The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

ELSEVIER

THE EFFECTS OF FOLLICULAR FLUID ON IN VITRO MATURATION, OOCYTF FERTILIZATION AND THE DEVELOPMENT OF BOVINE EMBRYOS KS.Kim’, N.Mitsumizo’, K.Fujita2 and K.Utsumi’ ‘Department of Animal Science, College of Agriculture, Kyoto University, Kyoto 606 ?3higa Prefectural Experimental Animal Station, Hino-cho, Shiga, Japan 529-16 Received for publication: December 30, 1994 Accepted: October 24, I:?95 ABSTRACT

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 604 D (v/v ) was added to Medium 199 with Earle’s salts supplemented with 0.1 k g/ml estradiol-17 fl (E2, Experiment 1) or 0.1 g g/ml Ez and 100 IUiml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to >2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (PcO.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E2 had more cells than the controls (P
Theriogenology 45:787-799. 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691W96/$15.00 PII S0093691X(96)00008-2

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788 INTRODUCTION The capacity for fertilization and embryonic

development

depends on nuclear and

cytoplasmic maturation of the oocytes(5, 13). Mammalian oocytes can spontaneously undergo meiotic maturation in vitro(I1). Nuclear events, such as germinal vesicle breakdown and polar body formation appear to occur normally during this spontaneous maturation. Fertilization and the subsequent development of oocytes to the preimplantation embryo following in vitro maturation are often less successful than that of oocytes matured in vivo. It has been suggested that this is caused by incomplete cytoplasmic maturation of the oocytes(l8). To improve cytoplasmic maturation, several materials have been added to the medium. These include growth factors(6, 7), sera(23, 33), hormones(2, 32) and follicular fluid(4, 19). Docytes have also been co-cultured with follicular cells such as cumulus c&(15, 26) and mural granulosa cells{15 31). Nevertheless, these in vitro conditions do not always duplicate those in vivo. The estradiol and LH levels in bovine follicular fluid change with follicular development in viva(8), and the follicular fluid in the bovine developing follicle stimulates oocyte maturation(30). Supplementation of folhcular fluid from large follicles(l6) have supported the nuclear and cytoplasmic maturation of bovine oocytes in vitro, but most studies on the relationship between follicular fluid and the maturation of oocytes have been carried out for not bovine but porcine. Meiotic resumption inhibitor(28) and stimulator(34) are present in pig and bovine follicular fluid, and their levels change according to the developmental stage of the follicles(22). Usually, classification of follicular fluid is based upon the size of the follicles, but this procedure is inaccurate due to the sexual cycle of the animals. In our present study, we examined the effects of heparinized follicular fluid from bovine small follicles on the maturation of bovine oocytes supplemented with estradiol or estradiol and hCG, in terms of fertilizability, developmental capacity to the blastocyst stage and the number of cells in blastocysts. MATERIALS AND METHODS Preparation of Follicular Fluid and Maturation Medium Ovaries obtained at a local slaughterhouse from fattened Japanese Black cattle, 2.5 to 3 yr of age, were transported to the laboratory within 4 h in Ringer’s solution containing penicillin G(200 II-J/ml; Sigma Chemical, St. Louis, MO, USA) and streptomycin sulphate(200 k g/ml; Sigma) at 36 to 38 “c. The ovaries were pooled regardless of the stage of the estrous cycle of the donors. All ovaries were rinsed twice with warm Ringer’s solution. Bovine follicular fluid was aspirated from small follicles(1 to 5 mm), 0.1 mg/ml heparin(H3393, Sigma) was added to

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prevent coagulation. This was centrifuged at 1,500 x g for 5 mm, filtered through a 0.22- km membrane and stored at -30 c. The medium for oocyte manipulation was Medium 199(M199) with Earle’s salts (25, Gibco BRL, Life Technologies Inc., Grand Island, NY, USA) containing 31 u g penicillin G/ml and 100 mg streptomycin sulphate/ml. The medium for oocyte maturation (OM199) was Ml99 supplemented with 0.1 w g/ml estradiol (Ez, Experiment 1; Sigma) or 0.1 b g/ml Ez and 100 W/ml human chorionic gonadotrophin(hCG, Experiment 2; Teikoku Hormone Zoki, Co., Ltd., Japan). Follicular fluid(lO, 30 and 60% v/v ) and 3 mg/ml polyvinylpyrrolidone (PVP K-30, Nakarai, Japan) were respectively added to 0M199. A 200- ti 1 droplet of the medium was covered with warm paraffin oil (Paraffin liquid light, Nakarai, Japan) in a culture dish (Coming 25010, Chiba, Japan), and equilibrated for 24 h at 39 “C in 5% Cch in air before use for oocyte maturation. Oocyte Maturation In Vitro Cumulus oocyte complexes were aspirated from small antral follicles of 1 to 5 mm in diameter with an 18-g needle attached to a disposable lo-ml syringe and placed into Petri dishes (Coming 25011). The collected oocytes with intact and compact cumulus cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS, Gibco BRL, Life Technologies Inc., Grand Island, NY, USA) supplemented with 3 mg/ml bovine serum albumin (BSA; A4503; Sigma) then twice with OM199. Cumulus oocyte complexes in groups of 30 to 40 oocytes were transferred to 200- b 1 drops of maturation medium under paraffin oil and cultured for 24 h at 39 “c in 5% COZ in air. Preparation of Feeder Layer At oocyte collection, granulosa cells were recovered from follicles washed with PBS (3 mg/ml, BSA) and centrifuged at 100 x g for 5 min. Pelleted granulosa cells were resuspended with PBS(BSA-free) containing 0.4% hyaluronidase (H3506, Sigma), and dispersed for 7 to 8 min at 38 “c . The cell suspension was mixed with 5 ml of PBS (3 mg/ml, BSA), washed and centrifuged for 5 min at 100 x g. The pellets were washed once with Ml99 t 5% CS (calf serum; Gibco), centrifuged and resuspended at a density of 4 to 5 x10’ cells/ml into 200 p 1Ml99 t 5% CS. The suspensions were cultured for 30 h to form a feeder layer at 39 “c in 5% CO2 in air. In Vitro Fertilization and In Vitro Coculture Frozen-thawed semen(0.5 ml x 3) was washed twice with m-Tyrode’s balanced salt solution supplemented with 10 mM caffeine (sodium benzoate C4144, Sigma) similar to Brackett and Oliphant(1) centrifuged at 500 x g for 5 min. After decanting the supematant, pellets

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containing spermatozoa were resuspended at a density of 5 to 6 x lo6 cells/ml in 100 ,u 1of a 50 : 50 mixture of BSA-free m-Tyrode’s solution with 10 mM caffeine and m-Tyrode’s solution with 20 mg/ml BSA. The sperm suspension was incubated for 3 h at 39 “c in 5% COz in air. Oocytes cultured for 24 h were washed 3 times with m-Tyrode’s solution containing 10 mg/ml BSA, and then 5 to 10 complexes were placed into 100 b 1 sperm suspension for 6 h at 39 “c in 5% COZ in air. After 6 h, the fertilized embryos were removed from the droplets and freed from surrounding cumulus cells and accessory spermatozoa. The embryos were then transferred onto the feeder layer of granulosa cells and cultured for 9 d, with half of the volume of the medium exchanged every 48 h. To evaluate the maturation and fertilization rates, at 24 h after maturation and 20 h after fertilization, oocytes were fixed with ethanol-acetic acid (3:1), stained with 1% orcein and examined by phase contrast microscopy (x 400). Maturation was assumed by the presence of Metaphase II chromosomes with the first polar body, and fertilization was considered complete when 2 pronuclei were evident. The rates of the development of oocytes to >2-cell, 8 to 16-cell and blastocyst and hatched blastocysts at 2, 3, 8 and 9 days after insemination were morphologically estimated. Mean measurements from 5 replicates were taken to determine examined statistical differences between procedures using the Chi-square analysis. Assessment of Embryonic Quality To estimate embryonic quality, the numbers of cells in Day-9 blastocysts and hatched blastocysts obtained from Experiments 1 and 2 were analyzed by the method of Byun et al.(3), in Experiment 3. The embryos were treated with Tyrode acid solution containing 1 mg/ml PVP @H 2.5) to remove the zona pellucida. The denuded embryos were then mounted on glass slides, fixed for 5 min in ethanol-acetic acid (3:1), stained for 5 min with basic fuchsin (Nakarai, Japan), and rinsed with acetic glycerin (acetic acid:distilled water: glycerin=l:3:1) at room temperature. The cells were counted under a phase-contrast microscope (Olympus, Tokyo, Japan). The numbers were analyzed via Student’s t-test on the logit using CATMOD procedures. In Experiments 1 and 2, averages of 5 replicate trials for each procedure were examined for statistical differences using SAS. RESULTS Experiment 1 The effect of follicular fluid with E2 during IVM on the ability of oocytes to be fertilized

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and to develop was examined. The average number of oocytes recovcrcd from one pair of ovaries was 22.5 + 8.5, and with 46.5% being compact cumulus oocyte complexes, which were used in the experiments. No statistically significant correlation could be found between the yield of compact cumulus oocyte complexes and any of the following criteria: age of animal, estrous cycle stage, date of experiment, or number of small follicles (1 to 5 mm) visible on the ovary. A total of 1698 cumulus oocyte complexes were studied. The maturation and fertilization rates of the oocytes in Experiment 1 are shown in Table 1. The maturation and fertilization rates of the oocytes cultured in medium containing various concentrations of follicular fluid and Ez were similar to those of the unsupplemented (PVP) control.

Table 1.

Effect of bovine follicular fluid and estradiol (E2) in the medium on the in vitro maturation of bovine oocytes and their ability to be fertilized

Added to TCM-199” as maturation medium 3 mgiml 10 % 30 % 60 % a b

PVPb follicular fluid follicular fluid follicular fluid

No. of oocytes examined 40 38 31 31

Matured(%)

34(85 .O) 33(86.8) 27(87.1) 25(80.6)

No.of oocytes examined 39 38 39 40

Fertilized(%)

33(84.6) 32(84.2) 34(87.2) 34185.0)

E2

0.1 kg/ml. polyvinylpyrrolidone.

The development of fertilized oocytes to development >2-cells, 8 to 16-cells, blastocysts and hatched blastocyst is shown in Table 2. The rates of development of the oocytes to >2-cell stage at 2 d after insemination were 63.2, 52.3 and 44.7% in the presence of 10, 30 and 60% follicular fluid. The presence of 10% follicular fluid in the maturation medium did not affect the development of the fertilized oocytes compared with that of PVP (60.9%) . On the contrary, the development of fertilized oocytes matured in 30 and 60% follicular fluid decreased significantly (PcO.01) compared with that of PVP supplement. Similar to that of the >2-cell stage, development of fertilized oocytes to 8 to 16-cell stage was not affected by 10 and 30% of follicular fluid in the maturation medium, but significantly decreased in medium containing 60% follicular fluid when compared with those matured in PW (control). The rates of development of fertilized oocytes matured in 10, 30 or 60% follicular fluid to blastocysts and hatched blastocysts were similar to those of the controls (30.3 and 6.9%, 27.0 and 7.4%, or 16.3 and 3.5%, respectively).

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Development of fertilized oocytes matured in 60% follicular fluid was lower than that of the controls (PcO.05).

Table 2.

Effect of adding bovine follicular fluid and &radio1 medium on development of fertilized bovine oocytes

Added to

No. of

TCM-199’ as

oocytes

maturation medium

tested

3 mg/rnl PVPb 10 % follicular fluid 30 % follicular fluid 60 % follicular fluid

138 145 148 141

(E2)

in

protein-free maturation

No.(%) of embryos developing to z-2-cell stage at 2 days

8 to 16-cell stage at 3 days

blastocyst stage at 8 days

hatched.Bl stage at 9 days

84(60.9)” 92(63.2)’ 77(52.0)d 63(44.7)d

59(42.7)’ 67(46.2)’ 58(39.2)” 33(23.4)d

39(28.3)’ 44(30.3)’ 40(27.0)’ 23(16.3)d

7(5.1)” lO(6.9)’ ll(7.4)’ 5(3.5)c

’ E20.1 kg/ml. b polyvinylpyrrolidone. gd Different superscripts differ significantly within same column(cd:P
Experiment 2 Effect of follicular fluid with EUrCG during IVM on the ability of oocytes to be fertilized and develop was assessed in this experiment. The rates of maturation and fertilization of oocytes matured in medium containing follicular fluid, IS? and hCG or control medium were similar (Table 3). The development of fertilized oocytes matured in media containing follicular fluid, E2 and hCG to >Zcells, 8 to 16 cells, blastocysts and hatched blastocysts is shown in Table 4. At 2 d after insemination, development (73.5, 76.0 or 73.0% for of the 10, 30 or 60% follicular fluid in the medium)was similar to that of the control (77.4%). Development to the 8 to 16-cell stage was also similar for fertilized oocytes matured in 10 and 30% of follicular fluid, but significantly less in 60% (PqO.01) than that of the controls. Development to the blastocyst stage was 37.9 and 33.6% for fertilized oocytes matured in 10 and 30% follicular fluid, respectively. However, development of fertilized(24.8%) oocytes in 60% follicular fluid was significantly less than in the controls (35.9%; PcO.05).

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Effect of bovine follicular fluid and estradiol (Ez)/hCG in the medium on the in vitro maturation of bovine oocytes and their ability to be fertilized

Table 3.

No. of oocytes examined

Added to TCM-199’ as maturation medium 3 mg/ml 10 % 30 % 60 %

Matured( %)

31(91.2) 30(83.3) 26(89.7) 31(88.6)

34 36 29 3.5

PVPb follicular fluid follicular fluid follicular fluid

No.of oocytes examined

Fertilized(%)

32(94.1) 30(88.2) 32(91.4) 34(94.4)

43 34 35 36

a E2 0.1 kg/ml, 100 IU hCG/ml. b polyvinylpyrrolidone.

Table 4.

Effect of bovine follicular fluid and estradiol (Ez)/hCG in protein-free medium on development of fertilized bovine oocytes

Added to

No. of

TCM-199” as

oocytes

maturation medium

tested

3 mg/ml PVPb 10 % follicular fluid 30 % follicular fluid 60 % follicular fluid

142 132 146 137

maturation

No.(%) of embryos developing to >2-cell stage at 2 days

8 to 16-cell stage at 3 days

blastocyst stage at 8 days

hatched.Bl stage at 9 days

l_lO(77.4)C 97(73S)C 111(76.0)c lOO(73.O)C

89(62.7)c 78(59.1)e 84(57.5)” 63(45.9)“r

51(35.9)” 50(37.9)” 49(33.6)ef 34(24.8)’

lS(10.6)c 11( 8.3)ef 7( 4.8)’ 9( 6.6)e’ -

a E2 0.1 Mg/ml, 100 BJ/ml hCG. b polyvinylpyrrolidone. c-f Different superscripts differ significantly within same column (cd:P
Experiment 3 In this experiment we tested the effect of bovine fohicular fluid in the maturation medium of the oocytes on the number of cells in blastocysts 9 days after insemination. The number of ceils in embryos obtained from Experiments 1 and 2 were analyzed. The numbers of cells in blastocysts and hatched blastocysts at 9 days are shown in Tables 5 and 6. The numbers of cells in blastocysts and hatched blastocysts that developed from fertilized oocytes matured in vitro in 10 or 30%

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follicular fluid with E2 were 153.2 f 11.0 or 141.9 + 10.1, respectively. These embryos contained more cells than those matured with PVP (110.5 rf: 10.4, PcO.01). However, the numbers of cells (112.6 + 10.7) in blastocysts and hatched blastocysts that developed from fertilized oocytes matured with 60% follicular fluid were similar to the controls (Table 5). Even when hCG was added to the maturation medium containing follicular fluid and E2, cell numbers of the blastocysts developing from these fertilized oocytes were similar (Table 6).

Table 5.

Effect of bovine follicular fluid and estradiol (E2)in protein-free maturation medium on the cell numbers of bovine embryos in vitro 9 days after fertilization

Added to TCM-199” as maturation medium 3 mg/ml PVPb 10 % follicularfluid 30 % follicular fluid 60 % follicular fluid

No. of embryos tested 19 17 20 18

Numbers of cell in blastocyst at 9-days Mean no. f SE 110.5 153.2 141.9 112.6

+ 10.4c z!z ll.Od z!I lO.ld f 10.7c

range Cell 61-198 91-257 63-211 44-239

Relative cell numbers 100.0 138.6 128.4 101.9

a E20.1 fig/ml. b polyvinylpyrrolidone. Gd Different superscripts differ significantly within same column (PcO.01).

Table 6.

Effect of bovine follicular fluid and estradiol (Ez)/hCG to protein-free maturation medium on the cell number of bovine embryos in vitro 9 days after fertilization

Added to TCM-199” as maturation medium 3 mg/ml PVPb 10 % follicular fluid 30 % follicular fluid 60 % follicular fluid

No. of embryos tested 27 18 23 19

a Ez 0.1 ,u g/ml, 100 ID/ml hCG. b polyvinylpyrrolidone.

Numbers of cells in blastocyst at 9-days

Relative cell

Mean no. I!KSE

numbers

range Cell

114.8 f 7.5b

65-175

100.0

128.0 + 9.2b 106.0 zk 8.2b 113.9 + 9.0b

69-177 36-206 45-209

111.5 92.3 99.2

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Theriogenology

The percentage distribution of bovine embryos containing 5 ranges of cells per blastocyst and hatched blastocysts at 9 d after insemination when the fertilized oocytes were matured in follicular fluid, E2 (Experiment 1) or E;?/hCG (Experiment 2) is shown in Fig-l. Adding 10 and 30% of follicular fluid with E2 to the maturation medium improved the subsequent development of these fertilized oocytes with respect to cell numbers. More than 80% of these embryos had z-100 cells in the blastocyts and hatched blastocysts at 9 d after insemination and 20% of the oocytes matured in 10% follicular fluid with E2 developed to blastocysts containing >200 cells.

Range of cell number I O-50 I 50-100 I 100-150

PVP(Exp.1) FFl-10% FFl-30%

q

FFl-60%

0

PVP(Exp.2)

150-200 200-250

FF2-10% FF2-30% FF2-60% 0

60 70 80 90 10 20 30 40 50 Proportional distribution of cell numbers in embryos

100 %

Fig 1. Percentage distribution of embryos containing 50 cells in blastocysts and hatched blastocysts at Day 9 derived from the oocytes matured in vitro with follicular fluid.

DISCUSSION This study suggests that follicular fluid derived from small follicles may improve the quality of embryos after fertilization when added to the in vitro maturation medium of bovine oocytes. Homologous follicular fluid supports in vitro oocyte maturation and promotes the subsequent fertilization and development of these oocytes(9, 17). Kim et a1.(16) reported that a higher-dose (60%) of follicular fluid(from follicles 1 to 5 mm diameter) added to the maturation medium inhibits development as well as oocyte maturation, probably due to coagulating the cumulus cell mass by fibrin-like substance in the follicular fluid; whereas a lower-dose (10 %) stimulated both the maturation and developmental ability of the bovine oocytes. Naito et a1.(19, 20) reported that porcine follicular fluid(pFF) derived from small follicles(2 to 5 mm) induced porcine oocytes to mature, and that the developmental capacity of the oocytes matured in pFF seems similar to that of oocytes matured in vivo. Yoshida et al.(34) have also reported on effective nuclear maturation of immature oocytes and on the formation of male

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pronuclei in porcine oocytes following in vitro fertilization in the presence of 10% porcine follicular fluid instead of fetal calf serum (FCS) in the protein-free maturation medium. Funahashi and Day(14) demonstrated improved male- and female-pronuclei formation in porcine oocytes matured in medium containing 10 IU Ph4SG, 10 IU hCG, 1 lo g/ml Ez and porcine follicular fluid (4 to 6 mm diameter) compared with oocytes matured in FCS. These results indicated that follicular fluids can stimulate the cytoplasmic and nuclear maturation of oocytes in vitro. Sluss et a1.(27) showed that a fraction of porcine follicular fluid contains both follicle stimulating hormone agonistic and antagonistic activities. These findings suggest that some factors in the follicular fluid function either positively or negatively in the resumption of oocyte maturation according to hormonal background. The earlier reports were based on in vitro studies in which porcine follicular fluid was shown to inhibit the maturation of porcine(28), rat(29) and mouse(9) oocytes. Romero et a1.(21) reported that adding 40% high molecular (>lOO,OOO)weight fraction fluid from large bovine follicles at 20 h after the LH-surge stimulated the resumption of meiosis in the presence of hypoxanthin(meiosis inhibitor). Recently, Iarocca et al.(17) reported that fluid from follicles over 1.5 mm in diameter added to the culture medium ( concentration of 30%)during in vitro maturation of bovine oocytes improved the fertilization rate and resulted in a higher percentage of morula to blastocyst development 10 d after insemination. We also found that a low concentration (10%) of follicular fluid in the maturation medium increased the subsequent cleavage rate and the ability to develop into blastocysts and hatched blastocysts, whereas the a high concentration (60%) decreased development. Our results support those of Iarocca et a1.(17) with respect to the effect of follicular fluid on the developmental ability of the oocytes. We therefore considered that the cumulus cell-surrounded oocyte is stimulated in vitro in the same way as it is by the LH surge-like system in follicular fluid and by hormones such as estrogen and LH, so that any hormones or proteins that can counteract the follicular inhibitory action and the induce meiosis resumption are synthesized in the cumulus oocyte complex. There was not only a decrease in nucleus maturation and in the fertilization rate when a higher concentration of follicular fluid was used in culture medium but suppressed developmental ability as well after fertilization. This was probably due to insufficient cytoplasmic maturation during oocyte maturation, caused by inhibitory factors such as purines(e.g. adenosine and hypoxanthine) in the follicular fluid. Tsafriri and Charming(28) have demonstrated that porcine follicular fluid derived from small follicles affects the inhibition of the spontaneous maturation of isolated porcine oocytes. Bovine follicular fluid from l- to 5 mm follicles contains some inhibiting factors, preventing germinal vesicle breakdown during culture(24) in the mouse. Sirard(25) has also reported that bovine cumulus oocyte complexes cultured in whole follicular fluid from the small follicles of ovaries derived from a slaughterhouse decreased oocyte maturation and inhibited nucleus maturation. Cooperation of follicular fluid with cAMP might sustain the suppression to resumption meiosis(9). These results indicated that follicular fluid contains an inhibitory factor(s) originally termed the oocyte maturation inhibitor. However, these inhibitors seemed to decrease the

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process of follicle development and ability to ovulate. Our results indicate that the developmental abihty of the oocytes to >Zcells and blastocysts was improved by adding estradiol(O.1 k s/ml) and hCG (100 ID/ml) to the follicular fluid (Table 4) compared with estradiol alone (Table 3). As shown in Fig-l, the number of cells in the blastocysts 9 d after insemination increased significantly in embryos derived from oocytes matured in medium containing 10 or 30% follicular fluid and Ez (Experiment 3). The number of cells (153.2 + 11.0) in the embryos obtained this experiment was similar to that (113.8 i 9.3) of 8 d bovine blastocysts obtained in vivo(l0). Yoshida et al.(35) have also reported that a high molecular weight substance from fluid of porcine follicles 1 to 5 mm in diameter induces full maturation of oocytes into embryos. This implies that the fluid from small small follicles contains a factor(s) involved in the oocyte maturation process necessary for subsequent fertilization and development also improves the quality of the embryos developed from these oocytes. In conclusion,

we found that adding follicular fluid derived from small follicles to

maturation medium containing estradiol, may enable bovine oocytes to mature fully. This suggests a method for improving the in vitro production of bovine embryos. Embryos at the blastocyst stage as obtained using this system contain morecellsand may aid the viability of cryopreserved embryos or cell numbers with which to initiate embryonic stem cell lines. REFERENCES 1.

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