Effect of complement component 5 polymorphisms on mastitis resistance in Egyptian buffalo and cattle.

Mastitis is one of the costliest diseases affecting the world's dairy industry. The important contribution of complement Component 5 (C5) to phagocytosis, which plays a major role in the defence of the bovine mammary gland against infection, makes this component of innate immunity a potential contributor in defending udder against mastitis. The objectives of this study were to sequence and analyse the whole coding region of the C5 gene in Egyptian buffalo and cattle, to detect any nucleotide variations (polymorphisms) and to investigate their associations with milk somatic cell score (SCS) as an indicator of mastitis in dairy animals. We sequenced a buffalo C5 cDNA fragment of 5336 bp (KP221293) and a cattle C5 cDNA fragment of 5303 bp (KP221294), which included the whole coding region and 3-UTR. Buffalo and cattle C5 cDNA shared sequence identity of 99%. The predicted complement C5 proteins consist of 1677 amino acid residues in both animals, one amino acid less than in humans and three amino acids more than in mouse C5 protein. Comparing cDNA sequences of different animals revealed nine novel SNPs in buffalo and seven SNPs in cattle, with two of them being novel. The association analysis revealed that five SNPs in buffalo are highly associated with SCS; indicating the contribution of complement C5 variants in buffalo mastitis resistance. No significant associations were detected between C5 variants and SCS in cattle. This is the first report about C5 variants in buffalo and its association with SCS.

Complement component 3: characterization and association with mastitis resistance in Egyptian water buffalo and cattle.

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids-as in cattle-and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C>A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C>T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle.

Genome-wide genetic diversity and differentially selected regions among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep.

Sheep are among the major economically important livestock species worldwide because the animals produce milk, wool, skin, and meat. In the present study, the Illumina OvineSNP50 BeadChip was used to investigate genetic diversity and genome selection among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds from the United States. After quality-control filtering of SNPs (single nucleotide polymorphisms), we used 48,026 SNPs, including 46,850 SNPs on autosomes that were in Hardy-Weinberg equilibrium and 1,176 SNPs on chromosome × for analysis. Phylogenetic analysis based on all 46,850 SNPs clearly separated Suffolk from Rambouillet, Columbia, Polypay, and Targhee, which was not surprising as Rambouillet contributed to the synthesis of the later three breeds. Based on pair-wise estimates of F(ST), significant genetic differentiation appeared between Suffolk and Rambouillet (F(ST) = 0.1621), while Rambouillet and Targhee had the closest relationship (F(ST) = 0.0681). A scan of the genome revealed 45 and 41 differentially selected regions (DSRs) between Suffolk and Rambouillet and among Rambouillet-related breed populations, respectively. Our data indicated that regions 13 and 24 between Suffolk and Rambouillet might be good candidates for evaluating breed differences. Furthermore, ovine genome v3.1 assembly was used as reference to link functionally known homologous genes to economically important traits covered by these differentially selected regions. In brief, our present study provides a comprehensive genome-wide view on within- and between-breed genetic differentiation, biodiversity, and evolution among Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep breeds. These results may provide new guidance for the synthesis of new breeds with different breeding objectives.

L-arginine augments the antioxidant effect of garlic against acetic acid-induced ulcerative colitis in rats.

Garlic contains many sulfhydryl compounds that act as antioxidants. However, the role of nitric oxide (NO) in inflammation is controversial. The aim of the present study is to investigate the possible protective effect of garlic against acetic acid-induced ulcerative colitis in rats, as well as the probable modulatory effect of L-arginine (NO precursor) on garlic activity. Intra-rectal inoculation of rats with 4% acetic acid for 3 consecutive days caused a significant increase in the colon weight and marked decrease in the colon length. In addition, acetic acid induced a significant increase in serum levels of nitrate as well as colonic tissue content of malondialdehyde (MDA). Moreover, colonic tissue contents of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were markedly reduced. On the other hand, pre-treatment of rats with garlic (0.25 g/kgbwt, orally) for 4 consecutive weeks and 3 days during induction of colitis significantly reduced the increase in the colon weight induced by acetic acid and ameliorated alterations in oxidant and antioxidant parameters. Interestingly, oral co-administration of garlic (0.25 g/kgbwt) and L-arginine (625 mg/kgbwt) for the same period of garlic administration mitigated the changes in both colon weight and length induced by acetic acid and increased garlic effect on colon tissue contents of MDA and GSH. In conclusion, L-arginine can augment the protective effect of garlic against ulcerative colitis; an effect that might be mainly attributed to its NO donating property resulting in enhancement of garlic antioxidant effect. Further studies will be needed to determine which one of the active ingredients of garlic has the main antioxidant effect to be used with L-arginine.

Experimental diabetic nephropathy can be prevented by propolis: Effect on metabolic disturbances and renal oxidative parameters.

Oxidative stress may play a key role in the pathogenesis of diabetic nephropathy. Propolis and its extract have antioxidant properties. The effect of ethanolic extract of propolis against experimental diabetes mellitus-associated changes was examined. Diabetes was induced experimentally in rats by i.p. injection of streptozotocin (STZ) in a dose of 60 mg/kg bwt for 3 successive days. Blood urea nitrogen (BNU), creatinine, glucose, lipid profile, malondialdehyde (MDA) and urinary albumin were measured. Superoxide dimutase (SOD), glutathione (GSH), catalase (CAT) and MDA were measured in the renal tissue. The results showed decreased body weight and increased kidney weight in diabetic animals. Compared to the control normal rats, diabetic rats had higher blood glucose, BNU, creatinine, total cholesterol, triglycerides, low-density lipoprotein-cholesterol (LDL-C), MDA and urinary albumin and lower high-density lipoprotein-cholesterol (HDL-C) levels. Moreover, renal tissue MDA was markedly increased while SOD, GSH and CAT were significantly decreased. Oral administration of propolis extract in doses of 100,200 & 300 mg/kg bwt improved the body and kidney weights, serum glucose, lipid profile, MDA and renal function tests. Renal GSH, SOD and CAT were significantly increased while MDA was markedly reduced. These results may suggest a strong antioxidant effect of propolis which can ameliorate oxidative stress and delay the occurrence of diabetic nephropathy in diabetes mellitus.

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology.

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.

Stage-specific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subtractive hybridization.

Differential display RT-PCR (DDRT-PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage-specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT-PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real-time PCR. The result of quantitative differential screening was found to be in agreement with DDRT-PCR banding patterns. In the second approach, a blastocyst-stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin-3 and fibronectin. As revealed by real-time PCR, the mRNA level of galectin-3 was three times higher in the blastocyst stage than in the morula stage. DDRT-PCR and SSH are both powerful tools for the identification of stage-specific expressed gene in preimplantation bovine embryos. Real-time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest.