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Banat’s University of Agricultural Sciences and Veterinary
Medicine
Faculty of Animal Sciences and Biotechnologies
Phd student:
Eng. Ivan (Boleman) Alexandra
PhD THESIS RESUME
ASSESSMENT OF MAMMALIAN EMBRYOS
VIABILITY BY MORPHOLOGICAL
CRITERIA AND STAINING TESTES
Scientifically coordinator:
Prof.Eng. Păcală Nicolae PhD
2009
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ABSTRACT
Preimplantational embryo development depends on various factors with different
origins: maternal and embryonic. In vitro embryos in early developmental stages grows and
develops in the absence of endogens factors, but often they manifest heterogeneity and variability
of their developmental capacity, requiring a longer time for achieving the developmental stage
characteristic to the cultivation day and often the embryos presents a more reduce number of
cells, as an answer of the suboptimal cultivation conditions comparing with the in vivo embryos.
Assessment methods used for embryos changed during the last 20 years. Morphological
criteria used for embryo quality assessment improved in order to increase the selection probability
of the most suitable embryos for transfer. In bovine, at the moment, transfer of a single embryo in
the 3-th day after fertilization assures a gestation rate of 10-30% and 40-60% for embryos in
blastocyst stage (Mendoza, 2005). Increasing of gestation rates is strongly correlated with the
precision of estimation and selection of quality embryos.
The PhD thesis titled ASSESSMENT OF MAMMALIAN EMBRYOS VIABILITY BY
MORPHOLOGICAL CRITERIA AND STAING TESTES elaborated by Eng. Ivan (Boleman)
Alexandra under the supervision of Prof. Eng. Păcală Nicolae PhD, is composed by two parts,
structures on 8 chapters, to which were attached general conclusions and recommendations. The
thesis has 203 pages from which first part (bibliographical study) with 59 pages, and the second
part (personal researchers) with 144 pages.
First part, Bibliographical study, encloses 3 chapters:
In the first chapter, titled Fertilization and the preimplantational stages of the
mammalian embryos, were presented the early developmental stages of the mammalian embryos.
In chapter II, titled Mechanisms and signals involved in preimplantational
development of the mammalian embryos, were presented the most important mechanisms and
signals involved in mammalian embryos early development.
In chapter III, titled Morphological criteria and staining tests for mammalian
embryo quality assessment, were presented different systems used for mammalian embryos
quality assessment.
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In the second part of the thesis, titled Personal researches, were presented the aim of
the paper, experimental model used for achieving the work, material and methods, results and
discussions obtained after the experiments implementation.
The aim of the work was to test and evaluate the quality and viability of mammalian
embryos, in order to select the most objective, fast and reliable method for the future use in
practical activities developed in the Embryotransfer and biotechnologies embryo associated
laboratory and in the current works of embryotransfer in farm species.
In order to achieve the goals were completed several working steps as are presented:
1.
Embryo recovery. For these experiments were used mouse embryos (in most of
the experiments) in vivo produced and swine embryos in vitro fertilized;
2.
Embryos quality assessment by morphological criteria and morphometric
measurements in order to establish a correlation between the embryo quality
and certain morphological parameters like. Embryo diameter, the thickness of
pelucide zone, the blastomers diameter;
3.
Viability testing on groups of embryos using staining tastes. For this step we re
used 5 viability tests, using vital dyes:
4.

Trypan blue staining;

Neutral red staining;

Fluorescein diacetat;

Propidium iodide;

Fluorescein diacetat and Propidium iodide mix staining;
Viability assessment and developmental competence, in culture, of the mouse
embryos. For this step, the mouse embryos, in early developmental stages (2, 4
and 8 cells) were cultivated in different variants of culture media: KSOM,
M16, Ham’s Nutrient mixture 12 supplemented with 4% BSA (bovine serum
albumins) and Ham’s Nutrient mixture 12 without BSA. Embryo evaluation
was made on a 72 hours interval.
5.
Viability assessment of and the developmental competence of the mouse
embryos after staining with Trypan blue, Neutral red, Fluorescein diacetat and
Propidium iodide. The mouse embryos, evaluated by staining tests, were
cultivated for 48 hours in appropriated culture media (M 16 media). The
viability assessment of the embryos was made by the developmental capacity,
during cultivation, subsequent applying the staining tests.
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In vitro swine embryo producing and developmental capacity evaluation during
cultivation. For in vitro embryo producing were used oocytes recovered from
ovaries from slaughterhouse females. The immature oocytes were in vitro
maturated and fertilized by cultivation on specific media and than cultivated for
7 days on SOF cultivation media. The oocytes maturation assessment was
made microscopically based on the developmental degree of the feeder cells
and by viability testes: Trypan blue and Neutral red. Subsequent fertilization,
were made observations regarding the developmental stages of the embryos
produced.
The second part compiles 5 chapters, as are presented:
In chapter IV, titled In vivo obtaining of mouse embryos, were presented the recovery
technique of mouse embryos, aspects referring to preparation of manipulation and cultivation
media for mammalian embryos, the chemical composition of the media used in experimentation
and data processing.
In chapter V, titled Quality assessment of mouse embryos by morphological criteria,
were presented the material a method used for mouse embryos obtaining, from normal cycle
females, comparing with superovulated females and morphological criteria used for quality
assessment.
By embryo recovery from 20 donor females, Swiss bread, with normal cycle were
obtained 195 embryos, in average 9.75±1.58 embryos/donor females. From the females hormonal
treated (37) were obtained 741 embryos, in average 20.58±2.60 embryos/donor female.
Most of the embryos obtained from normal cycle females were in blastocyst stage (76),
in average 3.80±0.98 embryos/donor female, value slowly higher comparing with the number of
embryos, in the same developmental stage obtained from superovulated females (122), in average
2.45±1.75 embryos/donor female. Differences were statistical assured as significant (t test,
p≤0.05). From superovulated females, most of the embryos obtained were in morula stage (254),
with average of 6.86±1.76. The statistical comparison with the data’s obtained for the embryos in
the same developmental stage from normal cycles females (67), with the average of 3.35±1.15
showed very significant differences (t test, p ≤0.001).
By morphological criteria assessment and quality codes classification was observed
that most of the embryos obtained from the normal cycle females (45.12%) were assessed as qood
quality (quality code 2), in average 4.40±2.63 embryos/donor female. In what concerns the
embryos obtained from superovulated females, most of the embryos recovered (39.27%) were
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assessed as satisfying quality (quality code 3), in average 7.86±1.83 embryos/donor females.
According to the obtained data’s, the superovulation treatments allows obtaining of a
higher number of embryos comparing to the number of embryos obtained from the normal cycle
females, but often, a considerable proportion of the recovered embryos is classified as quality ode
4, with a poor and very poor quality.
In chapter VI, titled, Assessment of mammalian embryo viability by staining tests,
was assessed the mammalian embryo viability by different staining methods. The methods used
tested the cells capacity to exclude certain dyes as: Trypan blue and Porpidium iodide, and to
assimilate and transform certain dyes as: Neutral red and Fluorescein diacetate.
Results referring to mouse embryo viability assessment by Trypan blue staining
In the first experimental group (Trypan blue 0.1 g/ml in PBS media) the viability
percentage was 100%. In the same experimental group, based on morphological criteria, 40% of
the embryos were evaluated as poor quality (code 4). The difference was statistically assessed as
very significant (χ2 test; p ≤0.001).
In the second experimental group (Trypan blue 0.4 g/ml in PBS media) were observed
significant differences between the percentage of viable embryos (97.77%) and of the nonviable
embryos (2.23%). Based on morphological criteria, 28.88% of the embryos were assessing as
quality code 4. Differences statistically assured as very significant (χ2 test; p ≤0.001).
In the 3-th group, after applying the staining test (Trypan blue 0.1 g/ml in M2 media)
the proportion of viable embryos was 92.30% and 7.69% for the nonviable embryos. Based on
morphological criteria, 34.61% of the embryos were classified as 4 quality code. The differences
were statistically assured as very significant (χ2 test; p ≤0.001).
For the 4-th experimental group (Trypan blue 0.4%) were observed significant
differences in what concerns the percentage of viable (90.62%) and nonviable (9.37%). Based on
morphological criteria, the percentage of poor quality embryos (code 4) was 56.25%. the
differences were statistically assured as very significant (χ2 test; p ≤0.001).
In the 5-th group of the experiment (Trypan blue 0.4%) were observed significant
differences between the percentage of viable embryos (95.55%) and nonviable embryos (3.44%).
Assessing the embryo viability based on morphological criteria indicated a percentage of poor
embryo quality of 34.48% (code 4) (χ2 test; p ≤0.001).
Between the experimental groups, after applying the Trypan blue staining test, were
observed no statistical differences (χ2 test, p>0.05).
The Pearson coefficient for simple correlation indicated a strong positive, linear
correlation (r = 0.914) between the percentage of viable and non viable embryos, evaluated by
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Trypan blue staining test and of those classified as 1,2, and 3 quality code. For the nonviable
embryos was observed a slow decreasing of the correlation (r = 0.513),
Assessment of viable mouse embryos based on morphological criteria, comparing to
Trypan blue staining test, indicated significant correlations between the proportions of poor
quality, non viable embryos. After applying the Trypan blue staining test we observed that the
dye passage through the pelucide zone, inside the poor quality embryos, with a high degree of
fragmentation, was much reduced. The number of embryos evaluated as nonviable using this
method was much reduced, comparing to the number of embryos evaluated as unsatisfying
quality based morphological criteria methods. We consider that this effect is due the fact that
Trypan blue assess the integrity of cellular membrane, and in the poor embryos case, even if the
blastomers fragmentation took place, the pelucide zone remained intact for a longer period and do
not allowed the dye crossing inside the embryo.
Results referring to the mouse embryo assessment by Neutral red staining
For viability assessment using Neutral red were used 4 staining solutions, with different
concentration; 1%, 2%, 0.5% and 0.1%. The percentage of viable embryos observed after staining
with Neutral red, were very high for all concentrations used. The maximum vales were observed
for 1% (94.11%) and 2% NR (93.02%). By statistical analysis of the results between the 4-th
experimental groups the differences were insignificant (χ2 test, p>0.05).
No differences were observed, regarding the degree of cellular membrane penetration
when small dye concentrations were used (0.1% and 0.5%).Neutral red has a very high
penetration capacity for the cellular membrane, so the used of high concentrations of dye are not
justified (1-2%).
The use of Pearson coefficient indicated a very strong correlation (r = 0.988) between
the proportion of viable embryos, evaluated by Neutral red staining test comparing with the
embryos in 1, 2, and 3 quality code. For the embryos evaluated as nonviable and poor quality,
code 4, the correlation was much more reduce (r = 0,239).
The intracellular accumulation of the dye, takes place also for the nonviable embryos,
in more reduces quality and can induce errors in the evaluation process and increases the
subjectivity of the Neutral red staining as a test for viable embryo evaluation.
Neutral red is a cationic dye capable to penetrate the cellular membrane and
accumulates inside lysososomes. Recent studies showed that modifications of the membrane
permeability represent a starting event of cellular apoptosis (Wei Chen, et al., 2005). Based on
these considerrents, the capacity to bind the Neutral red at the lysosomale membrane is
decreasing in embryos with a high degree of fragmentation. The quantity of dye accumulating
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inside the poor quality embryos, even if is more reduce comparing to viable embryos, can easily
induce errors related to embryo quality assessment and increases the method subjectivity.
Results referring to mouse embryos viability assessment using Fluorescein diacetat
staining test
Based on morphological criteria, from 37 embryo teste, 18 (64.8%) were assessed as
poor/very poor quality, and after Fluorescein diacetate staining 15 embryos (35.2%) were
evaluated as nonviable. The difference was statistically assured as distinctly significant (χ2 test, p
≤0.01).
The Fluorescein diacetate staining test indicated presence of enzymatic activity in
embryos with high degree of fragmentation, which by morphological method were considered as
4 quality code. The Fluorescein diacetate viability test for esterasic activity detection inside the
cells is a simple, fast and reliable test for enzymatic activity detection and at the same time for
cellular membrane integrity detection.
Results referring to mouse embryo viability assessment by Propidium iodide staining
By Propidium iodide staining were evaluated 53 embryos, in 2 experimental groups (50
μl/ml Propidium iodide in PBS media and 50 μl/ml Propidium iodide in M2 media).
For the first experimental group, tested with 50 μl/ml Propidium iodide solution in PBS
media, the percentage of viable embryos was 82.14% and 17.86% for the nonviable embryos. The
difference was assured as very significant (χ2 test, p ≤0,001). For the second group (50 μl/ml
Propidium iodide in M2 media) the percentage of viable embryos was 80% and 20% for the
nonviable. The difference was statistically assured as very significant (χ2 test, p ≤0,001).
The Pearson coefficient indicated a very strong correlation (r= 0.989) between the two
methods used for mouse embryo viability and quality assessment and indicates the Propidium
iodide test as a reliable method for embryo viability assessment.
There were observed no statistical differences between the experimental groups which
indicate that the solvents used for staining solution preparation do not influence significantly the
results obtained (PBS media – group 1 and M2 media – group 2). Choosing the solvent remains
an economical considering.
The Propidium iodide staining allows detection of the nonviable cells, with no
metabolic activity inside. The Propidium iodide test is fast, simple and noninvasive for the viable
cells. The selective binding of Propidium iodide is made only to the death cells nucleus, in which
the metabolic activity has stopped. The propidium iodide test can be used for the mammalian
embryo viability assessment, without considering the problems associated with the cellular
toxicity, caused by dyes accumulation and cells incapacity to metabolize the substance used.
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Results referring to mouse embryo viability assessment by Fluorescein diacetat and
Propidium iodide
For embryo viability assessment by Fluorescein diacetate and Propidium iodide test
was used a mix of the two dyes which allows detection of both viable and nonviable cells.
Embryos considered as viable shoed green fluorescence when were exposed to fluorescence
source with a wave length of 494-518 nm, while the nonviable cells showed a red fluorescence
when were exposed at a fluorescence source with a wave length of 515-560 nm.
After applying the Fluorescein diacetate and Propidium iodide test from 28 embryos,
25 (86.22%) were assessed as viable (shoed green fluorescence when were expose to 494-518
nm) and 4 (13.79%) were assessed as nonviable (shoed red fluorescence when were exposed to
515-560 nm).
The statistical analysis of data’s indicated very significant difference between the
percentage of viable and nonviable embryos (χ2test, p ≤0,001).
The images 6.36, 6.37, 6.38, 6.39 indicates the advantages of using at the same theime
the two dyes (Fluorescein diacetate and Propidium iodide) for mouse embryo viability
assessment. At the same time, indicates the deficiencies of embryo viability assessment only by
morphological criteria. Assessment of embryo viability only by morphological criteria does not
offer information’s regarding the presence of enzymatic and metabolic activity within the cells.
Even if, from this consideration, the use of staining tests for viability assessment based on
dyes able to mark the presence and absence within the cells, seams to be more objective, the
staining dyes bring the problem of cellular toxicity.
In chapter VII titled Assessment of mouse embryo viability by short term in vitro
cultivation, we observed the developmental capacity of the mouse embryos after vital dyes
staining and the influence of different cultivation media (M 16, KSOM, HAM F 12 supplemented
with 4% BSA and HAM F 12 without BSA) on the developmental capacity. On the embryos
developed until the morula and blastocist stage were made several morphometric measurements
(thickness of pelucide zone, inner and outer diameter, inner and outer perimeter of the embryo).
Results referring to assessment of in vitro development of mouse embryos after staining
Staining methods, even if they are wanted to be noninvasive for the embryos, use certain
dyes that poses the capacity to penetrate the pelucide zone and to get inside the embryo were
accumulates or enter in different metabolic paths and are transformed (Fluorescein diacetate is
hydrolyzed under cellular esterase to fluorescein). The embryos were cultivated for 48 hours.
After 24 hours of in vitro cultivation on M 16 media, in all experimental variants the
embryos evolved to the morula stage. The differences between the proportion of embryos
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evolving to the morula stage were distinct significant (χ2 test, p ≤0.01), 56.66% for the control
group, 21.73% for the FDA and Pi group, 19.40% for the Neutral red group and 16.66% for the
Trypan blue group.
After 48 hours of cultivation, the percentage of embryos which developed was more
reduce compared to the retarded embryos (χ2test, p ≤0,001). Only for the control group embryos
evolved to the hatched blasctocyst stage (13.33%). For the rest of the experimental groups,
embryos evolved to the expand blastocyst (FDA and Pi - 4.34% and NR – 4.76%), blatsocyst
(FDA and Pi – 13.04%, Nr – 4.76%) and compact morula (4.76% for the NR group).
After cultivation, was observed that embryos stained with Neutral red evolved until the
expanded blastocyst stage (4.76%). The results indicate the capacity of the cells to metabolize the
staining dyes. Even if, the percentage of retarded embryos was higher for both staining testes
(FDA and Pi – 73.91%, NR – 80.95%) the differences were insignificant (p>0.05) compared to
the control group.
Unexpected was the percentage of retarded embryos, observed for the Trypan blue group
(87.55%) compared with the percentage of retarded embryos observed in the control group
(56.68%). Differences were statistical assured as significant (p≤0.05).
The embryos able to developed until the blastocyst stage, indicates the fact that the
embryos are able to metabolize the staining dyes (Fluorescein diacetate and Neutral red), but the
high percentage of retarded embryos indicates a slowing up to stopping the developmental
capacity of the embryos after staining.
Results referring to the mouse embryo viability assessment by short term in vitro
cultivation
The developmental capacity of the embryos, during in vitro cultivation, varied very
much, between the 4-th experimental variants (M16, KSOM, HAM F 12 supplemented with 4%
BSA and HAM F 12 without BSA). After 24 hours of cultivation, in two of the experimental
variants (HAM F 12 nutrient mixture supplemented with 4% BSA and M 16) were observed
embryos in early blastocyst stage (19.14% for the first variant and 8.51% for the second variant of
media).
After 48 hours of cultivation, in 3 of the experimental variants (KSOM, M 16 and HAM
F12 with 4% BSA) were observed very significant differences between the embryos reaching ]n
expanded blastocyst stage (χ2 test , p ≤0,001). The highest percentage of embryos in blastocsyst
stage was observed in M 16 media (34.02%) and the lowest was observed in HAM F 12
supplemented with 4% (6.38%).
After 72 hours of cultivation, the M 16 media was the only variant where 14.90% of the
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embryos were observed in hatched blastocyst stage.
In both HAM F 12 media variants (supplemented with 45 BSA and without BSA) after
72 hours of cultivation, all embryos were assessed as retarded or degenerated. High proportions
of degenerated embryos were observed also in KSOM media (63.04%).
Results referring to morphometric measurements of mouse embryos in vivo and after
in vitro cultivation
The parameters taken in consideration were: thickness of pelucide zone, inner and outer
diameter, inner and outer perimeter. The measurements were made using the Quick Photo Micro
2.2.
On in vitro cultivated embryos were observed increasing values of the measured
parameters. The thickness of pelucide zone varied from 9.8±1.7 µm for morula stage embryos
recovered at 72 hours from the vaginal plug, up to 11.1±1 µm for the in vitro cultivated embryos
stained with vital dyes, before cultivation. The differences were statistical insignificant (p>0.05).
A similar situation was observed for the rest of the studied parameters: inner diameter (from
98.4±3.3 µm to 111.6±3.2 µm), outer diameter (form 116.7±2.5 µm to 129.4±4.8 µm), inner
perimeter (form 302±0.5 µm to 338±0.4 µm) and outer perimeter (from 352±0.8 µm to 391.±0.4
µm). Similar differences were observed for the blastocyst stage embryos (6.2±0.5 µm for
blastocyst stage embryos recovered after 72 hours from the vaginal plug and 8.7±1 µm for the
embryos previously stained).
The values obtained for the embryos in vitro cultivated without staining were higher
comparing to the values observed for the in vivo embryos, recovered at 72 hours from the vaginal
plug (10.8±1.2 µm for the in vitro embryos and 9.8±1.7 µm for the thickness of the pelucide zone
measured at the morula stage embryos recovered at 72 hours from vaginal plug). The differences
were statistical insignificant.
Embryos previously stained had higher values, comparing to the in vitro cultivated
embryos without staining, but the differences were insignificant (10.8±1.2 µm for the morula
prelucide zone thickness previously stained and 7.3 ±1 µm and 8.7±1 µm for blastocyst stage
embryos).
In chapter VIII, titled Assessment of in vitro produced embryos, we evaluated the
viability of the in vitro embryo produced starting from recovered oocytes from slaughterhouse
female’s ovaries. The working protocol included oocytes in vitro maturation, fallowed by in vitro
fertilization and in vitro cultivation of the zygotes for 7 days in SOF media (Synthetic oviductal
fluid).
We observed that the cumulus ooforus oocytes (COC) are in vitro maturated in a higher
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proportion (73.68%) comparing to oocytes without cumulus ooforus (9.67%). Differences were
distinct significant (p ≤0,001).
Referring to the in vitro fertilization rates, most of the embryos obtained from the
cumulus ooforus (73.68%) comparing to the oocytes without cumulus (9.67%). Differences were
very significant (p ≤0,001).
After 72 hours from the in vitro fertilization process from 28 oocytes with cumulus
ooforus evaluated as fertilized were obtained 2 embryos in two cell stage (7.14%) and 9 embryos
in 4 cell stage (32.14%), 20 of the oocytes (71.43%) were evaluated as degenerated.
After 7 days of cultivation in SOF media, from 28 oocytes with cumulus ooforus were
obtained 2 embryos in morula stage (7.14%), 3 embryos in 8 cell stage (10.71%), 5 embryos in 4
cell stage (17,86%) and 1 embryo in 2 cell (3.57%) and 17 were degenerated (60.71%). The
differences between the degenerated and in development embryos were very significant (χ2 test, p
≤0,001).
The slow rates of development of the embryos indicates the SOF media as unsatisfying
for assuring the swine embryos requirements and determines a high percentage of degenerated
embryos (60.71%).
Results referring to swine oocytes viability assessment by Trypan blue and Neutral red
staining
The developmental capacity of the oocytes includes the ability to form viable embryos
after in vitro fertilization process. This ability is conditioned by the citolasmatic and nuclear
maturation of the oocyte. Under natural condition oocytes present a high developmental
competence, but when the natural environment is replaced with artificial conditions, and the
process is experimental, the developmental capacity of the oocytes is very much reduced despite
the optimizations brought in this area (Lucid, et al., 2003, Rath et al., 1995, Nimann, et al., 2001).
After 46 hours of in vitro maturation based on morphological criteria 42% of the
cultivated oocytes in the first experimental variants (TCM-IVM supplemented with 10% FSB)
and 50% of the cultivated embryos in TCM-IVM medium supplemented with 10% FSB and
hormones were appreciated as matured. The differences were insignificant (χ2 test, p>0,05).
After Trypan blue staining, from the total of 52 oocytes cultivated in TCM-IVM
maturation media supplemented with 10% FSB, 100% were assessed as viable (remained
uncolored after Trypan blue inclusion). A similar percentage (100%) was observed for the
oocytes cultivated in TCM-IVM media, supplemented with 10% FSB and hormones. Significant
differences were observed between the proportions of viable and nonviable oocytes. There were
observed no significant differences between the results obtained by applying the two maturations
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media variants.
After applying the Neutral red staining test, from 55 oocytes cultivated on TCM-IVM
media supplemented with 10% FSB, 39 (70.90%) were evaluated as viable and 16 (29.10%) were
evaluated as nonviable. The differences were very significant (χ2 test, p ≤0,001).
From 24 oocytes cultivated on TCM-IVM media supplemented with 10% FSB and
hormones, 19 (79.16%) were evaluated as viable and 5 (20.08%) were evaluated as nonviable.
The differences were very significant (χ2 test, p ≤0,001).
There were no differences between the proportions of viable oocytes, obtained in
maturation media supplemented with 10% FSB (70.90%) comparing with the proportion of viable
oocytes observed in maturation media supplemented with 10% FSB and hormones (79.16%).
General conclusions
1. Even if applying the superovulation treatments influence the quality of the obtained
embryos, most of the embryos (39.27%) were assessed as 3-th quality code, comparing to
most of the embryos obtained from the normal cycle females (45.12%), the hormonal
treatments allows obtaining of a much higher number of embryos, in average 20.58±2.60
embryos/donor female, comparing to the average number of the embryos recovered from
the normal cycle females 9.75±1.58 embryos/donor female.
2. The Fluorescein diacetate and Propidium iodide viability test can be successfully used for
mouse embryo viability evaluation. This method allows detection of both viable and
nonviable cells.
3. There were no differences between the experimental groups that used as a solvent PBS or
M2 media and indicated the fact that the type of solvent used for staining solution
preparation do not influence the results obtained so the solvent choice remains an
economical considering.
4. Referring to embryos stained by staining tests and short time cultivated (48 hours) in M
16 media, was observed that the percentage of embryos developing decreases after the
first hours of cultivation. Only to the control group were observed embryos in hatched
blastocyst stage (13.33%). The rest of the embryos in the experimental groups continued
development up to the expanded blastocyst stage (4.34% for FDA and Pi, 4.76% for NR).
The differences between the experimental groups and the control were insignificant
(p>0.05).
5. After applying the viability tests we observed that the FDA+Pi and NR allow the embryo
development up to the expanded blastocyst stage (4.34% and 4.76% respectively).
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Surprising, the embryos stained with TB stopped in development in morula stage
(12.5%).
6. Referring to the time interval for mouse embryo cultivation (24-72 hours) was observed
that a high proportion of embryos developing in the first hours of cultivation are
degenerated at 72 hours. After 48 hours of in vitro cultivation most of the retarded
embryos were observed in KSOM media (36.95%) and the lowest proportion in HAM F
12 media supplemented with 4% BSA (2.12%). The differences were very significant (χ2
test, p≤0.001). After 72 hours of cultivation, the percentage of degenerated embryos
increased in KSOM media (63.04%), Nutrient mixture HAM F 12 with 4% BSA
(46.80%) and was 100% in HAM F 12 without BSA.
7. Regarding the cultivations media used for mouse embryos (KSOM, M 16, HAM F 12
with 4% BSA and HAM F 12 without BSA) was observed that the M 16 media was the
only media that assured the embryo development up to the hatched blastocyst stage
(14.90%). A reduced number of embryos evolved up to the expanded blastocyst stage
8.69% in KSOM media and 6.38% in HAM F 12 supplemented with 4% BSA.
8. In vitro cultivation of mouse embryos on synthetic media increases the thickness of
pelucide zone. The values varied from 9.8±1.7 μm, on morula stage embryos recovered at
72 hours from the vaginal plug up to 11.1±1 μm for the morula stage embryos in vitro
cultivated after staining with vital dyes. The differences where insignificant (p>0.05).
9. The thickness of pelucide zone, for the in vitro cultivated embryos assessed by staining
had higher values comparing to in vitro cultivated embryos unstained, but the differences
were insignificant (p>0,05) (10.8±1.2 μm respectively 11.1±1 μm for the morula stage
embryos and 7.3±1 μm and 8.7±1 μm for blastocyst stage embryos).
10. Referring to in vitro fertilizations rates, most of the embryos were obtained from cumulus
ooforus oocytes (73.68%) comparing to oocytes without cumulus (9.67%). The
differences were very significant. (p ≤0,001).
11. Low rates of embryo development indicate the SOF media as unsatisfying for swine
embryo in vitro development and are correlated with a high degree of degenerated
embryos (60.71%).
12. The use of staining tests (Trypan blue and Neutral red) for oocytes viability assessment
can improve the objectivity of the morphological criteria method but can not totally
replace it. It must be taken in consideration the effect of vital dyes used for staining onto
developmental capacity of the future embryo.
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Recommendations
1. For Fluorescein diacetate working solution preparation we recommend the use of
acetone as a solvent, which assures a fast dissolving, in 90% proportion of Fluorescein
diacetate and prevents the remaining of particles in suspension.
2. For mammalian embryo viability assessment we recommend the use both of the
morphological criteria method and the viability test with Fluorescein diacetate (FDA)
and Propidium iodide (Pi) which increases the precision of viable (FDA) and nonviable
(Pi) cells detection.
3. We do not recommend in vitro embryo cultivation on simple, complex and synthetic
media on a longer period of 48 hours, after this interval was observed a decreasing of
embryo development. For the embryos previously stained we don not recommend in
vitro cultivation over a longer period of 24 hours, due to the negative influence of
artificial environment and the use of dye which was observed that decreases the embryo
viability.
4. Based on the results obtained we don not recommend the use of SOF media for in vitro
cultivation of swine embryo, the media can not assure the requirements of developing
swine embryos and induces a high percentage of degenerated embryos (60.71%).
5. For in vitro embryo producing recommend the use of cumulus ooforus oocytes with a
rate of 40% embryo producing. The use of oocytes without cumulus ooforus for in vitro
embryo producing determines low percentage embryos.
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