Introduction

In vitro technique allows us to obtain a large number of mammalian embryos for research, genetic improvement or commercial purposes. The successful culture and embryonic development of in vitro matured oocytes has been reported in bovine, sheep and other species (Eppig et al., 1996; Accardo, et al., 2004). However the ability to develop mammalian oocytes matured and cultured in vitro is significantly lower than those produced in vivo (Dielman et al., 2002; Rizos et al., 2002).

The components of the culture media and the conditions of culture can affect the meiotic regulation of mammalian oocytes (Downs and Mastropolo, 1997). Different media have been introduced and experienced in in vitro maturation of oocytes. The most commonly used system is a culture medium supplemented with hormones, serum or albumin (Bavister et al., 1992; Thompson, 2000). The formulation of a more clearly defined medium might help to further elucidate the metabolic and nutritional requirements of mammalian oocytes and embryos produced in vitro.

In these studies, the basic medium was supplemented with follicular fluid (FF) and different concentrations of serum.

However, serum is not a naturally occurring biological product but a pathological fluid formed by blood clotting, a process that may lead to chemical changes which have detrimental effects on embryo culture [Maurer HR, 1992]. As a consequence, a replacement obtained from plasma might produce better results than serum in terms of embryonic development.

Animal′s serum at estrus stage contains different hormones and proteins that are essential for oocyte maturation. Serum is extremely complex fluid containing variety of energy substrates, amino acids, vitamins and growth factors that may support survival and growth of mammalian cell in culture (Han and Niwa, 2003).  Also, Serum added to the oocyte culture medium provides a source of albumin that balances the osmolarity and acts as a free radical scavenger (Thompson, 2000).

Follicular fluid is primarily the transudation of plasma that contains specific constituents such as steroids, glyocosaminoglycans and many other metabolites synthesized by the cells of the follicle wall (Choi et al., 2001). Assuming that preovulatory follicular fluid could play an active role in the final oocyte maturation, it would be logical to consider it to be the medium of choice for in vitro maturation as well. In fact addition of diluted follicular fluid to maturation media has been used as an alternative to serum for in vitro production of embryos in the equine (Dell´ Aquila, Me, et. al, 1997), bovine, porcine and canine (Rodrigues and Rodrigues , 2003), while the developmental potential of oocytes exposed to pure follicular fluid during IVM has been described in the bovine and equine species (Aguilar et al., 2001; Gerritse et al., 1988).

Maturation of oocytes includes two aspects: nuclear and cytoplasm maturation (Sun and Nagai, 2003). In 1980s and early 1990s, the heat treatment of bovine serum (Fetal Bovine Serum) was routinely practiced in IVM medium (Gordon, 1997). It was essential to supplement the holding and culture media with 1 – 5% serum to prevent the zona pellucida hardening (Downs et al., 1986).

The purpose of this study was to determine the efficiency of different rat estrus serum concentrations and estrous sheep serum concentrations on in vitro maturation of bovine oocytes.

Materials and Methods

Collection of ovaries

Bovine ovaries were collected from a local Fars slaughter house (Barmshur) and transported to the Physiology Department ,College of Veterinary Medicine, Shiraz University laboratory within 2 h in saline (9g NaCl/l) at 35– 38°C supplemented with 100 IU penicillin G and 100 µg /ml streptomycin sulfate. Oocytes were collected by aspiration of follicles using a 20 gauge needle fitted with a 2.5 ml syringe. Cumulus-oocyte complexes were recovered and morphologically classified as the following grades: (1) darkly pigmented and completely surrounded by one or more layers of cumulus cells or (2) lightly pigmented with incomplete layers of cumulus cells (Hewitt et al., 1998). From a total of 3,488 COCs, 1820 were morphologically classified as Grade I and were selected for in vitro maturation. The COC’s were washed 4 times in a modified TCM-199 (Sigma, M2154) solution and supplemented with 50 μg/ml gentamycin (Sigma), and BSA 1 mg/ml (Gibco), and 14.3 mM Hepes (Sigma,) without any serum supplementation.

Follicular fluid collection and preparation

Mixed bovine follicular fluid was retrieved from slaughterhouse ovaries. Follicles with diameters between 3-15mm were aspirated by an 18 gauge needle attached to a 10 ml syringe. The pooled follicular fluid was then centrifuged at 4000rpm for 10 min to discard insoluble materials followed by sterilization with filter (0/22 µm, Millipore, Brussels, Belgium), The supernatant stored at -20°C.

Rat estrus serum collection and preparation

For the preparation RSS, After diagnosis estrous stages with the help of vaginal smear in rats, blood sampling of the heart and afterward heat-inactivated serum (56°C, 30min) pooled, filtered with a 0.22 µm membrane (Millipore, Brussels, Belgium) and frozen in 1.5mL vials until used.

Culture of oocytes

The COC’s were randomly divided into three groups. controlled group (n = 260) COC’s were taken fresh controlled and cultured in TCM-199 medium, HCG and follicular fluid (FF) without serum supplementation. Group 2 (n = 779) COC’s were washed five times and cultured in control medium supplemented with different concentrations (10, 15 and 20%) of estrous sheep serum. Group 3 (n = 781) COC’s were washed five times and cultured in control medium supplemented with different concentrations (10, 15 and 20%) of rat estrus serum. The COCs were divided randomly and cultured in 100 μl drops (four drops per dish) under mineral oil (Sigma) at 37oC in a humidified atmosphere with 5 % CO2 for 38-42 hours. Oocytes were denuded after 40 h following culture by treating with TCM-199 containing 0.1% hyaluronidase (Sigma) and passing them through a fine pipette. Oocytes were fixed for 24 – 48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for evidence of different stages of maturation under a phase contrast microscope. The different stages of maturation examined based on chromosomal configuration were assigned to germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase-I (M-I) and metaphase-II (M-II) categories. A chromosome configuration was designated as GV, when having a single large nucleus with uniformly distributed filamentous chromatin subsequently condensing to form a ring of condensed chromatin around the compact nucleus. In the GVBD category, the nucleolus and nuclear membrane disappeared and chromosomes appeared as condensed and coiled up filaments. The metaphase-I stage was recognized by the appearance of paired chromosomes, while in the metaphase-II emission of first polar body, resulting in the formation of haploid set of chromosomes in the oocytes was observed.

Statistical analysis

The percentages of various nuclear maturation stages of oocytes among the different treatments groups were analyzed by chi-square analysis. A confidence level of P < 0.05 was considered statistically significant.

Results

Nuclear morphology was evaluated on 1820 oocytes, that were fixed and stained after 40 hours of culture with their respective treatments. Sixty-eight oocytes were classified as degeneration or as possessing unidentifiable nuclear material. Oocytes degeneration was characterized by nuclear pycnosis, unidentifiable intracytoplasmic structures, or loss of the cytoplasmic membrane. As indicated in Table 1, there were no significant differences among treated groups at any stage of nuclear maturation (VG, VGBD, MI/AI, or MII).

The nuclear maturation rates at 10, 15 and 20% RSS increased supplementation, although the effect was not significant in comparison with the 10, 15 and 20% ESS supplementation.

A significant difference (p < 0.05) in the in vitro maturation rate was recorded between the serum supplemented and non supplemented media.

Table 1: Nuclear maturation status of oocytes after IVM for each treatment group

Significantly with control٭

GV, germinal vesicle; GVBD, germinal vesicle breakdown; M-I, metaphase-I; M-II,  metaphase-II.

The maturation rate of the bovine oocytes in medium without serum supplemented was 50% as shown by the control group. However, the serum supplemented in the different concentrations significantly (p < 0.05) increased the maturation in comparison with control group (Table 1).

Oocytes remained in the GV stage from the onset to 6 – 8 h of culture. The GVBD occurred between 7 and 9 h and the metaphase-I became established within 12 – 18 h. Finally most oocytes reach the metaphase-II stage after 27 h (at 38.5°C) Resumption of meiosis and subsequent sequential configurations are species specific.

Discussion

The maturation of immature bovine oocytes in vitro is probably the most important step affecting the success of IVF and the subsequent development rate (Chanson et al., 2001). It was shown that during IVM of COCs, the constituents of the media could have an extreme effect on the developmental capability of in vitro produced bovine embryos (Rose; TA and Bavister; BD. 1992). There have been many studies investigating the effects of protein supplementation of media used for IVP (Smitz et al., 2001). Sera (i.e. FCS and OCS) and BSA are the most common protein supplements for IVM media. The scientific reason for the beneficial effect of adding serum is not clearly understood but it is commonly accepted that one major biological role of serum is to compensate for whatever essential elements are missing from the medium by serving as a reservoir for many of the beneficial components, such as different energy substrates, steroids, amino acids, fatty acids, vitamins and growth factors. Serum also serves as a protective compound against scavenging ions and small molecules secreted from the developing embryo (28). The results of the present study have supported previous studies reporting that serum supplementation is beneficial for the IVM, IVF and subsequent development of bovine oocytes (Lorenzo et al., 1995; Roa et al., 2002; Ghasemzadeh-Nava and Tajik, 2000; Wani, 2002).

Experiments on in vitro maturation of caprain oocytes using TCM-199 supplemented with EGS, revealed a maturation rate of 50% (Pawshe et al., 1996) and 57.6% (Mogas et al., 1995). In a study by Songsasen et al. (2002), canine oocytes were successfully matured in vitro in a protein-free medium at comparable rates to those that used medium supplemented with protein. Despite suboptimal rates of maturation, Hewitt et al. (1998) previously showed that canine oocyte maturation was possible in serum-free medium. In other species uch as cat (Felis ; catus), in vitro maturation and subsequent fertilization were not affected by the absence of protein supplementation in the medium(Wood; et al., 1995). Mogas; et al. (1997) reported the addition of FSH, LH and estradiol-17 µl as well as EGS in TCM-199 medium to increase the maturation rate to 72.4 and 64.1% in adult and pre pubertal goats, respectively. Tajik and Shams Esfandabadi (2003) reported higher maturation rate of oocytes in culture medium containing EGS without hormones. Our results are in agreement with the finding of Ghasemzadeh-Nava and Tajik (2000) that compared the effect of FBS and ESS and concluded that ESS could support the in vitro maturation of ovine oocytes slightly better.

Otoi ;et al. (1999) observed that the addition of serum to maturation medium contributes to a high number of canine oocytes with unidentified nuclear material after staining. In the present study, the percentage of degenerated oocytes or those with unidentified nuclear material was not different in various treatments. This indicates that the presence or absence of serum does not influence the incidence of oocytes with those abnormalities.

Maturation of follicular oocytes is normally arrested at the prophase-I of the first meiotic division and the oocyte remain in the dormant stage that called a dictate nucleus. At this stage, nuclear material is enveloped and the resulting structure is called a germinal vesicle. The oocytes remain at this stage until the onset of puberty. Under the influence of gonadotropins and particularly in response to the LH surge, oocytes resume meiosis just before ovulation.

These results in the disappearance of the nuclear membrane and germinal vesicle breakdown, followed by chromosome condensation with the occurrence of the M-I stage. Subsequently, upon extrusion of the first polar body, the oocytes reach the M-II stage and remain at this stage until penetration by the spermatozoa. The configuration of meiotic chromosomes at the time of co-culture of oocytes with spermatozoa for in vitro fertilization has a direct influence on the final success of fertilization. The M-II stage which is also known as the second phase of meiotic arrest, is consider as having have completed nuclear maturation of the oocytes required for successful fertilization of oocytes. Obviously, nuclear maturation of oocytes along with cytoplasm maturation is important at the completion of meiotic division for success of fertilization. Therefore, the in vitro maturation process is supposed to be completed when the highest percentage of M-II oocytes observed.

To our knowledge, this report is the first paper about used RSS in maturation medium for bovine oocytes. The results indicated that no significant differences are between different concentrations of RSS serum compare with ESS serum.

Acknowledgment

This study was supported from Office of vice Chancellor for Research, Islamic Azad University, Sarvestan Branch. We are grateful for kind cooperation of Physiology Department, College of Veterinary Medicine, Shiraz University and we are thanks Dr. Ahmad Karbasi for their help.

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