 1.1 per ovary. The number of oocytes selected for culture from 3.0 to 6.0~mm follicles was 0.82.

Oocytes with 3 or more layers of cumulus cells showed only a slight superiority in their capacity for maturation than oocytes with 1 or 2 layers in follicles of 3.0 to 6.0 mm, but the differences were not significant (Figure 2).

Theriogenology 975

137

g- 135

a b 2 133 cu s

36.3; 1.29 +5.51 ze5.26

a, = 131 B

0

129

127 I I I 1 I

Figure 1.

Table 4.

75

65

25

15 1.0-t .9 2.0-2.4 2.0-2.9 2.5-2.9

Range of follicular diameters (mm)

3-O-6.0

Relation between oocyte diameter and meiotic competence according to follicular diameter. Follicular diameter and number (n) of oocytes measured: 1.0 to 1.9mm n=186; 2.0’ to 2.4mm n=175; 2.0 to 2.9mrn n=212; 2.5 to 2.9mm n=70; 3.0 to 6.Omn-r n=185. a,b,c significant differences (PcO.05).

Effect of follicular diameter on the number and class of recovered oocytes

Follicular No. of No. of % of No. of selected oocytes (%) diameter (mm) ovaries follicles selected

oocvtes Total CUM1 CUM2 d

1.0 to 1.9 178 372 60.75a 226

(100) (204:o)a 179

(79.20)

2.0 to 2.9 673 906 72.8gb 660 247 413

(100) (37.42)b (62.58)

3.0 to 6.0 761 843 73.67b 621 367 254

(100) (59.1 O)C (40.90)

Oocytes per ovary 0.82 0.48 0.33

aab~c Values in the same column with different superscripts differ significantly (PcO.05).

976 Theriogenology

% Metaphase II (No. oocytedtotal)

80

60

40

20

0 1.0 to 1.9 2.0 to 2.4 2.0 to 2.9 2.5 to 2.9 3.0 to 6.0

Follicular diameter (mm)

Figure 2. Effect of the oocyte class on the meiotic competence according to follicular diameter.

DISCUSSION

The percentage of nuclear maturation in prepubertal goats was slightly lower than for adult goats, although the differences were not significant. These results are better than those described in the case of the ovaries of prepubertal calves stimulated with FSH (%Metaphase 11550%; 8) or collected in the slaughterhouse (maturation rate 37 vs 65% in adults; 28). However, embryos with a similar development capability to those from oocytes of adult females have been obtained from the IVM-IVF of oocytes of prepubertal calves treated with hormones (1,17).

In the first experiment, the percentage of maturation of the oocytes of follicles between 3.0 and 6.0 mm (88.0%) was significantly higher than that obtained for follicles between 2.5 to 6.0 mm (72.47%). Prepubertal goat ovaries contain a great number of small follicles, and because all the follicles greater than 2.5 mm were dissected in the present experiment, the number of follicles collected is doubled when follicles from 2.5 to 6.0 mm are used instead of those from 3.0 to 6.0 mm (data not shown). Thus, approximately half of the follicles in the 2.5 to 6.0 group were follicles with a diameter between 2.5 to 3.0 mm. This suggests that differences in meiotic competence could exist between follicles with a diameter greater or less than 3.0 mm. In an earlier work in adult goats (lo), under exactly the same conditions of IVM, a percentage of maturation of 86% was obtained with oocytes from adult goats coming from follicles between 2 and 6 mm of diameter.

Before culture, the majority of the oocytes (85.42%) analyzed were in the germinal vesicle stage. The rest of the oocytes, almost all of which had only 1 or 2 layers of cumulus

Theriogenology

cells (data not included), had nuclear configurations that did not coincide with normal meiotic stages. This is probably related to pseudomaturation phenomena, which appear in the first stages of follicular atresia in the majority of species (14). Song and Iritani (32) also observed in prepubertal goats that 12 % of compact cumulus oocytes were degenerated. Leibfiied and First (19) also observed in the cow that the oocytes with the fewest cumulus cells came from most atretic follicles, and their nuclei remained in germinal vesicle stage in a lower proportion. In fact, we have found that the nuclei of oocytes with an expanded cumulus or with clear signs of degeneration had, for the most part, abnormal or even Metaphase I or II configurations, as occurs in the cow (26). In the case of the sheep, the nucleus of the oocyte seems to be more resistant and remains in the germinal vesicle stage in spite of atresia (16).

The proportion of oocytes in Metaphase II increased significantly with a corresponding increase in culture time from 24 to 27 h. In oocytes from adult goats treated with FSH, the percentage of Metaphase II also increases significantly when passing from 18 to 27 h of culture (18). Also, for oocytes of prepubertal goats, whether or not they were primed with PMSG, the percentage of Metaphase II increased significantly between 15 and 25 h of culture, but few differences were observed increasing the culture time up to 30 or 35 h (32). We can deduce from all these results that the time necessary for goat oocytes, independently of the physiological stage of the animal, to reach Metaphase II in vitro is about 27 h, a little more than in the sheep (24 to 26 h; 21) and the cow (24 h; 35,33).

Most of the oocytes from small follicles (1 .O to 1.9mm) were arrested in Metaphase I, as occurs in the oocytes of adult goats (11). This arrest disappears progressively as the follicular diameter increases up to 3 mm. This progressive acquisition of competence for the completion of nuclear maturation can be explained by the growth of the oocyte which accompanies the growth of the follicle. The diameter of the oocyte increases markedly on changing from follicles of 1.0 to 1.9 mm to 2.0 to 2.4 mm, which coincides with the marked increase in the percentage of Metaphase II reached (from 24.14 to 52.70%). From 2 mm, the increases both in the diameter of the oocyte and the percentage of Metaphase II are more gradual. This means that there are probably drastic changes in the growth and other physiological aspects of the oocyte in follicles smaller than 2 mm. De Smedt et al. (11) observed a germinal vesicle arrest after culturing oocytes of follicles smaller than 0.5 mm, and a GVBD arrest in follicles of 0.5 to 0.8 mm. This coincided with an intense synthesis of RNA in this type of oocytes and different patterns of protein synthesis according to follicular size. Overall, it can be concluded from these results that meiotic competence in the goat, independently of sexual maturity, is acquired in various steps, as occurs in the pig and the cow (24,25). The competence for GVBD, Metaphase I and Metaphase II would be reached, in follicles of 0.5 to 0.8 mm, 1 .O to 1.9 mm, and 23.0 mm, respectively.

Le Gal et al. (18) did not detect synthesis of new proteins during the transition from Metaphase I to Metaphase II in goat oocytes. Possible explanations of the incapacity of the growing oocytes to resume or complete meiosis could be the incomplete synthesis of RNA coding for the cytoplasmatic inducers or regulators of the meiosis (possibly the Maturation Promoting Factor), or even the presence of an inhibiting factor (23,27). In the pig and the cow it has been possible to relate the acquisition of meiotic competence to the appearance of a compact nucleolus which contains stored RNA (25,5). Preliminary studies also indicate the appearance of a compact nucleolus in totally competent goat oocytes (11).

978 Theriogenology

The correlation that exists between the diameter of the oocyte and the meiotic competence suggests that it is possible to select oocytes for IVM according to their diameter. The average diameter of the oocytes (excluding the zona pellucida) from 3.0 to 6.0 mm follicles, and which have presumably completed their growth, was 136.37 pm. This value is slightly greater than that described for oocytes which reach their maximum size in the ewe (110 pm; 4), or which acquire meiotic competence in the case of the cow (120 to -130 pm; 2530).

The percentage of Metaphase II obtained for oocytes collected from 3.0 to 6.0 mm follicles was similar in Experiments 2 and 3 (73.21 and 74.78%). These values were lower than that obtained in Experiment 1 for the same follicular class (88.0%). This could be explained by the different culture systems used. In a previous study we obtained an improvement in nuclear maturation rates in a granulosa cell co-culture system, as in experiment 1 (29). Despite the lower maturation rates, culture in absence of granulosa cells was preferred to study the factors affecting the nuclear maturation because healthy granulosa cells are difficult to obtain from prepubertal goat follicles, especially from small follicles in Experiment 3.

The quality of the oocytes, measured according to the quantity and aspect of the cumulus oophorus, increases as the follicular diameter increases. The system of dissection used respects the integrity of the cumulus cells, thus the proportion of healthy oocytes selected for culture, or the number of cumulus cell layers can be used to asses the degree of follicular atresia. Therefore, follicular atresia increases as follicular diameter decreases. This coincided with the observation of the appearance of the smallest follicIes. However, the number of layers of cumulus cells, and thus the degree of follicular atresia, did not have any effect on the nuclear maturation capacity. This result has also been observed in bovines (19,9). Follicular atresia did not have much influence on IVM of ovine oocytes, because, as occurs in our case, the innermost layers of the cumulus oophorus and the corona radiata, or the oocyte itself, are the last structures to be affected during follicular atresia (22).

From 76 1 ovaries only 843 follicles between 3 and 6 mm (1.1 per ovary on average) were found. After dissection, only 621 oocytes selected for culture (CUM1 f CUM2) were obtained (0.82 per ovary). This low oocyte yield was not due to the quality of the oocytes (73.67% of the follicles gave oocytes selected for culture), but to the low number of follicles larger than 3 mm. In our study not all follicles smaller than 2.5 mm were dissected, but their number was much greater. In this way it is easily possible to reach, or even exceed, the value of 22.7 oocytes with compact cumulus per pair of ovaries obtained by Song and Iritani (31) by selecting follicles over 1 mm, in goats of 2 to 4 months of age. However, the meiotic competence of these oocytes is limited. In the cow a better performance from slaughterhouse ovaries is currently obtained, and 8 or 10 selected oocytes per ovary can be collected in calves or cows, respectively (20,28,13), from follicles of 2 to 6 mm.

In conclusion, the majority of prepubertal goat oocytes reach meiotic competence in follicles of 3 mm or more diameter. These oocytes, collected from slaughterhouse ovaries are capable of maturing in vitro under the same conditions as the oocytes of adult goats, with similar percentages of nuclear maturation. Because of the low number of follicles larger than 3mm in the ovaries of prepubertal goats, a large number of ovaries is needed. Experiments are being carried out in order to evaluate the capacity of these oocytes for in vitro fertilization.

Theriogenoiogy 979

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980 Theriogenolog y

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