الفهرس 
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Abstract
SUMMARY AND RECOMENDATIONS
The present study was carried out in Genetics Department, Faculty of Agriculture, Fayoum University, Egypt, during the period from 2011 to 2016. Twenty four Saidi ewe producing twin and thirteen Ossimi twins producing ewes were tested. They were carefully selected from the records of the Egyptian Agricultural Research Centre. The aim of any animal breeder is to get maximum possible profit from the domestic livestock. This can be achieved by improving the genetic potential by appropriate selection methods. The selection aimed at increasing fecundity will ultimately improve the reproduction rate and production efficiency in small ruminants. But selection for reproduction traits is usually difficult to achieve due to their low heritability values, so alternative selection method (Marker Assisted selection) with higher accuracy in prediction of breeding value can be adopted for these traits. Therefore this study was carried out to investigate a number of genes (GDF9 and BMP15) that can act as marker influencing fertility in local Egyptian sheep breed (Saidi and Ossimi).
The results of the present study are summarized as follows:
Reproduction traits:
The production and fertility traits of 191 and 145 individuals of sheep breeds with different families from Saidi (black, brown and white) and Ossimi sheep respectively are studied and the data showed that:
1. There are no significant differences in the average number of ewes pregnant between Saidi and Ossimi breed sheep.
2. There are no significant differences between Saidi black and brown in the average number of ewes lambing single, ewes lambing twin, ewes lambing triplet and live lambs born.
3. There are significant differences between Saidi white and Ossimi sheep in the average number of ewes lambing single, ewes lambing twin, ewes lambing triplet and live lambs born.
4. The average number of dead lambs born and the average number of total lambs birth for Saidi black, Saidi brown and Ossimi sheep were significantly higher compared with Saidi white sheep.
5. The higher average number of dead lambs born (10) was found in Saidi black sheep and the fewer average number (1.58) was found in Saidi white sheep.
6. The Saidi sheep (black, brown and white) had average number of ewes lambing triplet of 0.67, 0.33 and 0.17, respectively, while there were no ewes lambing triplet in Ossimi sheep.
7. The litter size, twinning rate and triplet rate showed highly significant differences between Saidi and Ossimi sheep breeds, while, lambing rate and fecundity rate showed no significant differences.
8. The mean litter size of Saidi white sheep was significantly decreased compared to black and brown.
9. The litter size differs between and within Saidi and Ossimi sheep breeds.
Molecular studies of BMP15 gene, exon 2 (310 bp):
1. A single fragment of approximately 310 bp nucleotide sequences was amplified from each ewe individual (19 Saidi and 13 Ossimi) sheep breeds.
2. Alignments of four sequences from Saidi and Ossimi ewe that produce single or twins lamb revealed 100% similarity between them.
3. The nucleotide frequencies were 23% (A), 27% (T), 30% (C) and 20% (G).
4. The highest value of different transitional substitutions was 23.19 for substitution C with T nucleotides while, the highest value of transversion substitutions was 2.55 for substitution C with A or C with G nucleotides.
5. The topology of UPGMA tree of Saidi and Ossimi sheep breeds with 9 accession numbers of Ovis aries in the GenBank database represented a monophyletic group. The DNA sequences of BMP 15 gene successfully grouped Egyptian breeds and Ovis aries sheep into two main cluster. The first cluster is extremely diverse and consisted of two Ossimi breeds. The second cluster had two Saidi breeds and closely related with 9 accession numbers of Ovis aries.
PCR-RFLP analysis of BMP15 gene, exon 2 (310 bp):
1. The digested products by Pst1 or Msp1 of PCR product of BMP15 gene (exon 2) were run on 3% agarose gel electrophoresis, bands with 310 bp in length were observed in all 9 Saidi ewes and also in all 9 Ossimi ewes
2. Saidi and Ossimi ewes producing single and twins lamb had a single band at 310 bp position indicating absence of mutation in the FecX gene (FecX++).
3. It can be assumed that the cause of twinning in Saidi and Ossimi sheep breed might be due to the effect of other fecundity genes or may be a combination of gene products that stimulate/alter the ovulatory cycle.
Molecular studies of GDF9 (exon1) 462 bp:
1. A single fragment of approximately 462 bp nucleotide sequences was amplified from each ewe individual (24 Saidi and 13 Ossimi) sheep breeds.
2. Alignments of two sequences from Saidi or Ossimi ewe that produce single or twins lamb revealed 100% similarity between them.
3. The nucleotide frequencies are 21% (A), 28% (T), 28% C and 23% (G).
4. The highest value of different transitional substitutions was 25.43 for substitution C with T while, the highest value of transversion substitutions was 4.65 for substitution T with A or T with G.
5. The topology of UPGMA tree of Saidi and Ossimi sheep breeds with 6 accession numbers of Ovis aries in the GenBank database represented a monophyletic group. The DNA sequences of GDF9 (exon1) 462 bp gene successfully grouped Egyptian breeds and Ovis aries sheep into two main cluster. The first cluster is extremely diverse and consisted two Ossimi breeds. The second cluster had two Saidi sheep breeds and closely related with 5 accession numbers (HE866499, KT853039, KR063137, NM1142888 and FJ429111 whereas, the accession numbers (AF078545) were the most distant.
Molecular studies of GDF9 (exon1), 710 bp:
1. A single fragment of approximately 710 bp nucleotide sequences was amplified from each ewe individual (24 Saidi and 13 Ossimi) sheep breeds.
2. Alignments of two sequences from Saidi ewe that produce single or twins lamb revealed 99.86% similarity between them.
3. The base A in the sequence of Saidi ewes that produce single lamb was substituted by T in sequence of Saidi ewes that produce twins lamb.
4. Alignments of two sequences from Ossimi ewe that produce single or twins lamb revealed 100% similarity between them.
5. The nucleotide frequencies are 21% (A), 26% (T), 28% (C) and 25% (G).
6. The highest value of different transitional substitutions was 10.72 for substitution G with A nucleotides while, the highest value of transversion substitutions was 8.62 for substitution C with A or C with G nucleotides.
7. The topology of UPGMA tree of Saidi and Ossimi sheep breeds with 6 accession numbers of Ovis aries in the GenBank database represented a monophyletic group. The DNA sequences of GDF9 gene successfully grouped Egyptian breeds and Ovis aries sheep into two main cluster. The first cluster is extremely diverse and consisted of four accession numbers (KT853039, KR063137, NM1142888 and FJ429111). The second cluster had both Egyptian sheep breeds and closely related with one accession number (AF078545) whereas, accession number (HE866499) was the most distant.
PCR-RFLP analysis and genotyping of GDF9 (exon1):
1. Saidi and Ossimi sheep breeds were screened for Pst1 enzyme digestion where it revealed a monomorphic type of restriction pattern consisting two bands with 710 and 400 bp in Saidi or Ossimi sheep breeds.
2. Saidi and Ossimi sheep breeds were screened for Msp1 enzyme digestion where it revealed two types of restriction pattern:
- In Saidi sheep breed, polymorphic restriction pattern consisting one band with 710 bp for ewes producing single lamb and two bands with 710 bp and 600 bp for ewes producing twins lamb.
- In Ossimi sheep breed, monomorphic restriction pattern consisting one band with 710 bp were observed in ewe producing single or twins lamb.
3. The alleles (Fec+ and FecG) frequencies of GDF9 gene based on Msp1 restriction enzyme digestion in Saidi sheep breed are 0.73 and 0.27.
4. The alleles (Fec+ and FecG) frequencies of GDF9 gene based on Msp1 restriction enzyme digestion in Ossimi sheep breed are 1.0 and 0.0.
5. The Saidi sheep genotyps frequeinces Fec++, Fec+G and FecGG are 0.533, 0.394 and 0.073.
6. The Ossimi sheep genotyps frequeinces Fec++, Fec+G and FecGG are 1.0, 0.0 and 0.0.
The multiplex PCR of BMP15 (exon 2) and GDF9 (exon 1) genes:
1. The results of multiplex PCR showed that designed primers worked well and it was possible to trace each gene.
2. The fragments of 310 bp, 462 bp and 710 bp in Saidi and Ossimi sheep breeds were successfully amplified from the DNA of each sample (19 Saidi and 13 Ossimi) used in the present study.
3. Agarose gel electrophoresis of the multiplex PCR showed amplified 462, 710 and 310bp fragments from exon GDF9 (exon1) and BMP15 (exon2) genes respectively, of Saidi breed,
4. Agarose gel electrophoresis of the multiplex PCR amplified 462, 710 and 310bp fragments from exon GDF9 (exon1) and BMP15 (exon2) genes respectively, of Ossimi breed.
Recommendations
This study has highlighted the importance of molecular markers in selection programs of Egyptian livestock. Based on the results of this study, the following are recommended:
1. With the absence of FecX mutations in local Egyptian sheep breeds and polymorphisms, we cannot depend on BMP15 (exone 1) gene as a molecular marker for fecundity traits in Egyptian Sheep breeds.
2. The genetic factor affecting fecundity should be investigated further by other candidate gene of higher litter size.
3. Presence of polymorphism of the isolated GDF9 gene (exon 1) among Saidi ewes that produce single and twins lamb. Suggests that this marker should be used as molecular marker for twinning and it can be used as a test to distinguish the twin producing individuals among the local Egyptian sheep.
4. The present study also recommends future experiments directed to new mutants and its relations to fecundity in Egyptian sheep.