Introduction
The in vitro culture systems used for embryo production still do not reach the level of effectiveness obtained in vivo. Whilst in vivo embryonic development rates achieve 70% blastocyst rate, only about 85% of in vitro matured oocytes are fertilized and only 30 to 40% of these develop to the blastocyst stage (Gutiérrez-Adan et al., Reference Gutiérrez-Adán, Lonergan, Rizos, Ward, Boland, Pintado and De La Fuente2001). For this reason, the development of strategies to improve efficiency of in vitro production (IVP) of bovine embryos is sought. Among several factors associated with culture media and methods that influence this procedure, great importance is given to macromolecule supplements and atmospheric conditions.
The macromolecule supplements that are frequently employed in IVP are bovine serum albumin (BSA) and fetal calf serum (FCS). These supplements are colloid particles that facilitate fluid transport through biological membranes (Webster, Reference Webster1982) and improve maturation and embryo development (Carolan et al., Reference Carolan, Lonergan, Van Langenonckt and Mermillod1995). BSA and FCS quelate heavy metal ions, buffer pH and act as surfactants and reactive oxygen species (ROS) scavengers (Orsi & Leese, Reference Orsi and Leese2004; Stein, Reference Stein2007). BSA also increases intracellular free amino acids after its hydrolysis (Orsi & Leese, Reference Orsi and Leese2004) and serum possesses more than 1000 different compounds, including growth factors and hormones (Stein, Reference Stein2007). Because of those characteristics, it is difficult to obtain a suitable substitute for BSA and FCS in culture. However, as BSA and FCS are products of animal origin: (i) their composition is not completely known; (ii) they present high variation among producers and batches (Mckiernan & Bavister, Reference McKiernan and Bavister1992); and (iii) they offer the risk of disease transmission (Krisher et al., Reference Krisher, Lane and Bavister1999). Then the culture medium supplemented with BSA and FCS is semi-defined or undefined in composition and contributes to variability of the culture systems and the obtained results (Bavister, Reference Bavister1995; Krisher et al., Reference Krisher, Lane and Bavister1999).
Thus, aiming to standardize IVP procedures and also to avoid pathogens transmission, synthetic and defined supplement sources are frequently used, including polyvinyl alcohol (PVA; Fukui et al., Reference Fukui, Kikuchi, Kondo and Mizushima2000), polyvinyl pyrrolidone (PVP; Chung et al., Reference Chung, Tosca, Huang, Xu, Niwa and Chian2007), Ficoll (Kuleshova et al., Reference Kuleshova, Macfarlane, Trounson and Shaw1999) and serum replacers, as synthetic serum substitute (Sagirkaya et al., Reference Sagirkaya, Misirlioglu, Kaya, First, Parrish and Memili2007) and KnockoutSR (Moore et al., Reference Moore, Rodriguez-Sallaberry, Kramer, Johnson, Wroclawska, Goicoa and Niasari-Naslaji2007). Among them, PVA and PVP are the synthetic macromolecules most used in culture medium to replace BSA and FCS. Ficoll is a polysaccharide usually employed in vitrification solutions (Checura & Seidel, Reference Checura and Seidel2007). KnockoutSR is a protein source used for stem-cell culture and whose defined formula is protected by fabricant, but does not have serum in its composition (Goldsborough et al., Reference Goldsborough, Tilkins, Price, Lobo-Alfonso, Morrison, Stevens, Meneses, Pedersen, Koller and Latour1998). However, the embryos produced in fully defined media usually present a developmental block (Camous et al., Reference Camous, Heyman, Méziou and Ménézo1984) and reduced viability (Wright & Bondioli, Reference Wright and Bondioli1981) when compared with those embryos cultured in undefined or semi-defined media.
Atmospheric conditions also affect IVP because the oxygen tension usually employed in in vitro culture (5% CO2 in air, which corresponds to 20% O2) is higher than that existent in ovarian follicles, oviduct and uterus. In follicles, the oxygen from capillaries diffuses through the layers of granulosa cells and a gradient of O2 occurs from the follicle periphery to the central oocyte (Gosden & Byatt-Smith, Reference Gosden and Byatt-Smith1986). Early embryo development after fertilization begins in oviduct, where the O2 tension is lower than in the atmosphere (Mass et al., Reference Mass, Storey and Mastroianni1976). Thus, the higher oxygen concentration used for in vitro culture may be a factor in the production of ROS that provoke intracellular damage and are detrimental for embryonic development (Batt et al., Reference Batt, Gardner and Camero1991). However, the published results using 20% or 5% O2 during the IVP steps are still controversial. Some research in bovine demonstrates that culture in lower oxygen tension (from 5 to 10%) improves maturation and embryo development (Nakao & Nakatsuji, Reference Nakao and Nakatsuji1990; Thompson et al., Reference Thompson, Simpson, Pugh, Donnelly and Tervit1990; Voelkel & Hu, Reference Voelkel and Hu1992) whilst others observed better results when using 20% O2 (Oyamada & Fukui, Reference Oyamada and Fukui2004; Castro e Paula & Hansen, Reference Castro e Paula and Hansen2007). Moreover, evidences of interaction between culture medium and oxygen tension were also reported (Noda et al., Reference Noda, Goto, Umaoka, Shiotani, Nakayama and Mori1994; Castro e Paula & Hansen, Reference Castro e Paula and Hansen2007).
Among the steps in IVP, in vitro maturation (IVM) is highlighted (Brackett & Zuelke, Reference Brackett and Zuelke1993; Eppig, Reference Eppig1996; Sirard & Blondin, Reference Sirard and Blondin1996) because in this phase the oocyte accumulates the mRNA and proteins that are essential to progression to the embryonic genome activation stage and to the acquisition of developmental competence (Thibault et al., Reference Thibault, Szöllösi and Gérard1987; Sirard et al., Reference Sirard, Florman, Leibfried-Rutledge, Barnes, Sims and First1989).
Thus, the aim of this study was to evaluate the effects of macromolecule supplements (BSA, PVA, PVP, Ficoll, Knockout and FCS) in IVM and of oxygen tension of 5% or 20% during all IVP procedures (maturation, fertilization and development culture – IVMFC) on oocyte nuclear maturation and embryo development to hatched blastocyst stage.
Materials and Methods
Reagents, media and culture conditions
Chemicals were purchased from Sigma Chemical Co., unless otherwise stated.
The medium for in vitro maturation (IVM) consisted of TCM-199 (Gibco BRL) supplemented with 0.2 mM sodium pyruvate, 25 mM sodium bicarbonate, 75 μg/ml kanamycin (Gibco BRL), 0.5 μg/ml FSH (Pluset®), 100 IU/ml hCG (Profasi®), 1.0 μg/ml estradiol and one macromolecular supplement according to experimental design.
In vitro fertilization (IVF) medium consisted of Tyrode's albumin lactate pyruvate (TALP) supplemented with 0.2 mM sodium pyruvate, 6 mg/ml fatty acid-free BSA, 25 mM sodium bicarbonate, 13 mM sodium lactate, 75 μg/ml kanamycin, 4 μl/ml PHE solution (2 mM penicillamine, 1 mM hypotaurine and 250 μM epinephrine) and 10 μg/ml heparin.
In vitro culture (IVC) medium was synthetic oviductal fluid (SOF) supplemented with 0.2 mM l-glutamine, 0.34 mM sodium citrate, 2.8 mM myo-inositol, 2% MEM essential amino acid solution, 1% MEM non-essential amino acid solution, 0.2 mM sodium pyruvate, 75 μg/ml kanamycin, 5 mg/ml fraction V fatty acid-free BSA and 2.5% FCS.
Cultures (IVM, IVF and IVC) were carried out at 38.5 °C with maximum humidity and under an atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2) depending upon the experimental design.
Oocyte recovery and culture
Abattoir-derived ovaries were transported to the laboratory in saline solution at 30–35 °C. The follicles (2–8 mm) were aspirated using an 18-gauge needle attached to a 20 ml syringe. Oocytes with at least four layers of cumulus cells were selected for the experiments.
For IVM, oocytes were washed and cultured in IVM medium supplemented with 6 mg/ml BSA, 6 mg/ml PVA, 6 mg/ml PVP, 6 mg/ml Ficoll, 10% KnockoutSR (Gibco BRL), or 10% FCS (Gibco BRL). The IVM culture was performed in 100 μl droplets (20 oocytes per droplet) under mineral oil (Dow Corning Co.) for 24 h.
Assessment of nuclear maturation
The percentage of oocytes at germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stages were respectively recorded at 6, 18 and 24 h of IVM. For that purpose, oocytes were stripped from their cumulus cells by vortexing for 3 min in 0.2% hyaluronidase and then stained with 10 μg/ml Hoechst 33342 for 10 min, placed between slide and coverslip and visualized under epifluorescence microscopy (330–385 nm; at 200×magnification).
In vitro fertilization
Motile spermatozoa were obtained by centrifugation of frozen–thawed semen on a Percoll (Pharmacia) discontinuous density gradient (2 ml of 45% Percoll over 2 ml of 90% Percoll) for 30 min at 900 g at room temperature. The supernatant was discarded and the spermatozoa were counted on a haemocytometer and then resuspended in IVF medium to obtain a final concentration of 2 × 106 cells/ml. Finally, 4 μl of the sperm suspension were added to each droplet, for a final concentration of 1 × 106 sperm/ml. Oocytes and sperm were co-incubated for 24 h.
In vitro development culture and embryo evaluation
Following fertilization, the presumptive zygotes were transferred to IVC medium. Zygotes were incubated under mineral oil up to 48 h for assessment of cleavage rates under stereoscopic microscopy (at a 40× magnification), when 2- and 4-cell embryos were counted. Morulae, blastocyst and hatched blastocyst development rates were observed, respectively, at 144 (day 6), 168 (day 7) and 192 h (day 8) post insemination (hpi).
Experimental design
Experiment I: The effects of macromolecular supplementation of IVM medium and oxygen tension on oocyte nuclear maturation
Oocytes (n = 4129 in five replicates) were in vitro matured in medium supplemented with one of the following macromolecules: BSA (6 mg/ml), PVA (6 mg/ml), PVP (6 mg/ml), Ficoll (6 mg/ml), Knockout (10%), or FCS (10%); in two oxygen tension: 5% CO2 in air (20% O2) and 5% CO2, 5% O2 and 90% N2 (5% O2). Oocytes were assessed for nuclear maturation and data for GVBD, MI and MII were collected, respectively, at 6, 18 and 24 h of IVM.
Experiment II: The effects of macromolecular supplementation of IVM medium and oxygen tension during maturation, fertilization and culture on embryonic development
Oocytes (n = 1139 in six replicates) were in vitro matured for 24 h in the same conditions described for the Experiment I. After IVM, they were submitted to IVF and IVC in 20% or 5% O2 and data for cleavage (48 hpi) and development to morulae (144 hpi), blastocyst (168 hpi) and hatched blastocyst (192 hpi) were calculated over total number of oocytes, and were recorded.
Statistical analysis
In this study, data were reported as mean ± standard error (SEM). Data were arcsine transformed and analysed by ANOVA in which the effect of macromolecule, oxygen tension and interaction between macromolecule and oxygen tension were evaluated. When a statistical significant effect was found, multiple comparisons of means were determined using Tukey's test (SAS Program V.8). A p-value <0.05 was considered to be statistically significant.
Results
Experiment I: The effects of macromolecular supplementation of IVM medium and oxygen tension on oocyte nuclear maturation
At 6 h of IVM in 20% oxygen tension, GVBD rates in oocytes treated with FCS (49.8% ± 4.9) were higher (p < 0.05) than in PVP (30.7% ± 5.3), Ficoll (32.0% ± 5.1) and Knockout (31.1% ± 4.9) (Table 1). At 5% O2, the treatments PVA (43.0% ± 5.7), PVP (44.7% ± 5.9) and Ficoll (43.8% ± 7.6) were superior (p < 0.05) to BSA (25.2% ± 4.3), Knockout (22.3% ± 4.9) and FCS (24.1% ± 4.8) (Table 1). For all treatments, except BSA and FCS that presented higher (p < 0.05) results at 20% O2, the GVBD stage rates were similar (p > 0.05) between both oxygen tensions (Table 1).
Table 1 Oocyte nuclear maturation stages.
Germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII), respectively, at 6, 18 and 24 h of in vitro maturation in medium supplemented with bovine serum albumin (BSA; 6 mg/ml), polyvinyl alcohol (PVA; 6 mg/ml), polyvinyl pyrrolidone (PVP; 6 mg/ml), Ficoll (6 mg/ml), KnockoutSR (10%) and fetal calf serum (FCS; 10%), in oxygen (O2) tensions of 5% CO2 in air (20% O2) and 5% O2, 5% CO2 and 90% N2 (5% O2).
a–dValues with different superscript letters within the same column differ (p < 0.05).
At 18 h, MI rates (32.0 to 41.4%) were similar (p > 0.05) in all treatments at 20% O2 (Table 1). In 5% oxygen tension, higher (p < 0.05) M I rates were observed in groups PVA (58.5% ± 6.8), PVP (66.1% ± 5.6), Ficoll (60.4% ± 5.9), Knockout (63.6% ± 7.2) and FCS (50.5% ± 7.2) (Table 1). The treatments PVP, Ficoll and Knockout presented differences (p < 0.05) between both oxygen tensions, and showed higher (p < 0.05) results at 5% O2 (Table 1).
At 24 h, similar (p > 0.05) MII rates (54.4 to 67.6%) were observed among all treatments at 20% O2 (Table 1). At 5% O2, PVP (56.0% ± 5.2) was superior (p < 0.05) to all treatments, but similar (p > 0.05) to PVA (41.9 ± 6.4) (Table 1). PVP at 5% O2 was similar (p > 0.05) to all treatments at 20% O2. In a comparison of both oxygen tensions, PVP was the only treatment that showed similar (p > 0.05) MII rates at 20% and 5% O2, while the other supplements presented better results at 20% O2 (Table 1).
Experiment II: The effects of macromolecular supplementation of IVM medium and oxygen tension during maturation, fertilization and culture on embryonic development
Data for cleavage and embryonic development to morulae, blastocyst and hatched blastocyst after in vitro maturation in medium with different macromolecule supplements (BSA, PVA, PVP, Ficoll, Knockout and FCS) under two oxygen atmospheres (20% and 5% O2) are presented in Table 2. The results of the ANOVA showed that there was no effect (p > 0.05) of macromolecule supplements of IVM medium (BSA, PVA, PVP, Ficoll, Knockout and FCS) in any of the variables studied, such as cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). On the other hand, oxygen tension (20% and 5% O2) had a significant effect (p < 0.05) in all the variables studied. There was no interaction between macromolecular supplements of IVM medium and oxygen tension. Hence, the data for those variables were presented separately as independent variables (Tables 3 and 4).
Table 2 Cleavage and embryonic development to morulae, blastocyst and hatched blastocyst.
Cleavage (48 hpi) and embryonic development to morulae (144 hpi), blastocyst (168 hpi) and hatched blastocyst (192 hpi) in bovine oocytes in vitro matured in medium supplemented with bovine serum albumin (BSA; 6 mg/ml), polyvinyl alcohol (PVA; 6 mg/ml), polyvinyl pyrrolidone (PVP; 6 mg/ml), Ficoll (6 mg/ml), Knockout (10%) and fetal calf serum (FCS; 10%) and submitted to oxygen (O2) tensions of 5% CO2 in air (20% O2) and 5% O2, 5% CO2 and 90% N2 (5% O2) during maturation, fertilization and culture.
Table 3 Cleavage and embryonic development to morulae, blastocyst and hatched blastocyst in bovine oocytes in vitro matured in medium supplemented with bovine serum albumin, polyvinyl alcohol, polyvinyl pyrrolidone, Ficoll, Knockout or fetal calf serum.
Cleavage (48 hpi) and embryonic development to morulae (144 hpi), blastocyst (168 hpi) and hatched blastocyst (192 hpi) in bovine oocytes in vitro matured in medium supplemented with one of the following macromolecules: bovine serum albumin (BSA; 6 mg/ml), polyvinyl alcohol (PVA; 6 mg/ml), polyvinyl pyrrolidone (PVP; 6 mg/ml), Ficoll (6 mg/ml), Knockout (10%) and fetal calf serum (FCS; 10%).
a,bValues with different superscript letters within the same column differ (p < 0.05).
Table 4 Cleavage (48 hpi) and embryonic development to morulae (144 hpi), blastocyst (168 hpi) and hatched blastocyst (192 hpi) in bovine oocytes in vitro matured, fertilized and cultured under oxygen tensions of 5% CO2 in air (20% O2) and 5% O2, 5% CO2 and 90% N2 (5% O2).
a,bValues with different superscript letters within the same column differ (p < 0.05).
The macromolecule supplement used during in vitro maturation did not affect (p > 0.05) the rates of cleavage (51.0 to 67.9%) and development to morulae (20.8 to 30.5%) and hatched blastocyst (20.0 to 32.5%) (Table 3). Blastocyst development rates were higher (p < 0.05) in treatments PVA (35.6% ± 6.0) and FCS (38.7% ± 5.9) when compared to Knockout (22.7% ± 5.4) (Table 3).
Higher (p < 0.05) rates of cleavage and development to morulae, blastocyst and hatched blastocyst were obtained after in vitro culture in 20% O2 (67.4, 29.2, 40.8 and 34.0%, respectively) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively) (Table 4).
Discussion
Oocyte nuclear maturation involves meiosis resumption (germinal vesicle breakdown–GVBD), resulting in MI, first meiotic completion, polar body emission and progression to MII stage. These steps, together with the cytoplasmic maturation, prepare oocytes for being fertilized and to acquire embryonic developmental competence (Thibault et al., Reference Thibault, Szöllösi and Gérard1987; Sirard et al., Reference Sirard, Florman, Leibfried-Rutledge, Barnes, Sims and First1989). Then, culture conditions, which included medium composition and oxygen tension, used for IVM of mammalian oocytes influence subsequent embryonic development (Rose & Bavister, Reference Rose and Bavister1992; Stock et al., Reference Stock, Woodruff and Smith1997). Currently, the medium commonly used for IVM is TCM-199 supplemented with serum and hormones. However, several studies attempted to use defined macromolecule as supplements to replace serum in culture, thus aiming to avoid composition variations and the risk of disease transmission (Mckiernan & Bavister, Reference McKiernan and Bavister1992; Stock et al., Reference Stock, Woodruff and Smith1997; Krisher et al., Reference Krisher, Lane and Bavister1999).
For that reason, undefined (supplemented with FCS), semi-defined (BSA) and defined (PVA, PVP, Ficoll and Knockout) TCM-199 media were used for IVM of bovine oocytes under two oxygen tensions (5% and 20% O2) to assess their effects on nuclear maturation. The normal meiosis progression was evaluated as the percentage of oocytes at GVBD, MI and MII at 6, 18 and 24 h of IVM, respectively.
Under atmospheric oxygen concentration (20% O2) the number of oocytes at GVBD stage produced in the undefined medium with FCS was higher than that in defined media with PVP, Ficoll and Knockout. This finding may be caused by meiosis acceleration promoted by FCS, as reported for blastocyst development (Mastromonaco et al., Reference Mastromonaco, Semple, Robert, Rho, Betts and King2004). However, all macromolecule supplements were similar in MI and MII rates at 20% O2. At a lower oxygen concentration (5% O2) the defined supplements PVA, PVP and Ficoll were superior to BSA, Knockout and FCS in GVBD rates, but all treatments, except BSA, were similar in MI rates and PVP was superior in MII rates. From these results, we can observe that, for the earliest step of nuclear progression (GVBD), undefined (FCS) and semi-defined (BSA) media were more efficient at 20% O2, while defined media (supplemented with PVA, PVP, or Ficoll) were better at reduced oxygen tension (5% O2). Thus, the oxygen concentration influenced the macromolecule supplement used for IVM, similar to the oxygen tension effects observed with culture medium (TCM-199 versus SOF; Castro e Paula & Hansen, Reference Castro e Paula and Hansen2007) and with other supplements, such as glucose (Oyamada & Fukui, Reference Oyamada and Fukui2004) and melatonin (Papis et al., Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007). The positive effects of undefined/semi-defined media at 20% O2 and of defined media at 5% O2 became less pronounced as oocytes progressed in development.
Based on the findings of nuclear maturation (MII rates), all evaluated macromolecule supplements were suitable for use in IVM of bovine oocytes at 20% O2, but at 5% O2 comparable results were only obtained for PVP. Then, we support the hypothesis that IVM under the most frequently used oxygen concentration, 20% O2 (Harvey, Reference Harvey2007), is optimal for nuclear maturation independent of the macromolecule supplement used (defined, semi-defined or undefined). These results are contrary to the described detrimental effect of atmospheric oxygen tension (20% O2) during IVM on nuclear progression (Eppig & Wigglesworth, Reference Eppig and Wigglesworth1995; Smitz et al., Reference Smitz, Cortvrindt and Steirteghem1996). Moreover, we observed that reduced oxygen tension (5% O2) during IVM was detrimental for oocyte nuclear maturation, as previously reported (Ali & Sirard, Reference Ali and Sirard2002).
To assess the effects of macromolecule supplement and oxygen tension on embryonic development, oocytes were matured in IVM media supplemented with BSA, PVA, PVP, Ficoll, Knockout and FCS at 5% or 20% O2 and then submitted to in vitro fertilization (IVF) and culture (IVC) in the same atmospheric condition used for IVM. The zygotes were then evaluated for cleavage and development to morulae, blastocyst and hatched blastocyst stages.
Independently of macromolecule supplement and oxygen tension, the results obtained for cleavage (mean of 60.9%) and development to morulae (25.7%), blastocyst (29.4%) and hatched blastocyst (24.7%) were similar to the average results described in the literature.
All the macromolecule supplements used for IVM promoted similar rates of cleavage, morulae and hatched blastocyst. For blastocyst development, Knockout alone was inferior to IVM media supplemented with PVA or FCS, in agreement with the results that the Knockout serum replacer is not a beneficial supplement for maturation (Moore et al., Reference Moore, Rodriguez-Sallaberry, Kramer, Johnson, Wroclawska, Goicoa and Niasari-Naslaji2007). Few studies have evaluated the effects of different macromolecule supplements during maturation on embryonic development. Among these, it was observed that IVM in media supplemented with BSA (Fukui et al., Reference Fukui, Kikuchi, Kondo and Mizushima2000) and PVP in bovine (Ali & Sirard, Reference Ali and Sirard2002; Chung et al., Reference Chung, Tosca, Huang, Xu, Niwa and Chian2007) and Ficoll in mouse (Kuleshova et al., Reference Kuleshova, Macfarlane, Trounson and Shaw1999) produced similar results in embryonic development when compared with serum. Then serum, which is the most used supplement used for IVM, can be effectively replaced by BSA, PVA, PVP and Ficoll without detrimental effects on early embryonic development.
These results are in disagreement with the observed reduction in embryonic development when PVA (Fukui et al., Reference Fukui, Kikuchi, Kondo and Mizushima2000) was used for IVM. Also, we did not observe developmental block (Camous et al., Reference Camous, Heyman, Méziou and Ménézo1984) nor reduced viability (Wright & Bondioli, Reference Wright and Bondioli1981) of embryos when IVM was performed in defined media.
Analysing the two atmospheric conditions employed during all IVP procedures (including IVM, IVF and IVC), independent of macromolecule supplement for IVM, we observed that utilization of atmospheric oxygen tension (20% O2) resulted in higher rates of embryonic development (cleavage, morulae, blastocyst and hatched blastocyst) than a lower oxygen concentration (5% O2). Therefore, these results suggest that, when adopting only one atmosphere condition for all IVP procedure, it is preferable to use 20% O2 rather than 5% O2.
In constrast to other reports (Yuan et al., Reference Yuan, Van Soom, Coopman, Mintiens, Boerjan, Van Zeveren, Kruif and Peelman2003; Corrêa et al., Reference Corrêa, Rumpf, Mundim, Franco and Dode2008), we have obtained suitable development rates to blastocyst (40.8%) in IVC at 20% O2 in the absence of co-culture.
The detrimental effects observed for 5% O2 during IVP culture may be a consequence of the utilization of this atmospheric value, especially for IVM, as lower maturation rates were obtained under this oxygen concentration. It is well established that IVM conditions affect embryonic development (Rose & Bavister, Reference Rose and Bavister1992; Stock et al., Reference Stock, Woodruff and Smith1997) and that an oxygen tension lower than atmospheric in IVM reduces cleavage and blastocyst development (Betterbed & Wright, Reference Betterbed and Wright1985; Castro e Paula & Hansen, Reference Castro e Paula and Hansen2007). The superiority of a higher oxygen concentration during IVM may be provided by oxygen availability to oocyte, after its consumption by cumulus cells in cumulus–oocyte complexes (COCs; Castro e Paula & Hansen, Reference Castro e Paula and Hansen2007), as described for the oxygen gradient that occurs in ovarian follicles (Gosden & Byatt-Smith, Reference Gosden and Byatt-Smith1986). However, better embryo development after IVM under lower oxygen tension was also reported (Hashimoto et al., Reference Hashimoto, Minami, Takakura, Yamada, Imai and Kashima2000), which may reflect other effects of medium composition and culture conditions.
Some studies (Papis et al., Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007; Corrêa et al., Reference Corrêa, Rumpf, Mundim, Franco and Dode2008) assumed that higher oxygen tensions during IVC resulted in ROS production that was detrimental for embryo development. However, it was previously demonstrated that physiological concentrations of ROS may, therefore, play a key role in the process of oocyte maturation (Dalvit et al., Reference Dalvit, Cetica, Pintos and Beconi2005). Then, IVM culture of COCs under low oxygen concentration may have lead to oocyte hypoxia and impairment of a physiological concentration of ROS that is necessary for normal cellular growth and development (Dalvit et al., Reference Dalvit, Cetica, Pintos and Beconi2005).
In summary, we observed that oxygen concentration influences the macromolecule supplement used for IVM in nuclear maturation, as beneficial results especially in earlier steps of maturation were obtained for undefined (FCS) and semi-defined media (BSA) at 20% O2 and for defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, higher nuclear maturation rates were obtained at 20% O2 and this atmospheric oxygen tension was found to be optimal for IVM of bovine oocytes. To promote embryo development, considering macromolecule supplement and oxygen tension as independent variables, the supplements BSA, PVA, PVP and Ficoll can replace FCS for IVM of bovine oocytes, while Knockout was not a suitable macromolecular supplement for IVM. As improved results for embryonic development were achieved at atmospheric oxygen tension, when all the steps of in vitro production (IVMFC) of bovine embryos were performed using only one atmosphere condition, the choice of oxygen concentration of 20% O2 is preferred to 5% O2.
Acknowledgements
This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), Brazil, Grant #01/06137–0. V.S.D. Caiado Castro was recipient of studentship from CAPES, Brazil and L.S.S. Barretto was recipient of studentship from FAPESP, Brazil.
