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IN VITRO MATURATION OF BOVINE OOCYTES

FOR IN VITRO FERTILIZATION

Professor Ioan GROZA*, PhD

Lecturer Simona CIUPE*, PhD

Assistant Mihai CENARIU*, PhD

Emoke PALL*, PhD Student

Anamaria PETREAN*, PhD Student

Abstract

The objective of the present study was to asses the quality of various cultivation media used for the maturation of bovine oocytes 
that are prepared for IVF. Upon collection from slaughtered bovine ovaries and after morphological evaluation, a total number of 513 
viable oocytes have been selected for cultivation, being divided into 3 batches, 171 oocytes / batch. The oocytes belonging to batch 
1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH 20 μl/ml. The oocytes belonging to batch 2 were cultivated in TCM 199 
NaHCO3 + 10% FCS + HCG 2.3 x 103 UI/ml + FSH 8 μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml. The oocytes belonging to batch 
3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 1 μl/ml + FSH 20 μl/ml. The cultivation conditions, for all three 
batches, were: 24 hours at 39°C, 5% CO2. Spermatozoa have been prepared using the Percoll method and IVF of the matured oocytes 
has been performed. Embryonic development has been assessed 72 hours and then up to 10 days after IVF. The results showed the 
superior quality of the oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 UI/ml + FSH 8 
μl/ml + pyruvate 0.25 mM + 17β estradiol 1 μl/ml, as their use for IVF yielded the highest number of viable embryos.

Key words: bovine, oocyte, in vitro fertilization, maturation, TCM 199, cultivation

Introduction

Embryo transfer technology has been used commercially over the past 2 decades [1,5]. The available 

statistics published by IETS show that the use of embryo transfer technology has increased rapidly 

during the 1980’s and the early 1990’s [3,8]. However, embryo production has stabilized over the past 

5 years. Part of the reason for this plateau in the use of embryo transfer is the difficulty to harmonize 

this technology with the production goals of every herd [2,4,9]. The in vitro production of embryos 

(IVF) is an approach that may increase the efficiency of reproduction in a cow without compromising 

its production life. Combined with the technologies developed to control ovarian function, increased 

production of viable embryos through IVF of oocytes derived from growing follicles may prove a 

* University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Department of Veterinary Reproduction, 
Obstetrics and Gynecology, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania, e-mail: isgroza@yahoo.com

Cluj Veterinary Journal, 15(1)/2009, pp. 15-18



16

valuable method [6,7]. The objective of the present study was to asses the quality of various cultivation 

media used for the maturation of bovine oocytes that are prepared for IVF.

Material and Methods

The oocytes used in this study have been obtained from 

104 slaughtered bovine ovaries, recovered in sterile saline 

with 100 UI penicillin/ml, at 35°C. The interval of time that 

passed from the recovery of the ovaries until processing them 

in the laboratory has not been longer than 2 hours. 

The ovaries were washed with sterile saline in order 

to remove any trace of blood and the follicles between 1-

8 mm were punctured using an 18G needle attached to a 

10 ml syringe. The follicular fluid was aspired, passed in a 

plastic Corning tube with maintenance medium represented 

by TCM 199 (Gibco) + heparin 2 UI/ml and then filtrated 

in order to concentrate the oocytes in a smaller amount 

of liquid. The oocytes have subsequently been passed in 

a Petri dish and evaluated using a stereomicroscope at a 

20x magnification for counting and 60x for morphological 

evaluation. Only the adequate oocytes were selected, which 

presented homogenous cytoplasm and compact cumulus, with at least 3 layers of cells surrounding 

the oocyte (figure 1).

After morphological evaluation, a total number of 513 viable oocytes have been selected for 

cultivation, being divided into 3 batches, 171 oocytes/batch. 

The oocytes belonging to batch 1 were cultivated in TCM 199 NaHCO3 + 10% FCS + FSH (FSHp, 

Shering) 20 µl/ml. 

The oocytes belonging to batch 2 were cultivated in TCM 199 NaHCO3 + 10% FCS + HCG (APL) 

2.3 x 103 UI/ml + FSH (Folltropin-V) 8 µl/ml + pyruvate 0.25 mM + 17β estradiol 1 µl/ml. 

The oocytes belonging to batch 3 were cultivated in TCM 199 NaHCO3 + 10% FCS + 17β estradiol 

1 µl/ml + FSH (FSHp, Shering) 20 µl/ml. 

The cultivation conditions, for all three batches, were: 24 hours at 39°C, 5% CO2.

The spermatozoa were thawed (5 s at room temperature and 30 s in water at 37°C) and added to 

a conical tube, on top of 2 ml of 90% Percoll solution and 2 ml of 45% Percoll solution. The mixture 

was centrifuged at 1000xg for 10 minutes, the spermatozoa were resuspended in 10 ml TALP and 

then centrifuged again at 200xg for 5 minutes.

The matured oocytes were passed into 4-wells culture dishes, in fertilization medium (FERT-

TALP + 55µg/ml heparin) at a density of maximum 30 oocytes/well. The spermatozoa were added at 

a concentration of 1.3 x 106 spermatozoa / ml and finally the microdrops were covered with mineral 

oil (Sigma). 

The dishes were vortexed at 18-20 hours and further incubated for 72 hours at 39°C, in 5% CO2. 

After this interval, the embryonic development was assessed recording the number of cells presented 

by every embryo as well as the presence of non-fertilized oocytes. The non-fertilized oocytes were 

discarded, the medium was changed and embryonic development was followed until day 10 after 

in vitro fertilization.

Fig. 1. Adequate oocytes, suitable 
for in vitro maturation



17

Results and Discussions

After evaluation of the embryonic development at 72 hours after IVF, the results obtained were 

as follows (table 1, chart 1 and figure 2):

Table 1. Results of the in vitro fertilization after 72 hours of culture

Batch number Total number of oocytes Non-fertilized oocytes 2-8 cells embryos
Batch 1 171 51 (29.82%) 120 (70.18%)
Batch 2 171 42 (24.56%) 129 (75.44%)
Batch 3 171 46 (26.90%) 125 (73.10%)

The figures presented above show small differences between the three batches. The maturation 

medium used for batch 2 yielded the best results as it contained FSH, HCG, pyruvate and β -estradiol. 

All these hormones, together with the pyruvate offered the best conditions for oocyte maturation as 

well as its preparation for in vitro fertilization.

When embryonic development was assessed at 10 days of culture after the in vitro fertilization, 

the following results have been obtained (table 2, chart 2 and figure 3):

Table 2. Results of the in vitro fertilization after 10 days of culture

Batch number Total number of embryos 
cultivated over 72 hours Expanded blastocysts obtained

Batch 1 120 16 (13.33%)
Batch 2 129 21 (16.27%)
Batch 3 125 19 (15.20%)

Batch 1
Batch 2

Batch 3

Non-fertilized oocytes

2-8 cells embryos

120 129
125

51
42 46

0

20

40

60

80

100

120

140

160

Chart 1. Results of the in vitro fertilization 
after 72 hours of culture Fig. 2. Embryos obtained at 72 hours after IVF

Batch 1
Batch 2

Batch 3

Expanded blastocysts

Embryos cultivated over 72 h

120 129 125

16 21
19

0

20

40

60

80

100

120

140

160

Chart 2. Results of the in vitro fertilization 
after 10 days of culture Fig. 3. Blastocysts obtained at 10 days after IVF 



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The figures shown above also show small differences between the three batches, but again, the 

highest number of expanded blastocysts has been obtained in batch number 2, proving the better 

quality of the oocytes matured using this maturation medium and subsequently the better quality of 

the embryos obtained from them.

Conclusions

After performing the research, the following conclusions have been drawn:

1. The evaluation of the best maturation conditions for oocytes has been performed, comparing 

various combinations of additives for the maturation medium. 

2. At 72 hours after the fertilization, satisfactory results have been obtain for all three batches, even 

though the maturation medium used in batch number 2 yielded the best results, leading to the 

highest number of embryos obtained after IVF.

3. The results obtained at 10 days after the in vitro fertilization confirm the superior quality of the 

oocytes belonging to batch 2 and matured using TCM 199 NaHCO3 + 10% FCS + HCG 2.3x103 

UI/ml + FSH 8 µl/ml + pyruvate 0.25 mM + 17β estradiol 1 µl/ml.

4. The supplementation of the maturation medium with gonadotropins and estradiol led to the 

increase of the percentage of fertilized oocytes, favoring the maturation of the cumulus cells and 

stimulating the metabolism of the oocyte.

5. We recommend the use of the medium containing TCM 199 NaHCO3 + 10% FCS + HCG 2.3 x 

103 UI/ml + FSH 8 µl/ml + pyruvate 0.25 mM + 17β estradiol 1 µl/ml for the in vitro maturation 

of bovine oocytes destined for in vitro fertilization.

References

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that affect in vitro maturation and development of bovine oocytes. Theriogenology 45, 943-956.

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and Endocrinology 169, 39-42.

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271.

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7. Sirad, M.A., R.D. Lambert, P. Guay, D.P. Menard, M. Bedoya, 1985, In vivo and in vitro development 
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8. Yang, X., C. Kubota, H. Suzuki, M. Taneja, P.E.J. Bols, G.A. Presicce, 1998, Control of oocyes maturation 
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