Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages

Animal Reproduction Science 95 (2006) 1–15

Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages S.S.D. Santos a,∗ , F.C. Biondi b , M.S. Cordeiro b , M.S. Miranda b , J.K. Dantas b , J.R. Figueiredo c , O.M. Ohashi b a

c

Departamento de Histologia e Embriologia, Centro de Ciencias Biologicas, Universidade Federal do Par´a, Bel´em, PA 66 075-000, Brazil b Laborat´ orio de Fertiliza¸ca˜ o In Vitro, Centro de Ciencias Biologicas, Universidade Federal do Par´a, UFPA, Bel´em, PA, Brazil Laborat´orio de Manipula¸ca˜ o de O´ocitos e Fol´ıculos Pr´e-antrais, LAMOFOPA, Faculdade de Veterin´aria, UECE, Fortaleza, CE, Brazil

Received 14 May 2004; received in revised form 19 July 2005; accepted 4 August 2005 Available online 2 May 2006

Abstract The aim of the present study was to determine the most desirable ovarian tissue section thickness to isolate preantral follicles (Experiment I), determine follicular density (follicles/mm2 of cortex) of ovaries of fetal buffalo of different ages (Experiment II), and cultivate preantral follicles of buffalo fetuses (Experiment III). In Experiment I, ovary sections with different thicknesses (25, 50, 75, and 100 ␮m) had 415.0 ± 285.2, 457.5 ± 341.9, 585.0 ± 309.3, and 685.0 ± 278.8 isolated preantral follicles, respectively. In Experiment II, the follicular density of 46 buffalo fetuses with ages between 3 and 8 months was estimated to be between 0 and 7220, with means of 0.0, 2070.7 ± 2190.3, 2570.8 ± 1796.6, 2298.1 ± 2286.5, 1277.5 ± 1074.9, and 643.6 ± 543.9 throughout the age range studied. The follicular density of 5-month-old fetuses was greatest, coinciding with the largest number of follicles isolated at this age. In Experiment III, preantral follicles isolated from the ovaries of buffalo fetuses aged from 5 to 9 months old were cultivated individually for 7 days in four different media: basic medium (Minimal Essential Medium (MEM), 10% SFB, kanamycin, pyruvate, glutamine, hypoxanthine) with additional ITS and FSH 0.5 mg/ml (treatment 1); basic medium with FSH and EGF 100 ng/ml (treatment 2); basic medium with additional ITS, FSH, and EGF (treatment 3); basic medium supplemented with ITS and EGF (treatment 4). Integrity and morphological features, viability, and increase in diameter of follicles cultured in vitro were evaluated individually with an inverted microscope and an ocular micrometer. The results showed that follicle structure and form were maintained during culture. Growth and survival rates of treatments 1, 2, and 3 over 7 day culture were 23.25 ± 17.06, 33.75 ± 26.19, and 43.75 ± 31.73 ␮m, and 31.3 ± 22.7, 22.06 ± 8.13, and 28.92 ± 21.32%, respectively. However, neither ∗

Corresponding author. E-mail address: [email protected] (S.S.D. Santos).

0378-4320/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.anireprosci.2005.08.012

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growth nor survival was observed in treatment 4. In conclusion, this study showed that preantral follicles of buffalo fetuses can be cultured in vitro, and that FSH is essential for follicle survival. © 2005 Published by Elsevier B.V. Keywords: Buffalo-preantral follicles; In vitro follicle development; Fetus

1. Introduction Females of all domesticated species, primates included, have a finite stock of germinal cells established during fetal life that is used in subsequent folliculogenesis. The formation of this stock usually occurs during fetal life in a series of stages such as migration of primordial germinative cells to the gonadal ridge, proliferation and initiation of prophase I, blocking at diplotene stage, and finally, the formation of primordial follicles. Although there are thousands of primordial follicles in mammalian ovaries, almost all (about 99.99%) are eliminated in vivo by follicular atresia. Enzymatic isolation methods of preantral follicles have been used in rats (Daniel et al., 1989), mice (Eppig and Schroeder, 1989; Torrance et al., 1989; Nayudu and Osborn, 1992), rabbits (Maresh et al., 1990), fetus and adult cattle (Figueiredo et al., 1993), humans (Roy and Treacy, 1993), pigs (Lazzari et al., 1992), cattle fetus (Car´ambula et al., 1999), and adult buffalo (Gupta et al., 2001). Despite being efficient, enzymes can have cytotoxic effects and alter cell structure (Nicosia et al., 1975), depending on enzyme treatment time and concentration, and the kind of tissue involved. The mechanical method of isolating preantral follicles has been used with several species such as cattle (Figueiredo et al., 1993; Hulshof et al., 1992), goats (Lucci et al., 1999), sheep (Amorim et al., 2000), and buffalo (Gupta et al., 2001). It has a number of advantages in comparison with the enzymatic method, being relatively simple, inexpensive, fast, and less harmful to follicles (Figueiredo et al., 1993). The development of techniques for the culture of ovarian follicles of primordial fetuses and prepubertal animals has been considered in many studies (Telfer, 1998), and a number of applications have been proposed, including animal production, transgenesis, conservation of rare species, and formation of genetic material banks. Preantral follicle culture has been carried out by many researchers (Eppig, 1977; Daniel et al., 1989; Eppig and Schroeder, 1989; Qvist et al., 1990; Nayudu and Osborn, 1992) to obtain oocyte growth, maturation, and in vitro fertilization. Among the domesticated animals, this procedure has been conducted with adult cattle (Figueiredo et al., 1994; Hulshof et al., 1995; Wandji et al., 1996; Ralph et al., 1996; Katska and Rynska, 1998; Saha et al., 2000; Itoh and Hoshi, 2000; Gutierrez et al., 2000), cattle fetuses (Wandji et al., 1996), pigs (Telfer, 1996), domestic and wild cats (Jewgenow, 1996; Jewgenow and Stole, 1996), Rhesus monkeys (Younis et al., 1993), goats (Huanmin and Yong, 2000), and buffalo (Gupta et al., 2002). However, the culture of buffalo fetal preantral follicles has not been reported in the literature. Danell (1987) found a relatively small number of primordial follicles in slaughtered buffalo (10,000–19,000), in comparison with those of cattle (about 150,000), as well as a smaller number of antral follicles and a greater incidence of atresia (82–92%) in ovaries (Kumar et al., 1997; Palta and Chauhan, 1998; Gupta et al., 2001). This indicates that more studies are necessary for perfecting follicle isolation and culture techniques for the production and transfer of a larger number of embryos in commercial scale, and their use in nuclear transfer, transgenics research, and in folliculogenesis studies of this species.

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Although the buffalo is very important in tropical countries, in situ and in vitro studies of preantral ovarian follicles of this species are scarce. The aim of the present study was the isolation of preantral follicles, the estimation of follicular density of buffalo fetuses of different ages, the development of a technique for the culture of preantral follicles for folliculogenesis studies, as well as in vitro culture, maturation, and fertilization of buffalo oocytes. 2. Materials and methods 2.1. Ovary collection Ovaries of buffalo fetuses of different ages were collected according to Abdel-Raouf and El-Naggar (1968), 20–30 min after slaughter. After collection, the ovaries were washed in 70% ethanol and a 0.9% sodium chloride solution containing 200 UI/ml penicillin and 200 ␮g/ml streptomycin. The ovaries were then stored in sterile vials containing phosphate buffered saline with 200 UI/ml penicillin and 200 ␮g/ml streptomycin, and transported to laboratory in isothermal containers. 2.2. Experiment I—best ovarian section thickness for preantral follicle isolation To evaluate whether the number of preantral follicles isolated is influenced by ovarian tissue section thickness, six ovaries of 9-month-old buffalo fetuses were collected. The ovaries were cut into four equal parts and stored in Minimal Essential Medium (MEM) with Hepes. Each part was placed in a tissue chopper for a series of longitudinal, transversal, and diagonal sections of different thicknesses (25, 50, 75, and 100 ␮m). This material was placed in Teflon vials containing 3 ml of MEM with Hepes, 10% fetal bovine serum, 0.23 mM pyruvate and antibiotics (200 UI/ml penicillin, 200 ␮g/ml streptomycin). It was then homogenized 40 times with a 1000 ␮l automatic pipette for follicular dissociation. The resulting suspension was filtered through 100 ␮m pore nylon mesh, and four samples of 100 ␮l were obtained for counting isolated preantral follicles. The total number of isolated follicles was estimated by multiplying the average number of the four samples by the final volume of the solution (3 ml). Isolated follicles were classified as either primordial, or primary, or secondary, according to Hulshof et al. (1992). 2.3. Experiment II—isolation of preantral follicles and determination of follicular density Both (to clarify the use of pair ahead) ovaries of 46 buffalo fetuses with ages varying from 3 to 8 months were collected and weighed, and one of each pair was selected for follicular isolation as in Experiment I using a 75 ␮m section. The remaining ovaries were fixed and processed for routine histology with serial section thickness of 7 ␮m and H.E. staining. Quantitative analysis was conducted by estimating the mean cortex area and counting the follicles of the 5th, 15th, and 25th histological sections of each ovary following the method used by Yamada (1995) for primate visual system cells. This procedure provides an estimate of follicular density (primordial follicles/mm2 of cortex). Only follicles having visible oocyte nuclei were counted. Follicular diameter was measured using an ocular micrometer with an inverted photomicroscope. Only rounded follicles presenting oocyte nuclei were measured. Measurements of the total area of the ovarian sections and of the cortex and medulla were conducted using a digital planimeter together with AutoCAD software.

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2.4. Follicle culture 2.4.1. Ovary collection Twenty-six ovaries of buffalo fetuses aged from 5 to 9 months were collected according to Abdel-Raouf and El-Naggar (1968) and used for isolation and culture. 2.4.1.1. Follicle isolation. After three washes in sterile medium, the ovaries were placed in 2 ml tubes containing 500 ␮l of MEM with Hepes, 10% SFB, 0.23 mM pyruvate, 200 UI/ml penicillin, and 200 ␮g/ml streptomycin (wash medium), and were completely fragmented with a pair of 12 cm straight-bladed (f/f) scissors. Fragments were homogenized 40 times in 3 ml of medium in a polystyrene tube using a 1000 ␮l automatic pipette to separate follicles. This homogenate was filtered through a 100 ␮m nylon mesh. Under a stereomicroscope, spherical follicles with a single layer of rounded cells and without apparent signs of degeneration were selected for culture, washed three times in wash medium and once in MEM with 10% SFB, 10 ␮g/ml kanamycin, 0.23 mM/ml pyruvate, 2 mM/ml glutamine, and 2 mM/ml hypoxanthine (culture medium). A sample was stained with 0.4% trypan blue to evaluate the percentage of viable follicles after isolation. Preantral follicles (n = 433) were individually cultured on collagen gel matrix (20 ␮l of agar 2% dissolved in culture medium) in 96-well plates with 60 ␮l of culture medium and additional compounds, according to four different experimental treatments, as follows. 2.4.2. Treatments Treatment 1 included 6.25 ␮g/ml insulin, 6.25 ␮g/ml transferrin, 6.25 ng/ml selenium (ITS), and 0.5 ␮g/ml FSH (Folltropin-V—Vetrepharm). Treatment 2 included 0.5 ␮g/ml FSH and 100 ng/ml EGF (Human Recombinant). Treatment 3 included ITS, 0.5 ␮g/ml FSH, and 100 ng/ml EGF. Treatment 4 included ITS and 100 ng/ml EGF. 2.4.3. Ovarian follicular culture conditions Follicle culture was conducted at 37 ◦ C under 5% CO2 and high humidity for 7 days. Each treatment was repeated five times. All products were from Sigma and Gibco. 2.5. Morphological analysis of follicles Integrity and morphology analyses were conducted according to Hulshof et al. (1995). Follicles with a clear oocyte surrounded by a clear granulosa layer were considered morphologically normal. In vitro increase in follicle diameter was estimated using an inverted photomicroscope equipped with a micrometric ocular. Follicles were measured on days 1 and 7 of culture. Follicle development and survival were evaluated through microscopic observation of morphological features. In vitro growth rates were estimated according to the difference between initial and final follicle diameters. 2.6. Statistical analysis Data were analyzed statistically with Student’s t for pairwise comparisons, ANOVA, and linear correlation and regression, using BioEstat 2.0 software (Ayres et al., 2000).

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Fig. 1. Linear regression between ovarian weight and fetal age in the buffalo.

3. Results 3.1. Experiment I—section thickness for preantral follicle isolation The average number (mean ± S.D.) of preantral follicles isolated using section thicknesses of 25, 50, 75, and 100 ␮m were 415.0 ± 285.2, 457.5 ± 341.9, 585.0 ± 309.3, and 685.0 ± 278.8, respectively. Differences between the results for different thickness sections were not statistically significant (p > 0.05). 3.2. Experiment II—isolation of preantral follicles and determination of follicular density Ovarian weight strongly correlates (r = 0.9678) with fetal age, although the increase is gradual (y = 13.163x − 29.412) (Fig. 1). No significant correlation was found between the number of isolated follicles and the mean areas of ovary, cortex, and medulla sections. The mean cortex area did not show significant differences (p > 0.05) between the fetal ages studied; however, at 6 months, it presented an exceptional variation, which was probably due to the presence of antral follicles (Table 1). The medulla area grew gradually and the total ovary area increased (p < 0.05) at 6 and 7 months (Table 1) and increased again at 8 months. No preantral follicles were isolated from 3-month-old fetal ovaries (Table 2). At this age, histological sections contained a number of oogonia, indicating that folliculogenesis had not yet been initiated. The number of follicles isolated varied from 0 to 7220, 150 to 5430, 390 to 7140, 320 to 3670, and 45 to 1630 at months 4, 5, 6, 7, and 8, respectively. The average number of Table 1 Weight of ovary, and mean area of buffalo fetus cortex, medulla, and ovarian sections (mean ± S.D.) at different ages Fetal age (months) (ovaries)

Ovary weight (mean ± S.D.) (g)

3 (n = 5) 4 (n = 4) 5 (n = 5) 6 (n = 6) 7 (n = 4) 8 (n = 3)

0.0157 ± 0.005a

0.0231 ± 0.005a 0.0240 ± 0.005a 0.0532 ± 0.036b 0.0651 ± 0.034bc 0.0768 ± 0.017c

Mean ± S.D. of area in mm2 (% of total) Cortex 5.57 5.50 5.33 10.8 5.34 4.40

± ± ± ± ± ±

Medulla 1.44a 0.92a 1.85a 9.28a 0.64a 1.59a

(74%) (55%) (51%) (51%) (28%) (20%)

2.12 4.37 5.57 8.89 14.73 21.80

± ± ± ± ± ±

Ovary 0.88a

(26%) 0.75a (45%) 2.8ab (49%) 6.69b (49%) 4.14c (72%) 6.5d (80%)

8.14 9.88 10.90 19.66 20.07 26.21

± ± ± ± ± ±

1.96a (100%) 1.37a (100%) 3.56a (100%) 9.09b (100%) 4.58b (100%) 5.27c (100%)

Different lower case letters in the same column (a–d) represent a significant difference (p < 0.05) between values.

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Fetal age (months)

Isolated follicles (mean ± S.D.) Follicle density (variation) Antral follicles (mean ± S.D.)

3

4

5

6

7

8

0 0 0

2070.7 ± 2190.2a

2570.7 ± 1796.6a

2298.1 ± 2286.4a

1277.5 ± 1074.8b

16.6 (0–276) 0

35.8 (37–510) 0

6.1 (17–157) 1.16 ± 0.83

16.6 (44–155) 0.66 ± 0.88

643.5 ± 543.8b 18.8 (6–167) 0

Different lower case letters in the same row (a and b) represent a significant difference (p < 0.05) between values.

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Table 2 Number (mean ± S.D.) of isolated preantral follicles, follicular density, and antral follicles of buffalo fetuses of different ages

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Table 3 Percentage distribution (mean ± S.D.) of primordial, primary, and secondary follicles isolated from buffalo fetuses of different ages Type of follicle

Fetal age (months) 4

Primordial (mean ± S.D.) Primary (mean ± S.D.) Secondary (mean ± S.D.)

5

17.57 ±

15.78a

6

16.26 ±

13.45a

7 9.57 ±

6.27ab

8 2.16 ±

4.17bc

1.6 ± 3.6c

64.25 ± 29.34a

75.65 ± 18.03a

55.76 ± 22.79ab

79.05 ± 6.27b

34.08 ± 28.34c

5.69 ± 7.59a

8.12 ± 6.51a

34.63 ± 24.34ab

18.77 ± 6.47b

64.31 ± 31.52c

Different lower case letters in the same row (a–c) represent a significant difference (p < 0.05) between values.

preantral follicles isolated from fetuses of 4–6 months of age were greater in comparison with those of 7 and 8 months of age (Table 2). The greatest follicular density was at 5 months, and antral follicles were observed at 6 and 7 months of age (Table 2). Data for the percentage distribution of primordial, primary, and secondary follicles isolated from buffalo fetuses of different ages are included in Table 3. With increasing fetal age, particularly from the 7th month on, there is a marked decrease in the proportion of primordial and primary follicles, and a concomitant increase in secondary follicles. The mean number of follicles isolated at different stages of development, and at different fetal ages is generally characterized by considerable individual variation, as evidenced by the relatively large standard deviations. Morphology of preantral follicles isolated from buffalo fetuses is included in Fig. 2. 3.3. Follicle culture 3.3.1. Post-isolation viability From the total of 1497 follicles stained by trypan blue, 60.34 ± 9.16% were considered viable, 27.32 ± 11.91% were dead, and 11.97 ± 5.77% had dead oocytes (Fig. 3). 3.3.2. Preantral culture Greater that 100 follicles were observed in each treatment group, with a total of 433 (Table 4). The mean initial follicle diameter differed (p < 0.05) between treatments 1 and 3. This variation was due to the different follicle sizes, even though all follicles showed a single cell layer (primary Table 4 Initial and final diameter, growth, and survival rates (mean ± S.D.) of buffalo fetal preantral follicles after 7 days of culture Treatment

Initial diameter (␮m) (mean ± S.D.) Final diameter (␮m) (mean ± S.D.) Growth (␮m) (mean ± S.D.) Survival (%) (mean ± S.D.)

FSH + ITS (n = 107)

FSH + EGF (n = 112)

FSH + EGF + ITS (n = 106)

54.79 ± 9.75aA 78.04 ± 18.2aB 23.25 ± 17.04a 31.73 ± 22.77a

63.78 ± 22.16abA 97.53 ± 30.4bB 33.75 ± 26.19ab 22.06 ± 8.13a

70.76 114.55 43.75 28.92

± ± ± ±

20.81bA 31.85cB 31.73b 21.32a

EGF + ITS (n = 108) 63.09 ± 22.01ab – – 0.0

Different lower case letters in the same row (a–c) indicate a significant difference (p < 0.05). Different capital letters in the same column (A and B) indicate significant difference (p < 0.05).

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Fig. 2. Preantral follicles isolated from buffalo fetuses: (A) primordial and primary follicles (200×) and (B) secondary follicles (100×).

follicles). Mean diameter increased (p < 0.05) in all treatment groups except 4, where follicles did not remain viable. Mean diameters also differed among treatments 1–3 (p < 0.05), with the largest diameters being with treatment 3, and the smallest with treatment 1. Mean growth was greater with treatment 3 (p < 0.05) in comparison with treatment 1, but treatment 2 was similar to both 1 and 3. Survival rates were similar for all treatments except treatment 4 and with this treatment follicle viability was not sustained. With treatment 4, follicles became darkened, and non-refringent. Figs. 4–6 show living follicles before culture, normal cultured follicles, and degenerated follicles after culture, respectively. 4. Discussion The present study has shown that the tissue chopper used by Figueiredo et al. (1993) for the isolation of preantral follicles of cattle can be successfully used for the same purpose with buffalo

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Fig. 3. Preantral follicles from buffalo fetuses stained with trypan blue: (A) live; (B) stained oocyte and granulosa cells; (C) degenerated oocyte (100×).

tissue, and that section thickness does not influence number of follicles obtained. Bem et al. (1997) obtained similar results with tissue from zebu cattle. Although the absolute area of fetal ovarian cortex did not vary significantly between 3 and 8 months, its proportion of the total area decreased progressively with fetal development from 74% at month 3 to 20% at month 8 as the medulla increased in size. This pattern may be due to either the observed reduction in follicular population or increase in medullar area concomitant with the growth of the ovary as a whole, according to Kumar et al. (1997). It was previously reported that 38.1% of the ovarian area in prepubertal buffalo (up to 3 years old) is occupied by follicles (Kumar et al., 1997). Between 3 and 12 years of age, this proportion is 45%, and at the age of 12 years, it decreases to 28.7%. The larger cortex observed in some animals at 6 months of fetal age is almost certainly due to the presence of antral follicles.

Fig. 4. Buffalo preantral follicles before culture (100×).

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Fig. 5. Buffalo preantral follicles after culture (100×).

The present study, together with that of Kumar et al. (1997), has shown considerable variation in the ovarian area occupied by follicles. This appears to be caused primarily by waves of follicular growth and degeneration, indicating that folliculogenesis is a dynamic process, with cell growth and death occurring continuously. There are no published studies on the isolation and culture of buffalo fetal follicles with which to compare the results of the present study. Preantral follicles could not be isolated from the ovaries of 3-month-old fetuses, which indicates that folliculogenesis had not yet been initiated. This could be confirmed by classical histology, which revealed the exclusive presence of oogonia at this age. In addition, buffalo ovarian folliculogenesis is usually later than that of cattle, which starts at 2.5

Fig. 6. Degenereted buffalo preantral follicles after culture (100×).

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months of fetal age (Tanaka et al., 2001). In contrast with the present study, Car´ambula (1997) showed that primordial, primary, and secondary follicles can be isolated from 3-month-old cattle fetuses. The mean numbers of preantral follicles isolated in the present study are similar to those found by Gupta et al. (2001) for adult buffalo and less than those reported by Figueiredo et al. (1993) and Car´ambula et al. (1999) from fetuses of cattle. The data obtained in this and other studies varied due to differences in isolation procedures. Buffalo ovaries have fewer primordial follicles, varying from 10,000 (Danell, 1987) to 19,000 (Samad and Nasseri, 1979), in contrast with 150,000 for ovaries of cattle (Erickson, 1966). Ovaries from slaughtered buffalo also had fewer antral follicles and a greater incidence of follicular atresia (82 and 92%) (Kumar et al., 1997; Palta and Chauhan, 1998). Buffalo fetuses had primordial follicles at month 4, and antral follicles at month 6. These results are not consistent with those of previous studies with buffalo (El-Ghannam and El-Naggar, 1974) and cattle (Tanaka et al., 2001). These authors reported the formation of antral and primordial follicles at months 5 and 9 in buffalo and at 2.5 and 5 months in cattle, respectively. According to Tanaka et al. (2001), the increase in antral follicles in fetuses of cattle is associated with changes in FSH concentration, which also appears to occur in buffalo. In conclusion, this study was the first to show that preantral follicles can be successfully isolated from buffalo fetal ovaries using a mechanical procedure. Furthermore, the number of follicles isolated is influenced by fetal age, but not by section thickness. This study was also the first to show that buffalo fetal preantral follicles can be cultured in vitro, and that this process is affected by culture medium composition. Trypan blue staining of follicles was used for the ease of assessment of viability during isolation and culture. Only 60.39% of the preantral follicles appeared to be morphologically normal after isolation. This result may be due to either the relatively greater frequency of atresia, which is typical for buffalo (Kumar et al., 1997; Palta and Chauhan, 1998), or excessive manipulation during isolation, which Figueiredo et al. (1994) showed to have a negative effect on viability. All culture media tested in the present study contained pyruvate, glutamine, and hypoxanthine. Jewgenow (1998) found that these compounds increase the percentage of morphologically normal follicles in culture. In this case, follicle growth was supported by the use of agar, which is similar to ovarian tissue. This prevents the formation of the granulosa cellular monolayer, which can cause follicle deformity, according to Figueiredo et al. (1994). However, this did not suffice in consistently successful results as indicated by previous studies of a number of different species (Eppig and Schroeder, 1989; Nayudu and Osborn, 1992; Figueiredo et al., 1994; Wandji et al., 1996; Jewgenow, 1996; Katska and Rynska, 1998; Saha et al., 2000; Gutierrez et al., 2000; Huanmin and Yong, 2000). In the present study, all treatments promoted follicle growth and survival, except the one without FSH. These results are consistent with those of Ralph et al. (1996) and Wandji et al. (1996), who reported that FSH is the most effective factor in maintenance of follicle viability. The treatment of preantral follicles with FSH stimulates the proliferation of granulosa cells, formation of antrum, steroidogenesis, and lactate production (Nayudu and Osborn, 1992; Ralph et al., 1995). However, Nuttinck et al. (1996) observed that treatment with FSHp induced the degeneration of preantral follicles of cattle, and that this degeneration may be attenuated by treatment with FGF-2, which increases follicular DNA synthesis, in contrast with FSH. These contradicting results may be due to differences in isolation and culture methods used. Growth rates for media with ITS and FSH (treatment 1) and with FSH, EGF, and ITS (treatment 3) are similar to those observed by Gupta et al. (2002) for adult buffalo follicles. However, survival

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rates were considerably less. These differences may have been brought about by either the different follicle diameters used or by other factors such as genotype and breed. Growth rates in media with ITS and FSH (treatment 1) and with FSH, EGF, and ITS (treatment 3) were similar to results obtained by Gupta et al. (2002) for adult buffalo; however, the survival rate was much less. These differences may result from either the different follicle diameters used or others factors such as genotype, species, and nutritional condition. In the absence of EGF (treatment 1), the growth rate is less than that for treatment 3, which has the most efficient medium in terms of follicle growth. The absence of EGF may have reduced growth rate by decreasing cellular proliferation. This agrees with the study by Hsue et al. (1994), who showed that EGF modulates preantral follicle cell growth and differentiation and has an antiapoptotic effect on in vitro granulosa cells. However, the survival rate for treatment 1 is similar to those for treatments 2 and 3, and was not affected by the absence of EGF. This agrees with Jewgenow (1996), where it was observed that growth factors modulate follicle cell differentiation, proliferation and survival, and gonadotrophin interaction, despite the important role peptidegrowing factors, i.e. EGF, have in folliculogenesis, being involved in cellular proliferation and differentiation, and in steroidogenesis. In treatment 2, the absence of ITS did not affect either follicle growth or survival, which were similar to those for treatments 1 and 3. This may be due to follicle diameter used. Saha et al. (2000) reported that follicles with a diameter of 40–80 ␮m have fewer active receptor sites for ITS, and that either FSH or FSH and EGF combined increase during follicle development. The addition of EGF and ITS to the culture medium prevents structural alterations, reduces the proportion of atresia, and increases the proliferation rate of granulosa cells (Saha et al., 2000). Final follicle diameter and growth were greater when treatment 3 was used in comparison with treatment 1. This probably occurs due to the interaction of FSH, EGF, and ITS, which can be also influenced by the greater initial follicle diameter with treatment 3. However, the presence of these substances or the initial follicle diameter did not alter survival rate, which was similar to those observed for treatments 1 and 2. The results obtained show that FSH and EGF favor in vitro growth and the survival of buffalo fetus preantral follicles, in agreement with the studies of Hulshof et al. (1995), Ralph et al. (1996), Wandji et al. (1996), Telfer (1998), Katska and Rynska (1998), Gutierrez et al. (2000), Saha et al. (2000), and Gupta et al. (2002). Mean growth rates at day 7 of culture (23.25, 33.75, and 43.75 ␮m) were similar to the results obtained by Nuttinck et al. (1993) and Saha et al. (2000) for cattle, and Gupta et al. (2002) for buffalo. However, Katska and Rynska (1998) and Huanmin and Yong (2000) reported greater growth rates for cattle and goats, respectively. Hulshof et al. (1995), however, reported lesser growth rates for bovines cattle. The differences may have been due to either nutritional or genetic factors, differences in isolation and culture methods, or even differences in follicle diameter at the beginning of culture. The follicle survival rates (31.73, 22.06, and 28.92%) recorded in the present study are less than those obtained by Wandji et al. (1996) for cattle fetuses, by Nuttinck et al. (1993) for adult cattle, and by Huanmin and Yong (2000) for goats, but are similar to results by Gupta et al. (2002) for buffalo. The differences may have been due to the greater rate of atresia in buffalo ovaries (Le Van Ty et al., 1989; Kumar et al., 1997; Manik et al., 1998; Palta and Chauhan, 1998). This is probably due to the greater nutritional needs of buffalo follicles, as is the case for their embryos in comparison to that of cattle (Takahashi et al., 1992). Other possible factors include species differences, variation in follicle diameter, media and supplements used, and nutritional and physiological

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conditions of the animals. Figueiredo et al. (1994) found that the latter can influence follicle viability. From the results obtained in the present study, we conclude that buffalo fetus preantral follicles can be cultured in vitro. However, these follicles have lesser growth and survival rates in vitro. In addition, FSH was identified as a major factor for follicle survival in vitro. Acknowledgement The present study was supported by Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq). References Abdel-Raouf, M., El-Naggar, M.A., 1968. Biometry of the Egyptian buffalo foetus. U.A.R.J. Vet. Sci. 5, 37–43. Amorim, C.A., Rodrigues, A.P.R., Lucci, C.M., Figueiredo, J.R., Gonc¸alves, P.B.D., 2000. Effect of sectioning on the number of isolated ovine preantral follicles. Small Ruminant Res. 37, 269–277. Ayres, M., Ayres Jr., M., Ayres, D.L., Santos, A.S., 2000. BioEstat 2.0—Aplicac¸o˜ es Estat´ısticas nas a´ reas das Ciˆencias Biol´ogicas e M´edicas. Sociedade Civil Mamirau´a, Bras´ılia, CNPq, 272 pp. Bem, A.R., Amorim, C.A., Rodrigues, A.P.R., Lucci, C.A., Figueiredo, J.R., 1997. Efeito do intervalo de corte do tecido ovariano no tissue chopper sobre o n´umero de fol´ıculos pr´e-antrais isolados a partir de ov´arios de vacas nelore. Arq. Fac. Vet. UFRGS 25 (1), 178. Car´ambula, S.F., 1997. Resgate de fol´ıculos pr´e-antrais de ov´arios de fetos bovinos e ovinos. Dissertac¸a˜ o de Mestrado. UFSM, Santa Maria, RS. Car´ambula, S.F., Gonc¸alves, P.B.D., Costa, L.F.S., Figueiredo, J.R., Wheeler, M.B., Neves, J.P., Mondadori, R.G., 1999. Effects of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries. Theriogenology 52, 563–571. Danell, B., 1987. Oestrus Behaviour, Ovarian Morphology and Cyclical Variation in the Follicular System and Endocrine Pattern in Water Buffalo Heifers. Tese de doutorado, Universidade de Uppsala, Sweden. Daniel, A.J., Armostrong, D.T., Gore-Langton, R.E., 1989. Growth and development of rat oocyte in vitro. Gamete Res. 24, 109–121. El-Ghannam, F., El-Naggar, M.A., 1974. The prenatal development of the buffalo ovary. J. Reprod. Fertil. 41, 479–483. Eppig, J.J., Schroeder, A.C., 1989. Capacity of mouse oocyte from preantral follicles to undergo embryogenesis and development to live young after growth, maturation and fertilization in vitro. Biol. Reprod. 41, 268–276. Eppig, J.J., 1977. Mouse oocyte development in vitro with various culture systems. Dev. Biol. 60, 371–378. Erickson, B.H., 1966. Development and radio-response of pre-natal bovine ovary. J. Reprod. Fertil. 10, 97–105. Figueiredo, J.R., Hulshof, S.C.J., Van Den Hurk, R., Nusgens, B., Bevers, M.M., Ectors, F.J., Beckers, J.F., 1994. Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro. Theriogenology 41, 1333–1346. Figueiredo, J.R., Hulshof, S.C.J., Van Den Hurk, R., Ectors, F.J., Fontes, R.S., Nusgens, B., Bevers, M.M., Beckers, J.F., 1993. Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal calf and adult bovine ovaries. Theriogenology 40, 789–799. Gupta, P.S.P., Nandi, S., Ravindranatha, B.M., Sarma, P.V., 2001. Isolation of preantral follicles from buffalo ovaries. Vet. Rec. 148 (17), 543–544. Gupta, P.S.P., Nandi, S., Ravindranatha, B.M., Sarma, P.V., 2002. In vitro culture of buffalo (Bubalus bubalis) preantral follicles. Theriogenology 57, 1839–1854. Gutierrez, C.G., Ralph, J.H., Telfer, E.E., Wilmut, I., Webb, R., 2000. Growth and antrum formation of bovine preantral follicles in long-term culture in vitro. Biol. Reprod. 62 (5), 1322–1328. Hsue, A.J.W., Billig, H., Tsafriri, A., 1994. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr. Rev. 15 (6), 707–723. Huanmin, Z., Yong, Z., 2000. In vitro development of caprine ovarian preantral follicles. Theriogenology 54, 641–650. Hulshof, S.C.J., Bevers, M.M., Van der Donk, H.A., Van den Hurk, R., 1992. Isolation and characterization of preantral follicles from foetal bovine ovaries. In: 12th International Congress on Animal Reproduction, vol. 3, pp. 336–338.

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