الفهرس 
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Abstract
The experimental work of this study was carried out in poultry farm of the Department of Animal Production, Faculty of Agriculture, Benha University, Egypt with cooperation of Genetic Engineering and Biotechnology Research Institute (GEBRI), University of Sadat City and Life Science Institute University of Michigan, USA from 2013 to 2014.The present work was carried out to study:I. Applicability of the modern biological technology for molecular genetics and genetic engineering to isolate, cloning and sequencing of chicken IGF-1 gene:-IGF-1 gene isolation and cloning from chicken (cobb 500 broilers) as a high producing exotic strain in growth traits and sequencing analysis compared to sequence reference which published in ncbi to transfer it to some local chicken strains.II. The effect of strain and IGF-1 gene transfer using SMGT techniques on some semen characteristics:A total of 400 hens and 96 cock from Mandarah and Silver Montazah strains (200 hen and 48 cock in each strain) were used in IGF-1 gene transfer by zero, 5, 10 and 15 micro gram dose.Semen traits (Individual motility, live sperm and abnormal sperm percentage) were studied in different groups. Then, this semen was used in artificial insemination and egg collected with different groups to incubation.III. The effect of strain and IGF-1 gene transfer using SMGT
techniques on incubation and after incubation traits:Fertility, hatchability, early and late embryonic mortality and abnormal chicks percentage were studied in different incubation
groups. A total of 2000 chicks were produced from incubation
program and growth traits, carcass traits, hormonal profile,
histological study in ileum and thyroid gland and rate of calcium
absorption studied in pure and transgenic chicks.
Results obtained could be summarized as follow:
I. Applicability of the modern biological technology for
molecular genetics and genetic engineering to isolation,
cloning and sequencing of chicken IGF-1 gene:-
1. Total RNA was extracted from the chicken cobb-500 liver
samples which was the active part in the total DNA in
somatic cell.
2. RNA was reverse transcribed into first strand cDNA to
complete other steps for IGF-1 gene isolation.
3. Specific primers which complementarities with sequence IGF-1
gene and plasmid structure were used to amplify fragment
from total cDNA.
4. Amplification of cIGF-1 from cDNA using specific primers by
polymerase chain reaction (PCR).
5. cIGF-1 product was cloned with pCR3.1 vector between Hind
III and EcoR1 in restriction site and insertion IGF-1 gene into
plasmid mapping using legation enzyme.
6. Plasmid containing IGF-1 gene transformed into E.Coli
bacterial colony as a template to increase amount of
modification plasmid.
7. The transformed cells were grown and plasmid extraction from
E.Coli bacterial colony using Miniprep plasmid.
8. The recombinant plasmid (pCR3.1-cIGF-1) was sequenced
using T7 common sequencing by the Sanger sequencing
method at DNA sequencing core (life science institute, University of
Michigan, USA, http://seqcore.brcf.med.umich.edu) and amino
acid sequences translated from the cDNA sequence were
compared with sequences in the Gen Bank public database, by
using Bioedit software v7.1 which obtained that plasmid
containing chicken IGF-1 gene.
II. The effect of strain and IGF-1 gene transfer using SMGT
techniques on some semen characteristics:-
1. Before semen incubation, the effect of strain was highly
significant (P<0.001) differences on percentage of individual
motility, live sperm and sperm abnormalities and semen of
Mandarah strain was higher in percentage of individual
motility and percentage of live sperm (56.62 and 79.80 %,
respectively) than Silver Montazah strain (50.20 and 75.30%,
respectively). However Silver Montazah was the best strain in
percentage of sperm abnormalities (8.97 %) when compared
with Mandarah strain (10.42%).
2. After semen incubation using SMGT techniques, the effects of
strain was non significant on percentage of individual motility
and live sperm, however it was found to be highly significant
(P<0.001) on percentage of sperm abnormalities. The average
of percentage of individual motility, live sperm and percentage
of sperm abnormalities were 31.03, 68.28 and 17.13 % in
Mandarah and were 50.20, 75.30 and 8.97 % in Silver
Montazah, respectively.
3. Effect of IGF-1 gene doses were highly significant (P<0.001)
on percentage of individual motility, live sperm and sperm
abnormalities and zero dose was highest average for
percentage of both individual motility and live sperm and the
lowest average of percentage of sperm abnormalities when
compared with other doses.
4. Interaction between strains and IGF-1 gene doses were
significant differences (P<0.05) effect on percentage of
individual motility, live sperm and sperm abnormalities. The
highest average was 53.25 % and lowest was 21.25 % appeared
with Silver Montazah in zero dose and Mandarah in 15 μg
dose of IGF-1 gene, respectively. The highest average of
percentage of live sperm was 85.75 % and lowest was 56.35 %
appeared with Silver Montazah in zero dose and Silver
Montazah in 10 μg dose of IGF-1, respectively. The highest
averages of abnormal sperm% were 19.37 % and lowest (the
best) were 10.90 % appeared with Mandarah in 10 dose and
Mandarah in zero dose, respectively.
5. Concerning groups that were treated by IGF-1gene doses, 15μg
was best dose affected in percentage of live sperm and
percentage of sperm abnormalities with Mandarah strain
however, 5μg was best dose affected in all semen traits
(percentage of individual sperm, live sperm and sperm
abnormalities) with Silver Montazah strain, respectively.
III. The effect of strain and IGF-1 gene transfer using SMGT
techniques on incubation and after incubation traits:-
1. In natural mating before using SMGT techniques, the effect of
strain was non significant on percentage of fertility,
hatchability, early and late embryonic mortality and abnormal
chicks.
2. After semen incubation using SMGT techniques, the effects of
strain was significant effect (P<0.05) on the percentage of
fertility and late embryonic mortality however it was non
significant effect on hatchability, early embryonic mortality
and abnormal chicks.
3. IGF-1 gene doses were significant effect (P<0.05) on the
percentage of fertility, hatchability, abnormal chicks, late
embryonic mortality and abnormal chicks and it was non
significant effect on the percentage early embryonic mortality.
4. Interaction between strains and IGF-1 gene doses were
significant differences (P<0.05) effect on percentage of
fertility, hatchability, late embryonic mortality and abnormal
chicks however it was non significant effect on percentage of
early embryonic mortality.
5. In natural mating before using SMGT techniques, the effect of
strain was non significant difference on body weight BW at 4
weeks (163.08 and 154.66 g) in Mandarah and Silver
Montazah, respectively. However, strain affected significantly
(P< 0.001) on BW at hatch, BW at 8, BW at 12 and BW at 16
weeks of age which recorded 32.94, 633.43, 744.62 and
1109.33 g in Mandarah and 30.28, 680.07, 851.67, and
1169.98 g in Silver Montazah, respectively.
6. In artificial insemination using SMGT techniques, the effect of
strains were highly significant effect (P<0.01) on the body
weight at hatch, body weight at 4 weeks, body weight at 12 and
body weight at 16 weeks and Mandarah was higher strain
(35.12, 175.15, 900.56 and 1339.01 g) than Silver Montazah
(34.48, 165.21 886.05 and 1246.79 g) however Silver
Montazah was higher in BW at 8 weeks (735.42 g) in than
Mandarah strain (728.48 g).
7. IGF-1 doses were highly significant effect (P<0.001) on the
body weight at hatch, body weight at 4 weeks, body weight at
8 weeks, body weight at 12 weeks and body weight at 16
weeks. The highest body weight at 16 weeks was 1339.34 g
appeared with 10 μg IGF-1 gene dose and lowest average was
1196.32 g appeared with zero dose.
8. Interaction between strains and IGF-1 doses were highly
significant effect (P<0.001) on body weight at hatch, body
weight at 4 weeks, body weight at 8 weeks, body weight at 12
weeks and body weight at 16 weeks. The highest body weight
at 16 weeks was 1429.36 g appeared with 10 μg IGF-1 gene
dose with Mandarah strain and 1304.96 g appeared with 15 μg
IGF-1 gene dose with Silver Montazah compared to lowest
average was 1208.64 g appeared in zero dose with Mandarah
strain, respectively.
9. In natural mating before using SMGT techniques, the effect of
strain was non significant on daily gain (g/day) on daily gain at
hatch to 4 weeks (DG 0-4). However showed highly significant
effect (P<0.001) due to strain on DG4-8, DG8-12, DG12-16
and DG0-16 weeks of age.
10. In artificial insemination using SMGT techniques, the effect of
strains were highly significant effect (P<0.001) on the daily
gain (DG) from hatch to 4 weeks, from 4 to 8 weeks, from 8 to
12 weeks, from 12 to 16 weeks and from hatch to 16 weeks
(g/day) of age.
11. IGF-1 gene doses were highly significant effect (P<0.001) on
DG from hatch to 4, DG from 4 to 8, DG from 8 to 12, DG
from 12 to 16, DG from hatch to 16 weeks of age.
12. Interaction between strains and IGF-1 gene doses were highly
significant effect (P<0.001) on DG from 4 to 8, DG from 8 to
12, DG from 12 to 16, DG from hatch to 16 weeks of age and
significant effect (P<0.05) on DG from hatch to 4 weeks of
age. 10μg IGF-1 gene was best dose in DG from hatch to 16
weeks with Mandarah (11.61 g/day) and 15μg IGF-1 gene with
Silver Montazah strain (10.56 g/day) compared to zero dose
(9.61 g/day), respectively.
13. In natural mating before using SMGT techniques, the effect of
strain was non significant differences on percentage of growth
rate GR from 12 to 16 and GR from hatch to 16 weeks,
however, it was found significant effect (P<0.05) on
percentage of growth rate from hatch to 4 weeks of age and
highly significant effect (P<0.001) on GR from 4 to 8 and GR
from 8 to 12 weeks. GR from hatch to 16 weeks was 195.70%
in Mandarah and 197.08% in Silver Montazah strain,
respectively.
14. In artificial insemination using SMGT techniques, the effect of
strain was highly significant effect (P<0.001) on growth rate %
(GR) from hatch to 4, GR from 4 to 8, GR from 8 to 12, GR
from 12 to 16 and GR from hatch to 16 weeks of age.
15. IGF-1 gene doses were highly significant effect (P<0.001) on
growth rate % (GR) from hatch to 4, GR from 4 to 8, GR from
8 to 12, GR from 12 to 16 and GR from hatch to 16 weeks of
age.
16. Interaction between strains and IGF-1 gene doses were highly
significant effect (P<0.001) on growth rate % (GR) from hatch
to 4, GR from 4 to 8, GR from 8 to 12, GR from 12 to 16 and
GR from hatch to 16 weeks of age. 10μg IGF-1 gene was best
dose in GR from hatch to 16 weeks with Mandarah (207.88%)
and 15μg IGF-1 gene with Silver Montazah strain (201.54%)
compared to zero dose (194.81%), respectively.
17. In natural mating before using SMGT techniques, effect of
strain was non significant differences on carcass traits
(percentage of dressing, giblets, total edible parts and
abdominal fat).
18. In artificial insemination using SMGT techniques, the effect of
strain was significant (P<0.05) on percentage of abdominal fat.
However, strain showed non significant effect on percentage of
dressing, giblets and total edible parts.
19. IGF-1 gene doses were non significant differences on
percentage of abdominal fat. However, it were highly
significant effect (P<0.001) on percentage of dressing, giblets
and total edible part.
20. Interaction between strains and IGF-1 gene doses was non
significant effect on percentage of abdominal fat however, it
was significant effect (P<0.05) on percentage of dressing,
giblets and total edible part. The highest percentage of
dressing, giblets and total edible part were 72.61, 4.96 and
77.57 % appeared in 10 μg dose with Mandarah strain,
respectively. On the other hand, the lowest percentage of
dressing (63.56%) appeared in zero dose with Silver Montazah
strain while the lowest percentage of giblets and total edible
parts (3.56 and 68.78%, respectively) showed in zero dose with
Mandarah strain. Also abdominal fat showed the best
percentage (0.48%) appeared in zero dose with Mandarah
strain.
21. In natural mating before using SMGT techniques, strain
showed non significant effect on serum level of IGF-1, GH,
insulin and TSH and it was 7.00 ng/ml, 9.66 ng/ml, 9.33,
mg/ml and 0.48 ng/ml in Mandarah strain respectively, while it
was 7.33 ng/ml, 8.66, mg/ml, 11.00 ng/ml and 0.80 ng/ml in
Silver Montazah strain, respectively.
22. In artificial insemination using SMGT techniques, strain
showed non significant effect on serum level of IGF-1, insulin
and TSH however, it was significant effect (P<0.05) on serum
level of GH.
23. IGF-1 gene doses were significant effect (P<0.05) on serum
level of IGF-1, GH and TSH however, it was non significant
effect on level of insulin.
24. Interaction between strains and IGF-1 gene doses was non
significant effect on serum level of IGF-1 and insulin however,
it was significant effect (P<0.05) on GH and TSH. The highest
level of IGF-1 and GH were 12.00 ng/ml and 15.08 ng/ml
appeared in 5 μg IGF-1 gene dose with Silver Montazah and
the highest level of insulin and TSH were 15.66 ng/ml and 2.46
ng/ml appeared in 10 μg dose with Silver Montazah and 15 μg
dose with Mandarah strain, respectively. The lowest level of
IGF-1 (7.66 ng/ml), insulin (10.67 ng/ml) and TSH (1.04
ng/ml) appeared in zero dose with Mandarah and the lowest
level of GH (9.33 ng/ml) appeared in 10 μg IGF-1 gene dose
with Mandarah strain.
25.In natural mating, section of ileum in pure strains (Mandarah
and Silver Montazah ) showing thickness of mucosa layer and
villi and crypt of liberkuhn was thin and relative by columnar
epithelium tissue containing few of goblet cells, aggregation of
small lymphatic cells and increasing thickness of muscular
layer.
26.In artificial insemination using SMGT techniques, section of
ileum in transgenic strain by IGF-1 gene (transgenic Mandarah and Silver Montazah strains) showing higher thickness of mucosa layer and increasing of number goblet cells rather than columnar cells and higher profilation epithelial and lymphatic cell and decreasing thickness of muscular layer than pure strain.27.In natural mating, section of thyroid gland in pure strains Mandarah and Silver Montazah) showing parenchyma formed of follicles which are filled of colloid and lined by flat follicular cells with some cubical cells. In addition, diameter of follicular, heights of follicular epithelium and activity of colloid were decreasing case. 28.In artificial insemination using SMGT techniques, section of thyroid gland in transgenic strain by IGF-1 gene (transgenic Mandarah and Silver Montazah strains) showing reached in thyroid follicales lined by cubical cells and appearance of empty space in colloid and some follicales have no colloid due to dissolving. In addition, diameter of follicular, heights of follicular epithelium and activity of colloid were increasing case.29. Improving case of calcium absorption per centimeters and dry matter/hour for duodenum, jejunum and ileum in treated groups than control groups.In conclusion, chicken IGF-1 gene was effected in all physiological traits and productive performance. The best dose and strain was 5 μg IGF-1 gene dose with Mandarah in individual sperm motility, fertility and embryonic mortality. In addition Mandarah strain and 10 μg IGF-1 was best in body weight, daily gain, growth rate, rate of calcium absorption and carcass traits than other studied doses. Molecular genetics and SMGT techniques were advanced applications to improve the physiological and productive performance.