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Histological and morphometric characteristics of chicken embryos with

different genotypes

Article  in  The Journal of Eurasian Research · March 2020

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 EurAsian Journal of BioSciences
Eurasia J Biosci 14, 719-725 (2020)

Histological and morphometric characteristics of chicken

embryos with different genotypes
A. Z. Alabdallach 1, A. A. Nikishov 1, N. A. Volkova 2, A. N. Vetokh 1,2, N. Y. Rebouh 1,
V. I. Semenova 1, A. K. Petrov 1, E. O. Rystsova 1, M. V. Bolshakova 1, E. A. Krotova 1,
S. G. Drukovsky 1, A. Norezzine 1*
1
Peoples` Friendship University of Russia, RUSSIA
2
L.K. Ernst Federal Science Center for Animal Husbandry, RUSSIA
*Corresponding author: assissnor@gmail.com

Abstract
Background: The GFP gene is commonly used in biotechnology and is used as an indicator.
Chicken embryos are also the most important experimental animals used in modern research for
easy access to embryos, as well as easy gene injection at an early stage. Study design: Study of
the influence of genetic engineering manipulations on the histological and morphometric parameters
of organs and tissues of chicken embryos. Methods: We took the three groups: - Normal (H),
Transgenic (G) and Crossbred (HxG). Research stage was divided into two parts :The first step was
to study the internal and external characteristics of the eggs. At the second stage, morphological and
histological changes in organs and tissues in embryos were studied. Results: The incubation eggs
of the studied genotypes met the regulatory requirements for morphometric indicators of egg quality.
Eggs from transgenic chickens (group 2) had statistically significantly lower values of the quality index
of dense protein - units of Hough. Compared to the eggs of the 1st and 3rd groups, the difference
was 4.8 and 3.6 Hau units, respectively (P≥0.95). On the 14th day of incubation, the embryos of the
1st and 3rd groups were statistically significantly superior the weight of group 2 embryos was 17.4%
and 13.5%, respectively (P≥0.95). By day 18, differences in the weight of the embryos persist.
Embryos from eggs from genetically modified chickens (group 2) on the 18th day of incubation had
the smallest absolute values according to linear indices of individual parts of the body. Genetic
engineering manipulations affect the morphology of the internal organs of embryos. Embryos of the
2nd group of 18 days of age have a heart mass of 7.6 and 5.7% more than peers from the 1st and
3rd groups, and liver mass is 13.0 and 19.3% less, respectively. the mass of the stomach is 4.4 and
20.8% less, respectively (P≥0.95). In group 2 embryos, the number of convoluted seminiferous
tubules in the field of view of the microscope is 1.7 and 1.5 times less compared to groups 1 and 2
(P≥0.95). Conclusions Genetic manipulations significantly reduce almost all morphological and
histological parameters in group 2 embryos and cause negative consequences in the development
of chicken embryos.

Keywords: GFP, chicken embryos, morphological changes, histological changes

Alabdallach AZ, Nikishov AA, Volkova NA, Vetokh AN, Rebouh NY, Semenova VI, Petrov AK,
Rystsova EO, Bolshakova MV, Krotova EA, Drukovsky SG, Norezzine A (2020) Histological and
morphometric characteristics of chicken embryos with different genotypes. Eurasia J Biosci 14:
719-725.

© 2020 Alabdallach et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.

INTRODUCTION sections of tissue from the brain, we could observe the

GFP is a ~ 27 kDa protein consisting of 238 amino
acids derived from crystalline jellyfish Aequorea victoria.
It has a wavelength of fluorescent radiation in the green
part of the visible spectrum (hence the name).
Fluorescent proteins are widely used as markers for the
positive identification and tracking of expression cells in
many in vitro and in vivo studies. Due to its intrinsic
fluorescence, GFP is commonly used as a molecular
label for studies of intracellular protein trade.
In the GFP study, expression was limited to the
peripheral and central nervous system (CNS). In
expression of GFP in the cell bodies of neurons, axons,
and dendrites. In particular, the dendritic fan and catfish
of Purkinje cells of the cerebellum and axons of
projection cells in the hippocampus were brightly
fluorescent (Vokova et al. 2018, Ribeiro et al. 2001). In
another study, when a lentiviral vector carrying the CMV
promoter was used to produce transgenic chickens,
transgene expression was mainly in the pancreas and to
Received: September 2019
Accepted: February 2020
Printed: March 2020

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EurAsian Journal of BioSciences 14: 719-725 (2020)
Table 1. Study design
Group Genotype The number of eggs,
pieces
Stage I – pre-incubation assessment of eggs
1 Normal (Н) 20
2 Transgenic (G) 20
3 Local (НхG) 20
II stage – egg incubation
1 Normal (Н) 30
2 Transgenic (G) 30
3 Local (НхG) 30
a lesser extent in the liver, skin, muscles and intestinal
lining (McGrew et al. 2004). In another study, GFP was
strongly expressed in tissue samples of the heart,
skeletal muscle, and the entire pancreas. As a rule, GFP
expression decreased in strength and frequency along
with organ development. Embryos 14E and 17E showed
strong fluorescence in tissue samples from the brain,
lungs, and liver. This fluorescence continued until birth,
but the number of fluorescent cells gradually decreased.
In adults, some brain neurons had strong GFP
expression, but only a weak expression in other cells.
The lungs and liver also showed little expression of GFP
(Koichi Takeuchi et al. 2003). This GFP was observed
only in the oviducts, without detection in the heart,
muscles, liver and intestines. This is the first study
reporting the expression of tPA in epithelial cells of egg
white and oviduct using an oviduct specific vector. GFP
was expressed only in the tissues of the oviduct, and
none of them were found in the heart, skeletal muscle,
liver and intestines. (Hubdar Ali Kaleri et al. 2011). GFP
expression has been found to be observed in the tissues
of the oviduct, but not in the heart, muscles, liver, or
intestines (Vokova et al. 2018, Scott and Carlos 2005(.
Some researchers have found the effect of the GFP
gene on morphological changes on the chicken embryo
(Agedah et al. 2015, Vetokh et al. 2018).
The aim of this work is to study the effect of genetic
engineering manipulations on the histological and
morphometric parameters of organs and tissues of
chicken embryos.
To achieve this goal, the following tasks were
performed:
- Assess the incubation quality of eggs of different
genetic origin;
- To study the morphological parameters of chicken
embryos of different genetic origin during the incubation
process;
- To study the morphological changes of some
internal organs (heart - liver - stomach - trachea) of
embryos of different genetic origin during the incubation
process;
- To study the histological changes of some internal
organs (heart - liver - stomach - trachea) of embryos of
different genetic origin during the incubation process.
720
Alabdallach et al.
Embryos Studied
age, days quantity, pieces
Exterior and interior characteristics of
the egg
7 10 Morphometric and histological
10 10 parameters of organs and tissues
14 10
18 10
MATERIAL AND RESEARCH METHODS
The research material was eggs obtained from
transgenic and non-transgenic chickens of the Shaver
Brown cross. The selection of eggs from chickens was
carried out taking into account generally accepted
methods for hatching eggs. The shelf life of the selected
eggs was no more than 3 days from the time of
laying.(Belay et al 2018)
The studies were carried out according to the
following scheme (Table 1).
The mass of the egg and its components was
determined on an HR-200 balance with an accuracy of
0.001 g, large and small diameters, and also the shape
index of the egg — using an IM-1 index meter.
After opening the eggs, quality indicators were
determined, such as:
• the height of the protein and yolk - using a spider
micrometer and a leveling table;
• diameter of protein and yolk - with a DIGITAL
CALIPEP ITE 1303 vernier caliper, with a
measurement accuracy of 0.1 mm;
Using the formulas were determined:
1. protein quality:
a) in units of Hough (according to the formula Hough
units = 100 log (H - 1.7 W0.37 + 7.57), where H is the
height of the dense protein, mm and W is the egg mass,
g,
b) the calculation of the indicator “protein index”
according to the formula:
where H is the height of the dense protein, mm; D is
the diameter of the dense protein, mm;
2. the quality of the yolk - by calculating the index
“yolk index” according to the formula:
where h is the height of the dense yolk, mm; d is the
diameter of the dense yolk, mm
Egg incubation was performed in a Suro 20
incubator.
The study was conducted on chicken embryos at the
age of 7-10-14-18 days of incubation.
Embryos were taken at 7, 10, 14, and 18 dyn
incubation, and the samples were fixed in Buen’s
solution. Histological preparation was prepared
according to the generally accepted method. histological
EurAsian Journal of BioSciences 14: 719-725 (2020) Alabdallach et al.

Table 2. Morphometric indicators of hatching eggs

Group
Indicator 1 2 3
M±m M±m M±m
Egg weight, g 63,9 ± 0,46 64,2 ± 0,78 65,4 ± 0,92
Big diameter (DB), mm 56,6 ± 0,53 56,9 ± 0,62 57,8 ± 0,52
Small diameter (MD), mm 43,0 ± 0,37 43,1 ± 0,59 44,3 ± 0,42
Form index 76,0 ± 0,28 75,8 ± 0,58 75,9 ± 0,42

Table 3. Interior Indices of the Quality of Hatching Eggs of Chickens

Group
Indicator 1 2 3 Norm (not less)
M±m M±m M±m
Hough units 84,9±1,21* 80,1±2,02 83,7±1,67* 80
Protein index 0,09±0,01 0,08±0,003 0,09±0,02 0,07
Yolk index 0,61±0,06 0,47±0,08 0,58±0,07 0,43
The ratio of the mass of protein to the mass of yolk 2,01 2,09 2,03 1,9
Note: here in after * - P≥0.95

Table 4. Dynamics of the mass of embryos, g

Group
Day incubation 1 2 3
M±m M±m M±m
7 0,58±0,057 0,62±0,052 0,66±0,032
10 1,66±0,129 1,76±0,121 1,99±0,426
14 8,91±0,775* 7,50±0,489 10,11±0,445*
18 21,78±0,355* 19,19±2,529 22,53±1,444

Table 5. Dynamics of body length of embryos, g

Group
Day incubation 1 2 3
M±m M±m M±m
7 25,45±0,561 26,31±0,355 26,07±0,442
10 36,95±1,575 36,13±0,762 37,60±0,090
14 59,48±2,191* 56,15±1,984 60,83±1,087*
18 90,04±0,789* 84,83±1,737 91,73±3,530*

Table 6. Dynamics of the body width of the embryos, g

Group
Day incubation 1 2 3
M±m M±m M±m
7 5,68±0,482 6,5±0,448 6,56±0,357
10 9,32±0,095 9,12±0,296 10,25±0,936
14 14,58±0,401* 12,89±0,532 15,10±0,395*
18 17,08±0,473 17,17±0,586 17,60±0,503

sections were obtained on a rotary microtome. The
resulting histological sections were stained with
hematoxylin-eosin. linear dimensions were determined
on a MBP-1C microscope using PC software (Celecron
HD) HD Digital Microsopelmager. Statistical processing
of digital material was performed using the updated
methods of the variational package of data analysis
Microsoft Excel 2010.
RESEARCH RESULTS
Morphometric Assessment of Hatching Eggs
As can be seen from the results of the preliminary
assessment of the geometric indicators of the hatching
eggs, the eggs of all groups in their morphometric
characteristics corresponded to the standard indicators
recommended by the industry standard and that we also
saw three groups very converging that were unreliable
to each other (Table 2).
Interior quality indicators of hatching eggs of
chickens. However, it should be noted that according to
the quality of the protein, expressed in units of Hough,
the eggs from transgenic chickens (group 2) had
statistically significantly lower values. Compared with
the eggs of the 1st and 3rd groups, the difference was
4.8 and 3.6 Hough units (Table 3).
Morphometric Assessment of Chicken
Embryos
When we moved on to the second stage, we studied
the morphological changes in the fetus.
Studying the weight and length of the fetus, we
observed in the group (1 and 3) large values of these
indicators, than in group (2) the difference is significant
and the head length is close to three groups (reliable)
with regard to the width of the head and the length of the
leg, it will be in group (3) is larger than group (1-2) on
day 18 (Table 4).
Also on day 18, the trunk length and wing length in
group (3) are greater than in group (1-2) (Table 5).

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EurAsian Journal of BioSciences 14: 719-725 (2020) Alabdallach et al.

Table 7. Morphometric indicators of chicken embryos, mm

Day incubation
Group
7 10 14 18
Head length
1 9,68±0,069 12,13±0,369 15,13±0,673 18,28±0,317
2 10,33±0,284 12,34±0,536 13,94±0,521 17,55±0,155
3 9,88±0,293 11,82±0,239 14,89±0,092 18,60±0,385
Head width
1 11,59±0,368 15,53±0,690 25,23±0,775 33,80±0,420*
2 12,43±0,457 15,67±0,542 23,05±0,434 29,52±0,589
3 11,78±0,509 15,33±0,307 26,1±0,248 32,68±1,270*
Torso length
1 11,56±0,665 17,25±0,625 33,48±1,016 48,86±0,507
2 11,47±0,560 17,40±0,645 29,71±1,155 49,38±0,737
3 11,91±0,367 19,12±0,254 33,4±0,907 53,45±1,758*
Leg length
1 5,26±0,529 16,56±1,109 38,72±0,746* 61,83±1,427*
2 6,03±0,600 15,24±0,676 34,31±0,967 52,77±1,065
3 6,27±0,920 15,73±0,363 37,92±0,571* 62,93±2,231*
Wing length
1 4,35±0,240 11,6±0,852 22,16±0,575 31,84±0,953
2 4,52±0,455 10,39±0,452 20,10±0,366 29,14±1,385
3 4,89±0,399 11,74±0,455 22,67±0,143 33,85±0,562*

Table 8. Dynamics of heart mass of embryos, g

Group
Day incubation 1 2 3
M±m M±m M±m
10 0,021±0,0031 0,029±0,0070 0,024±0,0021
14 0,075±0,0081 0,083±0,0125 0,098±0,0101*
18 0,172±0,0111 0,185±0,0108* 0,175±0,0084

Table 9. Dynamics of liver mass of embryos, g

Group
Day incubation 1 2 3
M±m M±m M±m
10 0,015 ±0,002 0,018 ±0,002* 0,021 ±0,001*
14 0,160 ±0,027 0,149 ±0,014 0,203 ±0,010*
18 0,478 ±0,041 0,423 ±0,032 0,505 ±0,036*

By 14 days of incubation, the maximum differences
in body width between the 1st, 3rd and 2nd groups are
observed. In transgenic animals, during this period, the
trunk is significantly narrower compared to the 1st and
2nd groups (Table 6).
The measurement results of individual parts of the
body are presented in Table 7.
Differences in embryo development during the first
half of incubation were not observed. Up to 14 days of
age, for all morphological parameters, the difference
was statistically unreliable. According to the length of the
body, the difference ranged from 0.3 to 5.6%, the length
of the head from 0.25 to 5.7%, and the length of the neck
from 0.35 to 5.8%. The lowest length for all parameters
was recorded in group 2 embryos.
From 7 to 14 days of incubation, the length of the
body increased on average 2.4 times, and the width 8.1
times. From 7 to 10 days of incubation, the length
increased on average by 30.7%, and the width - by
32.4%.
In the period from 14 to 18 days of incubation for
almost all morphological parameters, the difference was
statistically significant. Over the length of the body, the
difference ranged from 1.1 to 8.2%, head length from
4.25 to 4.7%, leg length from 17.1 to 18.8%, wing length
from 9.2 to 16.1% . The lowest length for all parameters
was recorded in group 2 embryos, with the exception of
the trunk length.
Analyzing the dynamics of the heart mass of the
embryos during the period from 7 to 14 days, it should
be noted that by the end of this period the largest mass
was observed in the 3rd group, and by the 18th day - in
the 2nd group (Table 8).
It was found that for the period from 10 to 18 days of
incubation in the 1st group, the liver mass increased by
31.7 times, in the 2nd group by 23.6 times and in the 3rd
group by 24.0 times. That is, the intensity of the increase
in liver mass in embryos in the 1st group (normal) was
on average 1.3 times greater than in groups with a
modified genotype (Table 9).
Analysis of the dynamics of growth of the stomach
showed that in the 1st group, the mass of the stomach
increased by 37.0 times, in the 2nd group by 44.8 times
and in the 3rd group by 31.1 times. That is, the intensity
of the increase in the mass of the stomach in embryos
in the 2nd group (transgenic) was on average 1.25 times
higher than in other groups (Table 10).
Histological assessment of organs and tissues. It
was interesting to study whether there are differences in
the structure of individual organs in embryos with
different genetic origins.

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EurAsian Journal of BioSciences 14: 719-725 (2020) Alabdallach et al.

Table 10. The dynamics of the mass of the stomach of the embryos, g
Group
Day incubation 1 2 3
M±m M±m M±m
10 0,026±0,0031 0,020±0,0051 0,036±0,0053
14 0,23±0,028 0,22±0,070 0,32±0,060
18 0,951±0,018* 0,911±0,036 1,101±0,033*

Table 11. The results of the measurement of the tracheal wall, microns
Group
Name of indicator Cut type 1 2 3
M±m M±m M±m
transverse 0,453±0,024* 0,353±0,019 0,415±0,054*
Slime layer
longitudinal 2,586±0,031 2,810±0,035* 2,023±0,033
transverse 3,483±0,052* 2,550±0,028 3,365±0,099*
Submucosal layer
longitudinal 3,326±0,019* 2,640±0,027 3,040±0,040
transverse 1,593±0,035 1,800±0,033 4,090±0,076*
Muscle layer
longitudinal 2,170±0,054 1,403±0,036 3,510±0,061*
transverse 0,196±0,047* 0,096±0,053 0,220±0,014*
Serous layer
longitudinal 0,216±0,031 0,230±0,027 0,180±0,022

Table 12. The results of the measurement of the wall of the stomach, microns
Group 1 (genotype Н)
Age, days
Name of indicator Type of stomach
7 10 14 18
M±m M±m M±m M±m
muscular 1,65±0,45 2,23±0,37 1,027±0,12
Slime layer
glandular 2,46±0,56 2,153±0,54
muscular 2,34±0,69 2,736±0,44 3,44±0,39
Submucosal layer
glandular 2,806±0,52 1,399±0,51
muscular 3,166±0,78 4,376±0,37 5,032±0,61
Muscle layer
glandular 1,176±0,22 2,186±0,33
muscular 0,21±0,02 0,336±0,056 0,28±0,024
Serous layer
glandular 0,328±0,042 0,14±0,009

Table 13. The results of the measurement of the wall of the stomach, microns
Group 2 (genotype G)
Age, days
Name of indicator Type of stomach
7 10 14 18
M±m M±m M±m M±m
muscular 0,90±0,07 1,766±0,34 1,876±0,528 3,80±0,915
Slime layer
glandular 1,612±0,32 1,626±0,563
muscular 2,29±0,68 2,383±0,61 2,105±0,641 2,85±0,545
Submucosal layer
glandular 1,316±0,43 7,836±0,661
muscular 0,553±0,051 3,613±0,38 4,842±0,475 4,766±0,387
Muscle layer
glandular 1,786±0,25 0,96±0,0714
muscular 0,066±0,002 0,293±0,081 0,266±0,035 0,16±0,080
Serous layer
glandular 0,132±0,012 0,203±0,014

Table 14. The results of the measurement of the wall of the stomach, microns
Group 3 (genotype НхG)
Age, days
Name of indicator Type of stomach 10
7 14 18
(measurements not taken)
M±m M±m M±m M±m
muscular 1,56±0,61 1,46 ±0,66 2,26±0,52
Slime layer
glandular 1,47±0,39 1,62±0,45 1,607±0,42
muscular 2,736±0,52 2,576±0,63 2,19±0,71
Submucosal layer
glandular 2,92±0,34 7,195±0,29 7,785±0,88
muscular 1,66±0,33 6,506±0,87 2,29±0,61
Muscle layer
glandular 2,82±0,24 1,65±0,33 0,992±0,43
muscular 0,183±0,035 0,203±0,08 0,1±0,01
Serous layer
glandular 0,21±0,04 0,22±0,016 0,172±0,02

Table 11 presents the results of histological studies
of the trachea of the embryos at 10 days of age.
The measurement results show that for all indicators
(except for the longitudinal section of the mucous layer),
tracheal samples taken from group 2 embryos were
smaller in their absolute values. The difference ranged
from 5.6-28.4%.
Tables 12-14 show the results of histological studies
of the gastric wall of the embryos at 10 days of age. On
histological sections studied the structure of the mucous,
submucous, muscle and serous layers.
A tendency toward an increase in the thickness of the
submucosal and muscular layers and, conversely, a
decrease in the thickness of the mucous and serous

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Fig. 1. Histological section of the muscular stomach in a 14- Fig. 3. Histological section of the neural tube in the embryo
day-old embryo 14 days old 14 days old (group 1)

Fig. 2. Histological section of the glandular stomach in the Fig. 4. Histological section of convoluted seminiferous
embryo 14 days old tubules in an embryo of 14 days old (group 1)

layers during the period from the 7th to the 14th day of
incubation in the stomachs of both types was noted
(Figs. 1 and 2).
When histological analysis of the formation of the
neural tube, significant differences in groups were not
found (Fig. 3).
The external signs show that the embryo of group 2
shows a significantly smaller number of convoluted
tubules in the field of view of the microscope.
And at the same time, there is a significant
predominance of parenchymal tissue.
CONCLUSIONS
Based on the research results, the following
conclusions can be drawn:
1. Hatching eggs of the studied genotypes
corresponded to regulatory requirements
morphometric indicators of egg quality.
2. Eggs from transgenic chickens (2nd group) had
statistically significantly lower values of the quality index
of dense protein - Hough units. Compared with the eggs
of the 1st and 3rd groups, the difference was 4.8 and 3.6
Hough units, respectively (P≥0.95).
3. On the 14th day of incubation, the embryos of the
1st and 3rd groups statistically significantly exceeded
the mass of the 2nd group embryos by 17.4% and
13.5%, respectively (P≥0.95). By the 18th day,
differences in the mass of embryos persist.
4. Embryos from eggs from genetically modified
chickens (group 2) on the 18th day of incubation had the
smallest absolute values according to linear indices of
individual parts of the body.
5. Genetic engineering manipulations affect the
morphology of the internal organs of embryos. Embryos
of the 2nd group of 18 days of age have a heart mass of
7.6 and 5.7% more than peers from the 1st and 3rd
groups, and liver mass is 13.0 and 19.3% less,
for respectively. the mass of the stomach is 4.4 and 20.8%
less, respectively (P≥0.95).
6. In group 2 embryos, the number of convoluted
seminiferous tubules in the field of view of the
microscope is 1.7 and 1.5 times less compared to
groups 1 and 2 (P≥0.95).

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