الفهرس 
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Abstract
Data for the present study were obtained from 1875 lactation records of Holstein cows, belonging to the Modern Agricultural Development Company (Dina farms), located about 82 km on Cairo-Alexandria Desert road, Egypt. Lactation records covered the period between 2013 and 2016. Blood samples were obtained from 38 cows (19 NA and 19 AF with CM). Data and laboratory analyses were performed at the Department of Animal Husbandry and Wealth Development. DNA sequencing was conducted at the laboratories of LGC Company, Germany. The aims of this work were to study: 1- the incidence and risk factors of clinical mastitis (CM) in Holstein cows, and its effect on productive and reproductive parameters. 2- The polymorphism of some TLR genes in Holstein cows, and its association with CM, milk somatic cell score (SCS), productive and reproductive parameters.
To achieve these aims, the following methodology was tried:
• Productive, reproductive parameters and previous history of mastitis were collected from the electronic herd records.
• Statistical analyses were performed for determining the incidence of CM and its impact on the productive and reproductive performance of Holstein cows in relation to some risk factors.
• Thirty-eight blood samples were collected for DNA isolation; 19 from Holstein cows without a previous lifetime history of mastitis (non-affected, NA) and 19 from Holstein cows with at least 3 previous episodes of mastitis (affected, AF).
• PCR was done for amplification of fragments of TLR2, TLR4, TLR6 and TLR9 with expected amplicon size of 501 bp, 682 bp, 515 bp, and 320 bp, respectively.
• Twenty four purified PCR products were sequenced: six products for each gene (three NA cows and three AF).
• The amplified DNA fragment was digested with selected restriction enzyme; Rsa1, MSP1, Hha1 and Taq1 for TLR2 gene; EcoR1, MSP1, Taq1, Hha1, HaeIII, and Rsa1 for TLR4 gene; MSP1, HaeIII, and Taq1for TLR6 gene; Rsa1, Hha1, and MSP1 for TLR9 gene.
• Statistical analyses were performed for testing the association of TLRs genes with milk production and fertility traits.
The obtained results are summarized in the following points:
• One third of cows (647/1875) had contracted at least one episode of CM throughout the entire lactation. Incidence of CM was high in low and moderate milk producing, pluriparous, and winter calving cows, and was more frequent during early lactation.
• Milk yield of affected cows averaged 301 kg/305 days lower than non-affected ones. CM adversely affected M305 of both low and high producing cows, in multiparous cows, and in those calved in summer months.
• CM significantly increased number of days to first estrus, days to first service, days open, and number of inseminations per conception. It was adversely affected the reproductive parameters of dairy cows in both seasons particularly winter calving and regardless of level of milk yield.
• The sequence analysis of TLR2 gene revealed three non-synonymous SNPs in affected cows compared to non-affected ones; 2102 T>G, 2105 T>G and 2107 C>T. The SNPs replacing 701 F>Cys, 702V>Gly and 703 P>S, respectively. This can be used for culling of calves with these SNPs in marker assisted selection program.
• Sequencing of TLR4 gene revealed of two non-synonymous SNPs; 8731 A> G and 8732 G> A which changed amino acids 322 S>D in affected cows.
• Analysis of TLR6 sequences revealed of two non-synonymous SNPs; 979 T>G and 980 G>T in affected animals leading to change of amino acid 174 Cys>V. these SNPs can be used as genetic marker in marker assisted selection.
• No SNPs were detected in affected animals in TLR9 gene. Phylogenetic analysis based on sequence data of TLR9 revealed 100% similarity among affected animals and 100% similarity among non-affected ones.
• The cutting pattern of Rsa1 in TLR2, TLR4 and TLR9; HaeIII in TLR6 and Msp1 in TLR9 are homozygous cutting pattern in affected and non- affected cows.
• Restriction analysis of PCR-RFLP of TLR2-MSP1 showed two different genotypes: AB (370 bp, 131 bp, 98 bp and 33 bp) which present only in the affected cows and can be used as a genetic marker for culling of the affected cows and BB (370 bp and 131 bp) in both AF and NA. But genotype AA is absent.
• Restriction analysis of PCR-RFLP of TLR2-Taq1 revealed two different genotypes: GT (501-bp, 475-bp, 390- bp, 85-bp, and 26-bp) in the AF cows and absent in NA, this genotype can be used in the culling of AF cows and TT (475-bp, 390- bp, 85-bp, and 26-bp), in both AF and NA. But genotype GG was absent.
• The effect of MSP1 restriction enzyme on TLR4 gene region (682-bp) in 19 susceptible cows resulted in three fragments (490 bp, 110 bp, and 82 bp) for BB and four fragments (682 bp, 490 bp, 110 bp, and 82 bp) for AB genotype.
• The effect of Taq1 restriction enzyme on TLR6 gene region (515-bp) resulted in two fragments (380 bp and 135 bp) for TT genotype and three fragments (515 bp, 380 bp, and 135 bp) for GT genotype.
• The effect of Hha1 restriction enzyme on TLR9 gene region (320 bp) resulted in three fragments (155 bp, 115 bp, and 50 bp) for CC genotype and four fragments (320 bp, 155 bp, 115 bp, and 50 bp) for TC genotype.
• The genotype AB in MSP1-TLR2, GT in Taq1-TLR2, AB in MSP1-TLR4 and TC in Hha1- TLR9 were only obtained in the affected cows. These genotypes can be used for culling of calves suspect to be infected.
• Affected animals had a heterozygous genotype associated with a cutting pattern for Taq1- TLR2, Taq1- TLR6 and Hha1-TLR9 which are repeated in four animals.
• The results of Fisher’s exact test of Taq1- TLR2 revealed a significant difference in genotypic frequencies between non-affected and affected animals.
• Cows of GT genotype at the Taq1-TLR2 locus tended (P <0.10) to have higher peak yield and fat percentage. Animals with the GT genotype of Taq1-TLR6 locus peaked at a higher level but were less persistent in lactation. Genotypes at both loci did not vary in SCS.
• Genotypes of Taq1-TLR2 or Taq1-TLR6 were not associated with fertility traits
In conclusion, mastitis is a highly recurrent disease in lactating dairy cows. Its incidence was high in low and moderate milk producing, in pluriparous cows, and was more frequent during early lactation. It negatively affects the productive and reproductive performance of dairy cows. Therefore, mastitis control programs should be a part of any production and fertility enhancing approach particularly during the critical early months of lactation.
The study revealed an association of Taq1-TLR2 gene polymorphism with clinical mastitis, fat % and peak yield, and Taq1-TLR6 with peak yield and lactation persistency. Repeated mutations that were reported in some mastitis animals in TLRs loci indicate that these genes could be used as a potential genetic marker for assisted selection for mastitis resistance in dairy cattle. This study provides researchers with data that can be used as a basis for further research to detect other novel SNPs and to analyze their correlation with mastitis resistance of Egyptian Holstein cows. Considering the economic importance of mastitis to the dairy industry, it appears clearly essential to further research on TLRs genes in Egyptian Holstein cows.