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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 3, Num. 2, 2005, pp. 79-82
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Iranian Journal of Reproductive Medicine Vol. 3, No. 2, Spring, 2005, pp. 79-82
The
effect of culture medium volume on in vitro development of mouse embryos
Abbasali Karimpor
Malekshah, Ph.D.1, Amir Esmailnejad Moghaddam, Ph.D.1
1 Department of Anatomy and
Embryology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Corresponding Author: Dr Abbasali Karimpor
Malekshah, Department of Anatomy and
Embryology, Faculty of Medicine, Mazandaran University of Medical Sciences,
Khazar Bulevard, Sari, Iran. E-mail: mohamed1@mazums.ac.ir
Code Number: rm05014
Abstract
Background: In the field
of mammalian embryo culture, the putative influence of autocrine/ paracrine
factor(s), produce by the embryos itself, is under investigation. A smaller
medium drop can prevent dilution of this factor(s).
Objective: The objective of this study was to examine
the effect of culture medium volume on in vitro development of mouse 2-cell
embryos.
Materials and Methods: The embryos were obtained from
female NMRI mice. To evaluate the effect of medium volume, groups of 16-20 late
2-cell embryos were cultured in 2, 5, 10, 20, 50 and 100 µl of drops of Hams F10 medium for 72 h.
Results: Development to
blastocyst stage in 50 and 100 µl of drop were significantly higher than this
in any other volume (p<0.001). Almost a similar pattern was also observed
for hatched blastocyst formation. However, the total number of cells in
blastocysts, developing in different volumes, were not significantly different.
Conclusion: These results indicate that the optimal
volumes of Hams F10 medium for mouse
early embryo development are 50 to 100 µl. However, volumes as small as 2 µl
can successfully support mouse 2-cell embryo development to blastocyst and
hatching stages.
Key Words: Mouse embryo, Culture medium, Incubation volume
Introduction
In order to support in
vitro development, different mammalian embryos have been empirically cultured
in drops of medium with different volumes (e.g., 10-500 µl) covered with
mineral oil. Recent studies showed that the volume of medium and embryo density
(number per unit volume) are important factors that influence early embryo
development (1-5). In IVF, different volumes of medium and numbers of embryos
per a definite of incubation drop, have been described in literature with
different results among authors, but only a few have studied these parameters
as variation.
Some
published data have showed that micro-cultures (small volume of medium), may
lead to improved early embryo development in different species (3,6). This
effect may be interpreted to suggest the secretion of autocrine or paracrine
growth or survival factors by the embryos to support itself and other embryos
in their development (3,6-8). A smaller incubation volume could prevent a
dilution of these specific embryo-derived factors. However, some other studies
could not support the idea of autocrine/paracrine stimulation of embryo
development (4,9,10), and even have shown that culturing embryos in small
volumes of culture medium is detrimental to their development (1,11). The
objective of this study was to examine the effect of culture medium volume on
in vitro development of mouse 2-cell embryos.
Materials and Methods
Embryo collection
The embryos were obtained from 6 to 8-weeks female NMRI mice, which exhibit the
two-cell block in vitro. The females were superovulated by an intraperitoneal
injection of equine chorionic gonadotrophin (7 IU; eCG: Sigma, St. Louis, MO), followed by human chorionic gonadotrophin (7 IU; hCG: Profasi, Serono
Laboratories, Inc.), 48hr later. They were then placed individually with males
of the same strain. The following morning, the presence of a vaginal plug was
confirmed. Late 2-cell embryos were obtained at 42-44 h after hCG
administration by flushing the oviducts. HEPES buffered human tubal fluid
medium (HTF; Irvine Scientific, Irvine, CA) medium was used for flushing and
embryos collection. The embryos were rinsed 3 times in HTF and another 3 times
in Hams F10 (Gibco, Grand Island, NY), then were transferred into the culture
treatments.
Experimental
design
To evaluate the effect of medium volume on early embryo development, 2, 5, 10, 20,
50 and 100 µl drops of Hams F10 with human serum albumin (5%)
(HAS,Vitrolife, Sweden AB, Kungsbacka, Sweden) were placed in a single 60mm
falcon dish (Falcon plastics, no. 1007) and covered with about 10ml mineral
oil. A microsampler was used to prepare the drops of medium. All medium drops
were equilibrated overnight at 37°C under 5% CO2 in air before
culture began. All collected embryos from different animals were harvested in a
100 µl drop of Hams F10 prior to distribution to treatment groups.
Harvested embryos were randomly allocated into the culture drops in groups of
16-20 and cultured for 72 h at 37°C in 5% CO2 in air. When embryos
were transferred into 2 and 5 µl drops, the culture medium volume in
each drop was maintained by removing a volume equal to the volume of transfer
medium added. Embryonic development was scored every 24 h and the proportions
of the 4 to 8-cell, morula, blastocyst and hatching stages were recorded.
A total of 7 replicate were conducted (including all experimental groups) to
standardize procedures and minimize experimental variations.
Determination of cell number
Embryos were placed into 0.9% (v/v) of sodium citrate (in water) for 10-20 min
and fixed with a solution of ethanol: acetic acid (2:1) for 2 min. The fixed
blastocysts were transferred to slides, air-dried and stained with 10% (w/v)
Gimsa (Sigma) and the cell numbers were determined under a microscope.
Statistical analyses
The proportion of 2-cell embryos developing to the blastocyst stage, and the
proportion of hatched blastocyst between groups were analyzed with the
chi-square test.
Data on cell number of blastocysts from different groups were analyzed by
ANOVA. Statistical analyses were conducted with the SPSS software package (SPSS
10).
Results
The proportions of the 2-cell embryos which developed to the blastocyst and
hatching stages in different volumes of culture medium are shown in table I.
No differences were observed for the rate of blastocyst stage between 2, 5, 10
and 20 µl volumes of culture medium (68.8, 62.1, 62.2 and 67.2%
respectively). However, significantly more embryos developed to blastocyst
stage in 50 µl (81.5%) and 100 µl (79.0%) volumes as compared to
any other volumes (p<0.001). A similar pattern was also observed for hatched
blastocyst formation, except that there was no difference between 20 µl
and 100 µl volumes and also there were significant differences between
20 µl volume and all smaller volumes (p<0.002). The mean (±SEM)
number of blastomere per blastocyst were 35.9 ± 4.6, 41.3 ± 12.3, 37.6 ± 13.8,
38.1 ± 11.1, 42.6 ± 12.9 and 37.5 ± 8.0 in 2, 5, 10, 20, 50 and 100 volumes
respectively. Which the differences were not significant.
Table I. Effect of the volume of Hams F10 medium
on mouse 2-cell embryo development after 72 h culture.
Volume (µl) |
N* |
Proportion (% ± SEM) of embryo developing to : |
Blastocyst |
Hatching |
2 |
128 |
68.8 ± 5.6a |
20.3 ± 6.2a |
5 |
132 |
62.1 ± 5.4a |
19.7 ± 2.1a |
10 |
135 |
62.2 ± 8.4a |
23.7 ± 16.5a |
20 |
134 |
67.2 ± 7.3a |
30.6 ± 11.3b |
50 |
135 |
81.5 ± 4.2b |
37.8 ± 16.3c |
100 |
138 |
79.0 ± 1.0b |
34.1 ± 11.6cb |
* Total number of
2-cell embryos cultured in 7 replicates; within each replicate 16-20 embryos
were cultured in each volume.
abc Different
superscripts differ significantly within the same column (first column: p<0.001,
second column: p<0.05).
Discussion
Some previous studies have shown that mammalian early embryo development and
quality were improved by culturing the embryos in reduced volumes and/or by
increasing embryo density
(the embryo medium volume ratio) (3,6,12,13). To interpret this effect, the
authors assumed that preimplantation embryos produce some mitogenic or
embryotrophic factor(s) which enhances their development, specially the
blastocyst and hatching rates (6-8,12,14). In smaller incubation volume the
concentration of these specific embryo-secreted factor(s) increases
sufficiently to exert its effect. However, other experiments have also shown
that depending on the culture conditions, single embryos can be produced with
the same developmental competence as embryos produced in groups (4,15,16) and
also developmental rates were significantly higher in large volume (100 µl)
than those in small volume of culture medium (1).
Our results does not support the idea of autocrine/paracrine stimulation of
embryo development. Because two-cell embryos cultured in small volumes (2-20µl)
of medium had significantly lower development than those cultured in 50µl
and 100µl volumes. These results may be interpreted to suggest either an
accumulation of toxic metabolites in the smaller volume medium (17) or
depletion of medium components essential for development (18). The interaction
between medium volume and culturing embryos can be divided into two aspects:
positive conditioning, representing embryo-derived beneficial factors that
stimulate their own development (Cooperative interaction), and negative
conditioning, that produces detrimental effects on embryo development via
accumulation of embryotoxic elements in the culture medium (11), or changing
some critical environmental conditions such as PH and the osmolarity of medium
drops. Variations in experimental conditions could account for some differences
between our data and those published by other groups. It can be supposed that
the reduced volume of incubation medium may show two adversed effect on embryo
development in comparing to higher volume: In well quality controlled
experimental conditions the positive cooperation of cultured embryos may acts
more effectiveness in smaller droplet of medium; on the contrary, in low
controlled conditions accumulation of the detrimental agents and changing the
optimal conditions over to embryos tolerance, occurred sooner in small drops
than larger one. Therefore, we suggest that the optimal incubation volume for
embryo culture of the same species is different depending to culture conditions
include type of medium and laboratory conditions.
The results of this study also showed that although large volumes of Hams F10
medium has advantage for mouse embryo development, interestingly, the
ultra-microdroplets (2-5µl) of medium could support early embryo development up
to blastocyst and hatching stages. More importantly, the quality of the
blastocysts were not different between the ultra-microdroplets and large drops
because the total number of cells per blastocyst were not different between two
groups. This data are on contrary to which reported by Kito et al (1997)
(1). Their study showed that 2µl volume of culture drop only support the first
cleavage and 5µl volume up to 3 cleavage of hamster zygotes. This controversy
may be caused by interspecies differences and also difference in culture
medium.
In conclusion, the results indicated that, medium volume influence the embryo
development in mouse and the optimal volumes of Hums F10 medium for blastocyst
development of mouse 2-cell embryos are 50 to 100 µl. However, volumes
as small as 2 µl could successfully support mouse 2-cell embryo
development to blastocyst and hatching stages.
Acknowledgements
This work was supported by Mazandaran University of Medical Sciences.
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