Buffalo Bulletin Vol.28 No.1 (March 2009) p. 25-28

INFLUENCE OF BUFFALO OESTRUS SERUM (BES) AND FETAL BOVINE SERUM (FBS) IN THE PRESENCE OF OESTRADIOL (E₂) AND FOLLICLE STIMULATING HORMONE (FSH) ON NUCLEAR MATURATION OF BUFFALO OOCYTES

S.A. Adlak, K.P. Khillare, C.H. Pawshe and S.W. Mude

Department of Animal Reproduction,

Post Graduate Institute of Veterinary and Animal Sciences, Akola 444104, India

ABSTRACT

The influence of buffalo oestrus serum (BES) and fetal bovine serum (FBS) in the presence of oestradiol and follicle stimulating hormone on nuclear maturation of buffalo oocytes was evaluated. The oocytes matured in the presence of 10 % BES or 10 % FBS showed higher maturation rates than those matured in the medium alone. The addition of follicle stimulating hormone (FSH) and oestradiol (E₂) in the presence of serum to the medium improved maturation rate over that of the serum alone. However, the maturation rate was higher (71.05 %) in Ham’s F-10 + FBS + E₂ + 10 µg /ml FSH than in Ham’s F-10 + BES + E₂ + 10 µg /ml FSH i.e. 51.16 %, but no significant difference was observed.

In the present study, FBS was found to be a more suitable protein supplement for in vitro maturation of buffalo oocytes in comparison with BES. Therefore Ham’s F-10 supplemented with 10% FBS in combination with E₂ and 10 µg /ml FSH proved to be the most efficacious medium for maturation of buffalo oocytes.

Keywords: buffaloes, hormone, FSH, oocytes, oestradio, oestrus, FBS, BES

INTRODUCTION

In mammals during in vitro maturation (IVM), the basic medium is supplemented with serum and hormones. The selection of protein supplements and hormones for in vitro maturation (IVM) play an important role in subsequent in vitro fertilization (IVF) and in vitro development (IVD).

For the successful fertilization and subsequent development, the medium must contain a blood serum or extract because full oocyte maturation involves not only acquisition of meiotic competency but also cytoplasmic maturation (Motlik and Fulka, 1986). The most common serum preparations used for bovine oocyte maturation are fetal calf serum (FCS), oestrus cow serum (ECS) and bovine serum albumin (BSA). Recent studies have shown that blood serum was slightly superior than BSA (Cross and Brinster, 1970). Blood serum enhanced cumulus and corona cell survival and it was likely that it has beneficial effects on meiosis resumption, which was indirectly mediated through corona cells.

Supplementation of media supplemented with blood serum resulted in a high frequency of nuclear maturation and cumulus expansion when FSH was added (Leibfried et al., 1987). The addition of FSH, LH and oestradiol cause synergistic enhancement of nuclear maturation of buffalo oocytes (Totey et al., 1993)

Our study was to determine the effect of bovine oestrus serum (BES) and fetal bovine serum (FBS) in the presence of follicle stimulating hormone (FSH) and oestradiol on nuclear maturation of buffalo oocytes.

MATERIALS AND METHODS

Buffalo ovaries were collected from the local slaughterhouse and transported to the laboratory in normal saline at 30-35 °C within 1-2 h of slaughter. Cumulus oocyte complexes (COCs) were isolated from the follicles by a slicing method. Only the oocytes with more than four layers of compact cumulus cells (COC_S) with evenly distributed cytoplasm were used for maturation. The isolated oocytes were washed thrice with TL-Hepes medium. The final two washings were given in the maturation medium. The maturation was studied in two different groups: 1) Ham’s F-10+10 % BES. 2) Ham’s F-10+10 % FBS. Each group consisted of six different treatments i) medium only (control) ii) medium+10 % serum (BES or FBS) iii) medium + 10 % serum + 1 µg/ml oestradiol (E₂) iv) medium + 10 % serum+1 µg/ml oestradiol (E₂)+1 µg/ml FSH. v) medium+ 10% serum+1µg/ml oestradiol (E₂)+10 µg/ml FSH. vi) medium+10 % serum+10 µg/ml oestradiol (E₂)+20 µg /ml FSH.

The COC_S were randomly allocated to different treatments within an individual group, and three replicates were carried out separately for each group. The COC_S were cultured for 24 h in air at 39 °C, 95 % humidity, and 5 % CO₂ and, at the end of the culture period, the cumulus cells of oocytes were removed by repeated pipetting using a fine bore pipette. The oocytes were fixed overnight with ethanol and acetic acid (3:1) and stained with 1 % aceto orecin stain. The oocytes were then evaluated for germinal vesicle (GV), metaphase I (MI), metaphase II (MII) stage and degeneration. The number of oocytes in the different stages were evaluated in different treatments.

RESULTS AND DISCUSSION

The buffalo oocytes were isolated from the ovaries and distributed among different treatment groups. The effect of sera (BES and FBS) in the presence of follicle stimulating hormone (FSH) and oestradiol on nuclear maturation was evaluated. The results are presented in Tables 1 and 2.

Table 1. Influence of buffalo oestrus serum (BES) in the presence of oestradiol (E₂) and follicle stimulating hormone (FSH) on nuclear maturation of buffalo oocytes.

S.N.	Media	Sera	Hormone concentration	Total no. of oocyte	Stages of Maturation
					GV (%)	MI (%)	MII (%)	Deg (%)
1	Ham's F-10	-	-	30	9 (30.00)	5 (16.66)	10 (33.33)	6 (20.00)
2	Ham's F-10	BES	-	33	9 (27.27)	6 (18.18)	12 (36.36)	6 (18.18)
3	Ham's F-10	BES	E2 (1 μg)	30	8 (26.66)	5 (16.66)	12 (40.00)	5 (16.66)
4	Ham's F-10	BES	E2 +FSH (1 μg)	39	6 (15.38)	7 (17.94)	17 (43.58)	9 (23.07)
5	Ham's F-10	BES	E2 +FSH (10 μg)	43	6 (13.95)	8 (18.60)	22 (51.16)	7 (16.27)
6	Ham's F-10	BES	E2 + FSH (20 μg)	37	5 (13.51)	7 (18.91)	18 (48.64)	7 (18.91)

Note: Means bearing similar superscripts show non-significant differences.

GV – Germinal Vesicle stage. MI – Metaphase I stage.

MII – Metaphase II stage. Deg –Degenerated oocyte.

Table 2. Influence of buffalo oestrus serum (BES) in the presence of oestradiol (E₂) and follicle stimulating hormone (FSH) on nuclear maturation of buffalo oocytes.

S.N.	Media	Sera	Hormone concentration	Total no. of oocyte	Stages of Maturation (%)
					GV (%)	MI (%)	MII (%)	Deg (%)
1	Ham's F-10	-	-	30	9 (30.00)	5 (16.66)	10 (33.33)	6 (20.00)
2	Ham's F-10	FBS	-	38	7 (18.42)	8 (21.05)	15 (39.47)	8 (21.05)
3	Ham's F-10	FBS	E2 (1 μg)	24	4 (16.66)	4 (16.66)	10 (41.66)	6 (25.00)
4	Ham's F-10	FBS	E2 +FSH (1 μg)	32	4 (12.50)	4 (12.50)	15 (46.87)	9 (28.12)
5	Ham's F-10	FBS	E2 +FSH (10 μg)	38	3 (7.89)	3 (7.89)	27 (71.05)	5 (13.15)
6	Ham's F-10	FBS	E2 +FSH (20 μg)	41	6 (14.63)	9 (21.95)	21 (51.21)	5 (12.19)

Note: Means bearing similar superscripts show non-significant differences.

GV – Germinal Vesicle stage. MI – Metaphase I stage.

MII – Metaphase II stage. Deg –Degenerated.

The percentage of oocytes reaching metaphase II in medium alone in the two groups was 33.33 %. The addition of protein supplement in the form of BES improved metaphase II percentage (36.36 %) over control but no significant difference was observed. However, FBS showed improved metaphase II percentage (39.47 %) over the control group.

The maturation rate was further increased after the addition of different concentrations of FSH and oestradiol (E₂). The increases with addition of oestradiol to the medium in the presence of BES and FBS were 40.00 % and 41.66 %, respectively.

When the medium was supplemented with BES+ E₂ (1 μg)+FSH (10 μg), the metaphase II percentage was about 51.16 %. Whereas, when the medium was supplemented with FBS + E₂ (1 μg) + FSH (10 μg), the metaphase II percentage was about 71.05 %. The metaphase II percentage was higher in FBS than BES, but the difference was not significant.

In the present study, the medium (Ham’s F-10) containing FBS showed a dramatic improvement in oocyte maturation in presence of oestradiol and FSH (10 μg) as compared to those in Ham’s F-10 containing BES + E₂ (1 μg) + FSH (10 μg)

In the present study, we found that maturation also took place in the medium alone; however, although maturation took place in the medium without any supplementation, oocytes matured under such conditions, fail to form normal pronuclei after sperm penetration due to incomplete cytoplasmic maturation (Thibault, 1977; Bavister, 1987). To improve the cytoplasmic maturation of cattle oocytes, some biological fluids like serum are essential (Saeki et al., 1990). The addition of sera to the culture medium during the maturation of oocytes promotes the rupture of the germinal vesicle and facilitates the oocyte maturation (Sanbuissho et al., 1990).

In the present study, we found that addition of 10 % FBS in the presence of 1 μg E₂ and 10 μg FSH gave an improved maturation rate as compared with 10 % BES supplemented group, but no significant difference was observed. Fetal calf serum improves the maturation and fertilization over those of BES because it contains some unidentified growth promoting components that were absent in the serum of adult animals or because fetal calf serum lacks components (hormones and immunoglobulins) present in the adult serum that retard the in vitro development of the cells (Mochizuki et al., 1991).

Allen et al. (1982) suggested that FCS is advantageous for use because it has some growth promoting components such as fetuin, which is higher in FCS than in adult oestrus serum.

Our results were in concurrence with the observations of Fukui and Ono (1989) who did not observe any significant difference between FCS and oestrus cow serum on bovine oocyte maturation.

In conclusion, the results of this experiment showed that medium alone supported the maturation of buffalo oocytes only partially. The type of serum used during in vitro maturation also plays a significant role in the improvement of maturation rate of buffalo oocytes. In the present study, FBS was found to be a more suitable protein supplement for in vitro maturation of buffalo oocytes in comparison with BES.

ACKNOWLEDGEMENT

Authors are thankful to Dr. D.B. Sarode, Associate Dean, Post Graduate Institute of Veterinary and Animal Science of Akola, for providing all necessary facilities.

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