Veterinary Research Communications, 20 (1996) 15-19 Copyright 0 Kluwer Academic Publishers bv- Printed in the Netherlands
Short Communication THE EARLY DEVELOPMENT OF MOUSE EMBRYOS IN VITRO IN MEDIUM SUPPLEMENTED WITH DIFFERENT BATCHES OF SERUM AND BOVINE SERUM ALBUMIN M.B. TORNESI AND J. ARCHER Department of Veterinary Pathology, WCVM, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N 5B4 ABSTRACT Tornesi, M.B. and Archer, J., 1996. The early development of mouse embryos in vitro supplemented with different batches of serum and bovine serum albumin. Veterinary Communications, 20 (1), 15-19 Keywords:
in medium Research
albumin, culture, embryo, hatch, mouse, nucleus, serum
Abbreviations: BSA, bovine serum albumin; DBS, donor bovine serum; FBS, fetal bovine serum; ECG, equine chorionic gonadotrophin; hCG, human chorionic gonadotrophin; IV, international unit
INTRODUCTION Mammalian embryos are generally cultured in medium supplemented with serum. Any use of serum involves the addition of a wide range of undefined proteins, hormones and other elements which may vary widely from batch to batch (Barnes and Sato, 1980). In many studies, serum has been replaced by bovine serum albumin (BSA) so as to reduce variability, since commercial BSA is 98% pure albumin. On the other hand, variation in batches of BSA has been suggested as the reason for variations in embryonic development when using the same type of media (Rorie et al., 1994). This may be due to the 2% of unassayed materials in commercial BSA. BSA binds biological substances such as fatty acids, hormones and many trace metals (McKierman and Bavister, 1992) and it is therefore not an inert constituent of culture media. The ability of different batches to support embryonic development could be related to these bound, unassayed, low-molecular-weight contaminants. Gray and colleagues (1992) identsed citrate as one low-molecular-weight embryotrophic factor. The objective of our study was to determine the effects on embryonic development of different batches of two types of sera - fetal bovine serum (FBS) and donor bovine serum (DBS, steer serum) - and of BSA.
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MATERIALS
AND METHODS
Animals and recovery of embryos
Prepubertal female mice of the CD-l strain, weighing 20-22 g, were kept in a controlled environment. Mice were induced to superovulate by an intraperitoneal injection of 5 IU of equine chorionic gonadotrophin (ECG, Equinex, Ayerst Lab., Montreal, PQ, Canada), followed 48 h later by 5 IU of human chorionic gonadotrophin (hCG; A.P.L., Ayerst Lab.). After the hCG injection, these females were caged with fertile males and examined for the presence of a vaginal plug, which was taken as evidence of mating. Forty-eight hours later, the mice were euthanized by cervical dislocation and embryos were collected by retrograde flushing of their oviducts (Mintz, 1967; Pratt, 1987).
Media for culture of embryos
All media were prepared using purified water (Barnstead, Thermnolyne Corp., Dunbunque, IA, USA). Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Grand Island, NY, USA), supplemented with an antibiotic+antimycotic mixture (penicillin, streptomycin, amphotericin B Antibiotic-Antimycotic, Gibco) and 15% FBS (Cansera Lab., Rexdale, ON, Canada), was used for the collection and handling of the embryos (collection medium). A BWW medium (Biggers et al., 1971) was used for the culture of the embryos. Just prior to establishing the culture, the BWW medium was supplemented to obtain a final concentration of 25.07 mmol/L NaHCOs and 0.25 mmol/L sodium pyruvate, and the antibiotic-antimycotic mixture was added. Serum was added to the BWW medium prior to culture as described in the following experimental design. Three batches of BSA (nos. 196971, 10818,11506), FBS (SF 900510 (l), SF-g03-16 (2), SF g-08-23 (3)) and three of DBS (SD 80804 (l), SD g-02-28 (2), SD 9-1102 (3)), all provided by Cansera, were used at concentrations determined to be optimal for each protein source (Tornesi et al., 1993). The albumin content of the serum was estimated and all the different batches of FBS and DBS were added in amounts such that the final media contained the same amount of albumin. BSA was added at 0.8%; FBS at 20 mg/ml (FBS: (1) 10.5%; (2) 8.3%; (3) 8.7%); and DBS at 19 mg/ml (DBS: (1) 5.5%; (2) 5.5%; (3) 4.9%). A control medium with no additions of serum or BSA was also included in each experiment. Studies were performed in 15 replicate experiments.
Culture
After collection, the embryos were examined under a stereoscopic microscope ( x 40) and classified according to their morphology and stage of development (Seidel and Stringfellow, 1990). Only good or excellent quality embryos (compact, uniform cytoplasm, uniform cell size) with 16 or more cells were used. These embryos were pooled, washed three times in collection medium and randomly assigned into the treatment groups.
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The embryos were incubated in 40 pl drops of culture medium under parafhn oil at 37°C in 5% CO2 in air with 95% humidity (Brinster, 1963). They were examined with an inverted bright light microscope at x 200 following 24 h and 48 h of incubation to determine their viability, cellular integrity and stage of development. Embryos were considered viable if they were at the compacted morula or early blastocyst stage at 24 h; and at the blastocyst, hatching blastocyst (beginning to leave the zona pellucida) or hatched blastocyst stage (completely free of the zona pellucida) at 48 h. Staining technique The cell number was determined by Tarkowski’s (1966) method for chromosome preparation in early embryos. After incubation in a hypotonic solution (1% sodium citrate), all the embryonic cells were spread on a coverslip, followed by fixation in freshly prepared acetic acid-alcohol (3: 1 v/v). The nuclei and also the number of cells in mitosis were identified after staining with 1% Giemsa.
Data analysis The results of the studies on the embryos are reported as percentages. Group differences were determined using a test for homogeneity (Daniel, 1978). Multiple chisquared test comparisons were made when an overall significant effect was found. A significant difference was assumed to exist when the probability of making a type I error was less than 5%. Results of the counts of the nuclei were analysed using a statistical analysis system (SAS, 1985). Data were subjected to a ‘general lineal models’ procedure to show whether treatment effects existed. Duncan’s multiple range test was used to determine where treatment differences lay. Differences were assumed to exist when the probability of making a type I error was less than 5%.
RESULTS Total embryonic survival was high in all the groups (> 90%), including the control serum-free group. There were no significant differences between the three batches of FBS or DBS, but the hatching rate was higher in the serum-supplemented (FBS and DBS) groups (>50% hatching) than in the serum-free group (26.2%). The three batches of BSA differed in their ability to support embryonic development. One lot (no. 11506) was significantly better than the serum-free group or the other two lots of BSA (Table I). After 48 h in culture, the nuclear numbers differed between the groups. The serumfree group had the lowest number of nuclei and was different from all the BSA and DBS groups but not from the FBS group. One lot of BSA (no. 11506) and one of DBS (no. SD 80804) caused the embryos to have higher numbers of nuclei than the other two respective lots (Table II).
18 TABLE I The percentage of mouse embryos which developed after 48 h in BWW culture medium alone or with different batches of BSA, FBS and DBS Treatment groups (lot no.)
Concentration of albumin (mg/ml)
n
0
149
94.0
26.2b
BSA (196971) BSA (10818) BSA (11506)
80 80 80
111 100 109
96.4 90.0 98.2
24.3b 24.0b 46.8”
FBS (SF900510) FBS (SF90316) FBS (SF80823)
20 20 20
109 109 108
94.5 92.7 94.4
53.38 54.1= 54.6”
DBS (SD80804) DBS (SD90228) DBS (SD91 102)
19 19 19
101 117 106
92.1 92.3 96.2
49.5” 54.8a 58.5”
Serum-free
Total survival W)
Hatching/ hatched (%)
“.bPercentages within a column with different superscriptsare significantlydifferent cp <
0.05)
TABLE II The average number of nuclei in the embryos after 48 h in BWW culture with different batches of BSA, FBS and DBS Treatment groups (lot no.)
Concentration of albumin (mg/ml)
n
0
43
65.5”
BSA (196971) BSA (10818) BSA (11506)
80 80 80
34 38 37
80.gb 77.0b 94.6a
FBS (SF900510) FBS (SF903 16) FBS (SF80823)
20 20 20
26 23 25
68.1” 73.9” 75.4”
DBS (SD80804) DBS (SD90228) DBS (SD91 102)
19 19 19
33 30 20
98.8’ 85.6b 77.2=
Serum-free
Number of nuclei
“*bWumbersof nucleiwithin a column with different superscriptsare significantlydifferent0, < 0.05)
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DISCUSSION The results demonstrated that serum improves the hatching rate of embryos in vitro compared to BSA or the defined medium alone, but embryos can develop from the morula stage to the expanded blastocyst stage without serum or BSA. The variability between lots was greater in media supplemented with BSA than in media supplemented with complete serum (FBS or DBS). The BSA used was 98% pure and we consider that the 2% of uncharacterized impurities are the probable source of the variability between batches of BSA and may also be important essential factors for the hatching process. As the nuclear counts did not correlate with the morphological evaluation of the embryos, they do not appear to be a reliable method for evaluating embryonic development in culture. Caution is advised when using commercial BSA to supplement culture media. It is clear that a medium containing BSA is not a detied medium. Testing each batch and the use of BSA with the same lot number is advisable if reproducible culture conditions are required.
REFERENCES Barnes, D and Sato, G., 1980. Serum-free cell culture: a unifying approach. CeZZ,22,649-655 Biggers, J.D., Whitten, W.K. and Whittingham, D.G., 1971. The culture of mouse embryos in vitro. In: J.C. Daniel and J. Freeman (eds.), Methods for Mummaliun Embryology, (W.H. Freeman and Co., San Francisco, CA, USA), 86116 Brinster, R.L., 1963. A method for in vitro cultivation of mouse ova from 2-cell to blastocyst. Experimental Cell Research,
32, 205-208
Daniel, W., 1978. Chi-square test of independence and homogeneity. In: W. Daniel (ed.), Applied NonParametric Statistics, (H. Mifflin, Boston, MA, USA), 160-184 Gray, C.W., Morgan, P.M. and Kane, M.T., 1992. Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate. Journal of Reproduction and Fertility, 94, 471-480 McKierman, S.H. and Bavister, B.D., 1992. Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In vitro Cellular and Developmental Biology, 28A, 154-156 Mintz, B., 1967. Mammalian embryo culture. In: F.H. Witt and N.K. Wessels (eds), Methods in Developmental Biology, (Thomas Crowell, New York), 21-27 Pratt, H.P., 1987. Isolation, culture and manipulation of preimplantation mouse embryos. In: M. Monk (ed.), Mammalian Development, A Practical Approach, (IRL Press, Oxford), 13-14 Rorie, R.W., Miller, G.F., Nasti, K.B. and McNew, R.W., 1994. In vitro development of bovine embryos as affected by different lots of bovine serum albumin and citrate. Theriogenology, 42, 397-404 SAS (1985). SAS Statistics Version 5. SAS User’s Guide, (SAS Institute, Inc, Cary, NY, USA) Seidel, SM. and Stringfellow, D.A., 1990. Certification and identification of the embryo. In: SM. Seidel and D.A. Stringfellow (eds), Manual of the ZETS, (International Embryo Transfer Society, Champaign, IL, USA), 12-59 Tarkowski, A.K., 1966. An air-drying method for chromosome preparations from mouse eggs. Cytogenetics,
5, 394-400
Tornesi, M.B., Palasz, A.T., Del Camp, M.R., Rousseaux, C.G., Archer, J.F. and Mapletoft, R.J., 1993. In vitro culture of preimplantation mouse embryos and day 12 limb-buds: Effects of serum and albumin. Reproductive
Toxicology,
(Accepted 7 August 1995)
I, 623-630
