Abstract: The objective of this study, was to compare the developmental competence of bovine in vitro fertilized embryos in three different culture methods; microdrop method (50 μL of medium under mineral oil in petri dishes) compared to tube methods (1 mL of medium in tubes) with or without oil overlay and to examine the influence of Fetal Bovine Serum (FBS) in culture methods. They were randomly allocated to one of following culture conditions: (Group 1) microdrop method supplemented with 10% (v:v) FBS at day 5 post-insemination (pi); (Group 2) microdrop method without FBS; (Group 3) tube method with oil overlay and FBS added at day 5 pi; (Group 4) tube method with oil overlay; (Group 5) tube method without oil overlay but FBS added at day 5 pi; (Group 6) tube method without oil overlay and FBS. There were no differences (p>0.05) in cleavage rates among the culture methods (78.3, 75.6 and 74.9% for microdrop, tube with oil overlay and tube method without overlay, respectively). However, regardless of serum addition, blastocyst rates in the microdrop method (30.5%) were significantly higher (p<0.05) than those in the tube method without oil overlay (9.8%). There was no difference (p>0.05) between with or without serum in blastocyst rate regardless culture methods. Numerically, the highest blastocyst rate was observed in the microdrop method with the FBS supplementation (Group 1; 36.6%). Overall, the microdrop method was the optimum culture method among the culture methods; however, serum supplementation did not significantly affect the blatocyst rate.

INTRODUCTION

Bovine embryo in vitro Production (IVP) has become an important tool for basic science research and for applications in animal biotechnologies (Lohuis, 1995; Galli et al., 2003). In support of this, a diverse array of in vitro culture methods (e.g., microdrop, tube and four well methods) have been developed and studied among different laboratories (Rombauts and Wood, 2000). In these component based IVP systems, bovine preimplantation embryos have been cultured from the zygote to blastocyst stages in various volumes of culture medium, and in the presence or absence of oil overlay with different culture methods and serum has been frequently added to culture medium as a protein source for in vitro Culture (IVC) (Bavister, 1995). Since, Brinster first developed the microdrop culture method (Brinster, 1963), this culture method has become the most commonly used culture method among IVF laboratories (Boatman, 1987; Nagy et al., 2003). The microdrop method is achieved by culturing a group of embryos in a small volume (10-100 μL) of culture medium covered with liquid oil (e.g. mineral, paraffin, or silicon oil) that prevents the microdrop of medium from rapid evaporation and controls the pH changes of the culture medium (Brinster, 1968; Miller et al., 1994b; Tae et al., 2006). Nevertheless, some studies have shown that oil overlay of culture medium results in interactions with steroid hormones such as oestradiol and progesterone present in the medium causing alterations in medium composition (Miller and Pursel, 1987; Shimada et al., 2002). Additionally, some batches or lots of oil in this method can be a source of toxic contaminants in embryo culture (Erbach et al., 1995; Provo and Herr, 1998; Van Soom et al., 2001; Otsuki et al., 2007). In order to avoid oil use, a group of embryos could be cultured in a relatively large volume (1-3 mL) of culture medium in either culture tubes or 4-well dishes. Culturing embryos in a large volume of medium, however, potentially has an adverse effect. It may dilute embryo-derived growth factors which may have beneficial effects on their development (Canseco et al., 1992; O'Neill, 1998).

Additionally, serum supplementation of the culture medium during the later stage (morulae or blastocyst stage) of embryo development is known to increase blastocyst and hatching rates and cell numbers of bovine embryos (Pinyopummintr and Bavister, 1991; Lim et al., 1994; Van Langendonckt et al., 1997; Yoshioka et al., 1997). However, the composition of serum is highly variable from different suppliers and even between batches or lots (Maurer, 1992). Moreover, some batches of serum contain non-defined molecules that could be detrimental to embryo development (Bavister, 1995).

These components of in vitro culture systems used for IVP influence the developmental competence of mammalian embryos (Bavister, 1995; Galli et al., 2003; Gardner and Lane, 2004). For this reason, success rates of the embryo culture vary among different protocols and the efficiency of culture methods also differs between laboratories and studies. Furthermore, a recent review by Gardner (2008) emphasized the necessity of prescreening batches and lots of serum in order to optimize the efficacy of culture systems. It is therefore, important to examine these components and culture methods affecting the development of in vitro derived embryos when applying routine IVP to new studies. In this study, we designed a simple and comparative experiment evaluating different culture methods and effects of serum on bovine embryo development. The developmental competence of bovine embryos were compared between the microdrop method with 50 μL of culture medium covered with mineral oil and the tube methods with 1 mL culture medium (with or without mineral oil overlay), which contained an equivalent number of embryos and to examine the influence of serum supplementation on embryos in in vitro culture.

MATERIALS AND METHODS

All the chemicals utilized in the present investigation were purchased from Sigma Chemicals (St. Louis, MO) unless otherwise indicated. Bovine Cumulus-Oocyte Complexes (COCs) were purchased from a commercial supplier (BOMED, INC., Madison, WI). Each week, approximately 600 bovine oocytes were shipped in 2 mL of the maturation medium in tightly closed tubes and were over-night shipped in a 39°C portable incubator. On arrival at the laboratory, COCs were removed from the shipping unit and were incubated at 39°C and 5% CO₂ in air for 24 h.

Sperm preparation and In Vitro Fertilization (IVF): The sperm preparation for IVF used the BoviPure™ gradient and was performed according to the protocol provided by the manufacturer as follows (Nidacon International AB, Gothenburg, Sweden). All the media used for sperm preparation were warmed at 39°C before use. In a 15 mL conical centrifuge tube, 2 mL of BoviPure™ Bottom Layer was placed and then carefully layered with 2 mL of BoviPure™ Top Layer on the top of the BoviPure™ Bottom Layer to make a gradient. Two straws of frozen semen from a single Angus bull were thawed in a 37°C water bath for 120 sec. The thawed semen were gently mixed with BoviPure™ Extender and brought to a 2 mL volume. The 2 mL of the prepared semen was then gently layered on top of the BoviPure™ gradient and centrifuged for 20 min at 500 x g. After the centrifugation, the fluid above the sperm pellet was carefully removed with a sterile Pasteur pipette. The pellet was resuspended with 5 mL of BoviPure™ Wash and centrifuged for 5 min at 300 x g. This pellet was resuspended again in 5 mL of warmed IVF-TL (Tyrode’s Albumine Lactate Pyruvate) medium (Millipore, Billerica, MA) and centrifuged for 5 min at 300 x g. IVF-TL medium was supplemented with pyruvate (0.2 mm), fatty-acid-free Bovine Serum Albumin (BSA-FAF, 6 mg mL^-1), penicillin (100 U mL^-1), streptomycin (100 μg mL^-1), heparine 10 μg mL^-1, hypotaurine 0.5 μg mL and epinephrine 0.5 μg mL^-1 (Miller et al., 1994a). The number of sperm was counted using a hemocytometer and the sperm pellet was diluted to a concentration of 50x10⁶ spermatozoa mL^-1. To make the 2x10⁶ final concentration, 2 μL of diluted sperm were added to each 50 μL drop of the fertilization medium (IVF-TL) containing 10 matured oocytes that had been washed previously in the fertilization medium (IVF-TL). Sperm and oocytes were co-incubated at 39°C and with 5% CO₂ in air for 18 h.

In vitro culture: Following fertilization, presumptive zygotes were denuded from cumulus cells by vortexing for 2 min and washed 3 times with HEPES-TL wash medium (Bioniche, Pullman, WA). They were cultured in Synthetic Oviductal Fluid (SOF) culture medium (Millipore, Billerica, MA) supplemented with pyruvate (0.4 mm), 100 x MEM (20 μ mL^-1), 50 x BME (10 μL mL^-1), BSA-FAF (8 mg mL^-1), penicillin (100 U mL^-1) and streptomycin (100 μg mL^-1) at 39°C and with 5% CO₂ in air (Edwards et al., 1997). All the culture medium was equilibrated at 39°C and with 5% CO₂ in air overnight either in the 35x10 mm sterile polystyrene disposable Petri dishes (Fisher Scientific, Pittsburgh, PA) or 12x75 mm sterile polystyrene disposable tissue culture tubes (Fisher Scientific, Pittsburgh, PA) before use. The sterile mineral oil (Cat. No. M5310; Sigma) was used for the culture and it was stored in the dark to avoid the production of embryo-toxic compounds (Provo and Herr, 1998). The 10% (v:v) of FBS (Fetal Bovine Serum, Gibco/Invitrogen, Carlsbad, CA) was added to selected treatment groups in the culture medium at day 5 pi (post insemination) in the culture (Lim et al., 1994). All the FBS and mineral oil that was used in the present study were from one batch (lot).

Experimental designs: To evaluate the effects of culture methods and serum supplementation in bovine in vitro culture, three different culture methods (microdrop method, tube method with oil overlay and tube method without oil overlay) and with and without serum supplementation were used in a 3x2 factorial experiment. A total of 1,696 presumptive zygotes were randomly distributed to the following six groups. A group of approximately 50-70 presumptive zygotes were cultured in Group 1: microdrop method (50 μL microdrops of culture medium covered with mineral oil in the petri dish) and with serum supplementation (10% (v:v) FBS added at day 5 pi); Group 2: microdrop method without serum supplementation; Group 3: tube method (1 mL of culture medium in the test tube) with mineral oil and serum supplementation; Group 4: tube method with oil overlay but no FBS supplementation; Group 5: test tube method without oil overlay and FBS added at day 5 pi; Group 6: tube method without oil overlay and no serum supplementation. The cleavage rate of the embryos was assessed and the culture medium in all groups was replaced with the fresh culture medium at day 2 pi. The blastocyst development was assessed at day 9 pi.

Statistical analysis: Experiments were repeated three times and data were analyzed by two-way ANOVA (culture methods and serum supplementation and their interation). All percentage data were subjected to arcsine transformation before statistical analysis. Data are expressed as mean±SEM. A probability of p<0.05 was considered to be statistically significant. When ANOVA revealed a significant effect, values were compared by Fisher’s PLSD post hoc test. All the analysis was conducted with the STATVIEW program (Abacus Concepts, Inc., Berkeley, CA). The percentages of blastocysts at day 9 pi were based on the total number of cleaved embryos determined at day 2 pi.

RESULTS AND DISCUSSION

The effects of the bovine embryo different culture methods (microdrop, tube with oil overlay and tube without oil overlay) and serum supplementation on the proportions of the cleaved embryos and blastocysts are shown in Table 1. While there were no significant differences (p>0.05) culture methods and serum supplementation interaction and serum supplementation in cleavage and blastocyst rates, means of bovine embryo developmental rate were averaged only in each culture methods for pair wise comparisons (Table 2). Regardless of serum supplementation, there were no significant differences (p>0.05) among culture methods in cleavage rates at day 2 pi (78.3±3.6, 75.6±2.5 and 74.9±2.1% for microdrop, tube-oil and tube methods, respectively). However, the microdrop method was superior (p<0.05) to the tube method without oil overlay for blastocyst formation (30.5±5.4 and 9.8±3.1%, respectively). There was also a trend (p<0.10) of increasing blastocyst rates when embryos were cultured in the microdrop method (30.5±5.4%) compared to the tube method with oil overlay (18.7±3.0). Additionally, the blastocyst rates of the embryos cultured in the tube method with oil overlay (18.7±3.0) tended to be higher (p<0.10) than those cultured in the tube method without oil overlay (9.8±3.1%). The Table 1 shows that the supplementation of the culture medium with 10% (v:v) serum at day 5 pi had no significant (p>0.05) effect on the blastocyst rates regardless of the culture methods. Numerically, the highest development rate of blastocysts was observed in the microdrop method with the serum supplementation (36.6±8.5%) and culturing embryos in the tube method without oil overlay and serum supplementation (6.3±3.5%) exhibited the lowest blastocyst rate (Table 1).

The current study, was conducted to evaluate bovine embryo culture conditions as affected by culture methods and serum supplementation. The microdrop and tube methods are generally used among IVF laboratories (Rombauts and Wood, 2000; Nagy et al., 2003). Major differences of these methods are the volume of culture medium and use of oil overlay. The microdrop with oil is the most commonly used embryo culture system because progress of embryo development is easily observed under the microscope and a small volume of culture medium appears to be a beneficial factor for embryo development. Many studies reported that the rates of blastocyst formation and cell numbers were increased when embryos were cultured in the microdrop method where a group of embryo is cultured in a small volume of medium in the mouse (Canseco et al., 1992; Gardner et al., 1994; Salahuddin et al., 1995; Gardner, 2008), sheep (Gardner and Lane, 2004) and bovine (Keefer et al., 1994; Palasz and Thundathil, 1998; de Oliveira et al., 2005). Paria and Dey (1990) interpreted that embryos secrete growth factors that may act in an autocrine or paracrine manner. Thus, the effect of culturing a group of embryos using the microdrop method is likely to be due to the minimization of the diffusion of growth factors secreted from the embryo themselves. The possible evidence of this effect has been demonstrated by culturing bovine embryos in conditioned medium derived from a roup of other embryos, which enhances the blastocyst formation rate of single cultured embryos (Fujita et al., 2006).

Table 1:	Effect of culture methods and serum supplementation on bovine embryo developmental rates
1Microdrop method: 50 μL of culture medium in petri dishes. 2Serum: 10% (v:v) fetal bovine serum was added at day 5 post insemination. 3Tube method: 1 mL of culture medium in test tubes. 4Number of total cleaved embryos developing to blastocyst (%)

Table 2:	Effect of culture methods on bovine embryo developmental rates
1Microdrop method: 50 μL of culture medium in petri dishes. 2Tube method: 1 mL of culture medium in test tubes. 3Number of total cleaved embryos developing to blastocyst. Superscripts differ: a,b(p = 0.08); a,c(p = 0.001); and b,c(p = 0.07)

Another assumption is that increasing embryo density (ratio of embryos to volume of culture medium) may deplete embryo-toxic substances in the medium or reduce the concentrations of naturally occurring inhibitors like glucose or ammonium (Bavister, 1995). Similar to these previous reports, the present results show that regardless of FBS supplementation, the microdrop method was superior to the tube method with the large volume of medium without oil overlay for blastocyst formation (Table 2). In addition, there was a trend of increasing blastocyst rates when embryos were cultured in the microdrop method compared to tube method with oil overlay. Fukui et al. (1996) similarly reported that the microdrop method resulted in a larger proportion of blastocyst formation than the 4 well method, where 500 μL of medium is covered with the oil in the well dish. Moreover, their study has shown that the renewal of culture medium at 48 h intervals in the microdrop lowered the rate of bovine blastocyst formation compared with non-renewal of medium. Thus, the large volume of culture medium and renewal of culture medium causes a decrease in the blastocyst rates presumably by diluting and removing embryo-derived growth factors from the culture (Canseco et al., 1992).

In the present study shown in Table 2, the blastocyst rates of the embryos cultured in the large volume of medium with oil overlay tended to be higher than those cultured in the large volume of medium without oil overlay. The primary function of using oil overlay in the embryo culture is that it prevents liquid evaporation which facilitates the maintenance of the appropriate pH and osmotic pressure of culture medium (Brinster, 1968; Boatman, 1987; Miller et al., 1994b; Gardner and Lane, 2000; Tae et al., 2006). Culturing embryos without the oil overlay can lead to low blastocyst formation even though they were cultured in a high humidity environment. Nevertheless, there were no significant differences in the cleavage rate at day 2 pi between the tube method with and without oil overlay groups (Table 2). Thus, the components in the media may have been concentrated during the 9 days of culture resulting in increased osmolarity. Also, Tae et al. (2006) have shown that sterilized-good quality oil absorbs the accumulated toxic components in the medium and resulted in an increased rate of development to the blastocyst formation. Nevertheless, other investigators have shown that unidentified toxic components from some particular batches of oil could be transferred into culture medium (Miller et al., 1994b; Erbach et al., 1995; Provo and Herr, 1998; Otsuki et al., 2007), or oil could absorb substances present in the medium such as oestradiol and progesterone (Miller and Pursel, 1987; Shimada et al., 2002). Thus, oil overlay could cause the alteration of medium composition. In this experiment, sterile-filtered, mouse embryo tested and light mineral oil (M5310; Sigma) was used for the embryo culture and apparently the mineral oil did not have either significant inhibitory or stimulatory effects on bovine embryo development in the present experiment.

We also investigated, the effects of serum supplementation in the culture medium on the developmental ability of the bovine embryos produced in vitro. The supplementation of the culture medium with FBS had no significant effect on the blastocyst rates. Serum contains a variety of known and unknown substances which may stimulate or inhibit embryo development in vitro (Maurer, 1992; Bavister, 1995). Studies have shown that serum supplementation during the early cleavage stages of culture can inhibit the first cleavage but enhances the formation of morulae and blastocyst when serum is added during the later stage of embryo development (Pinyopummintr and Bavister, 1991; Lim et al., 1994). Many investigators have reported the improved embryo development with serum-supplemented media (Pinyopummintr and Bavister, 1991; Yoshioka et al., 1997; Khurana and Niemann, 2000; Kim et al., 2004); yet Caro and Trounson (1984) did not find any improvement of the embryo development with the serum-supplemented medium vs. non-supplemented medium in the mouse. Serum is a pathological fluid formed by blood clotting, which may induce chemical alterations with possible harmful effects for embryo culture (Maurer, 1992). It has been reported that some batches of serum decrease the blastocyst rate and cell number and increase apoptotic cells on a particular culture system (Van Langendonckt et al., 1997). Fukui et al. (1991) reported that even the effectiveness of serum supplementation in the culture medium varies by different components of culture conditions used in vitro. Under these experimental conditions, supplementation with FBS, which was supplemented in either the microdrop or the tube methods, did not statistically improve the rate of blastocyst formation; although embryos derived from culture medium with FBS supplementation exhibited a numerical increase in apparent developmental competence (Table 1).

CONCLUSION

Numerically, the highest blastocyst rates were obtained by the microdrop method with FBS supplementation and overall, the microdrop method was the optimum culture system among the culture methods (microdrop, tube with oil overlay, tube methods). However, FBS supplementation did not significantly affect bovine embryo development in vitro. In the present study, we examined the interactions among culture methods with a constant number of embryos with presence or absence of oil overlay and FBS supplementation in bovine embryo culture. While there are many studies demonstrating positive or negative effects of these components, comparing studies and protocols has been a difficult process and it is necessary to examine exact culture methods and batches and lots of serum to be used for in vitro culture at the same time as has been described here relative to their impacts on bovine in vitro embryo development.

ACKNOWLEDGEMENT

This research was supported, by the Mississippi Agriculture and Forestry Experiment Station, the Department of Animal and Dairy Science, Facility for Organismal and Cellular Imaging (FOCI) and the USDA-ARS funded Biophotonics Initiative (#58-6402-3-0120).