II. The effect of strain and IGF-1 gene transfer using SMGT techniques on some semen characteristics:-

Transcription

1 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 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

2 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,

3 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 %, 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, and % in Mandarah and were 50.20, 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 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 % and lowest was % 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 % and lowest was % 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 % and lowest (the best) were % 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

5 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 ( and 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, , and g in Mandarah and 30.28, , , and 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, , and g) than Silver Montazah (34.48, and g) however Silver Montazah was higher in BW at 8 weeks ( g) in than Mandarah strain ( 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 g appeared with 10 μg IGF-1 gene dose and lowest average was 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,

6 body weight at 8 weeks, body weight at 12 weeks and body weight at 16 weeks. The highest body weight at 16 weeks was g appeared with 10 μg IGF-1 gene dose with Mandarah strain and g appeared with 15 μg IGF-1 gene dose with Silver Montazah compared to lowest average was 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

7 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 % in Mandarah and % 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.

8 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 % 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, 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

9 effect (P<0.05) on GH and TSH. The highest level of IGF-1 and GH were ng/ml and ng/ml appeared in 5 μg IGF-1 gene dose with Silver Montazah and the highest level of insulin and TSH were 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

10 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.