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This work aims to evaluate thecytotoxicity, antioxidant (SOD), histological changes in the testicular and sperm abnormality in male rats post exposure to lead acetate and the possible protective role of casein, which reducing heavy metal toxicities has raised worldwide. Animals divided to four groups for 45 days. Every group (n=5). Group1 controlGroup. treated with distal water. Group II, received 300mg/kg lead acetate for 45 days. Group III, received (300mg/kg) lead acetate + casein to 45days. Group IV, received casein 20mg/kg for 3 week and (300mg/kg b. wt./day) lead acetate with for 4 weeks. Cytotoxicity, antioxidant test, histological study on testis changes and sperm abnormally were estimated. The result showed clearly impact lead acetate on chromosomal aberration, antioxidant SOD, testicular tissue and sperm abnormality of male rats and degenerative changes in seminiferous tubules, vascular congestion, also depletion of the spermatogonia cells (some seminiferous tubules appeared with one or two layers of spermatogonia) for 45 days while and some abnormal sperm shape, the groups receiving casein after exposure for lead acetate were ameliorated the damaging effects. The lead causes a change in cytotoxicity ,physiological, histological; casein have ability to modifying physiological, histological effect induced by lead, the casein is good antioxidant compound and its mechanism of action based on metabolism and the free radical inside cells. [ABSTRACT FROM AUTHOR]

Milk yield, protein and fat content are regulated by milk protein genes. However, genetic makeup of different milk protein genes of cattle available in Bangladesh is not known. This study determined the genetic variants of kappa-casein (κ-CN) and a-lactalbumin (α-LA) gene in cattle. DNA was extracted from 50 blood samples collected from indigenous and crossbred cattle. Polymerase chain reaction (PCR) was performed with gene specific primers. For genotyping PCR products of κ-casein gene was digested with HindIII and HaeIII restriction enzymes while the PCR products of α-LA gene was digested with Bsp1286I. Overall from all the tested samples we could amplify specific DNA viz. 935 and 429 bp for κ-CN and α-LA genes, respectively. In case of κ-CN gene 30 samples were analyzed for genotyping and of these 66.67, 30.00 and 3.33% samples were genotyped as AA, AB and BB, respectively. AA genotype was found dominant for κ-CN gene. Allele frequency for A was 82%. AA genotype was found higher in indigenous (0.366) than crossbred (0.30) animals. Homozygous BB genotype was not found in crossbred animals. For a-LA gene 50 samples were genotyped and all the samples (100%) were belonging to AA genotype. Three genetic variants of κ-CN gene viz. AA, AB & BB and one genotype (AA) of α-LA gene were detected in tested samples. Alleles of both the genes are reported to be associated with higher milk, protein, and fat yields. [ABSTRACT FROM AUTHOR]

We developed knock-in vector system of human Noggin mature sequence with glutathione S-transferase (GST) containing factor Xa protease linker to facilitate the subsequent purification of recombinant protein. To achieve this, bovine ear fibroblast cells were isolated and transfection conditions were optimized by electroporation. To generate knock-in vector, human Noggin lacking its native signal peptide is fused to GST and foot and month disease virus 2A (F2A), and then inserted into bovine β-casein gene exon 3. We also generated enhanced green fluorescent protein (EGFP) expression vector of GST-human Noggin mature fused to β-casein signal peptide and F2A, and successfully detected recombinant human Noggin protein secreted into culture media, followed by factor Xa cleavage. Then, we co-transfected human Noggin knockin vector with single-guided RNA and Cas9 expression vectors into bovine ear fibroblasts and obtained the stably-integrated colonies by antibiotic selection. PCR screening analysis revealed that 26 out of 35 colonies positively integrated human Noggin knock-in vector into bovine β-casein locus. One of positive clones was subjected to chromosome analysis, presenting normal karyotypes. Our data may provide the additional purification guideline of recombinant proteins by tagging GST with a protease linker sequence in the upstream of target genes and a high efficiency of integration ratio into bovine β-casein locus. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the phenolic compounds and antioxidant activity of goat milk yogurt characterized by different αS1-casein genotypes and fortified with Rhus coriaria leaf powder. The αS1-casein genotype was determined by isoelectric focusing, total phenol content was determined by the Folin–Ciocalteu method, phenolic compounds were identified and quantified by HPLC-UV analysis, and antioxidant activity was measured using 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and ferric-reducing antioxidant power. The statistical analysis showed a significant effect of the studied factors. Comparing different genotypes it emerged that yogurt from goats with weak alleles at CSN1S1 loci (FF) showed the lowest phenolic compounds and therefore a lower antioxidant activity compared with yogurt from goats with strong alleles at CSN1S1 loci (AA, BB, AB). Rhus coriaria-fortified yogurt showed a significant increase in total phenolic compounds and antioxidant activity in comparison with plain yogurt. The FF-fortified yogurt presented the lowest total phenol content and antioxidant activity. This could be due to a greater capacity of proteins and peptides in this yogurt to form stable complexes with phenols. The different total phenol content detected in R. coriaria-fortified yogurt indicates that the αS1-casein genotype influenced the amount of added phenols that are bound to the caseins and, therefore, the part that remains free and that affects the biological capacity of the final product. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate genetic polymorphism of the alpha S1 casein gene and its association with milk production parameters (milk yield and milk composition) of indigenous Balkan goat breed reared in Montenegro. Two genetic variants (A and F) and three genotypes AA, AF and FF of alpha S1-casein have been identified. The analysis showed a prevalence of strong A allele (0.626) compared to the weak F allele (0.374), while the frequencies of genotypes were 0.398, 0.456 and 0.146 respectively. The effect of genotype of alpha S1 casein on the total milk yield, length of lactation and daily milk yield was not significant (p>0.05), while it is significantly influenced to the milk composition parameters. Goats with AA genotype of alpha S1 casein had significantly higher (p<0.001) average content of protein (3.54%) than AF and FF genotype (3.40% and 3.33%). The same genotypes order were for fat content and solid non-fat content (AA>AF>FF) with significant differences (p<0.05) of AA genotype in comparison to AF and FF. Genetic characterization of the alpha S1-casein gene contributes to the knowledge of the genetic structure of Balkan goat breed in Montenegro. These results could be important for establishing of the concept of conservation and sustainable use of this indigenous goat breed, as well as for selection scheme and breeding program. [ABSTRACT FROM AUTHOR]

The study was conducted to identify genetic marker for CSN3 gene related to higher casein yield in 78 pedigreed Karan Fries (HF crossbred) cattle population at ICAR-NDRI, Karnal. PCR-RFLP of exon 4 region of CSN3 gene, carried out using HindIII restriction endonuclease, revealed three genotypes--CC, CA and AA with frequencies as 0.18, 0.67 and 0.15 and frequency of C and A allele as 0.51 and 0.49. Eight SNPs (C296A, C380T, C383T, G394T, C419A, G480A, A488T and G529T) were revealed through sequencing. Using Regression model, SNP (C419A) in the targeted region was found to contribute an increase of 1.8 g in κ-casein yield (CY) which was 2.81% of CY (63.62 g) in average test day milk yield (ATDMY). After screening progenies for SNPs based on Daughter-Dam Design, SNP G480A was found to be a potential genetic marker, contributing an increase of 7.5 g in CY (11.79% of CY in ATDMY). [ABSTRACT FROM AUTHOR]

Relative concentrations of αS1-casein and αS2-casein (αS1-CN and αS2-CN) phosphorylation isoforms vary considerably among milk of individual cows. We estimated heritabilities for αS2-CN phosphorylation isoforms, determined by capillary zone electrophoresis from 1,857 morning milk samples, and genetic correlations among αS2-CN phosphorylation isoforms in Dutch Holstein Friesian. To investigate if phosphorylation of αS1-CN and αS2-CN are due to the same genetic mechanism, we also estimated genetic correlations between αS1-CN and αS2-CN phosphorylation isoforms as well as the genetic correlations between the phosphorylation degrees (PD) of αS1-CN and αS2-CN defined as the proportion of isoforms with higher degrees of phosphorylation in total αS1-CN and αS2-CN, respectively. The intra-herd heritabilities for the relative concentrations of αS2-CN phosphorylation isoforms were high and ranged from 0.54 for αS2-CN-10P to 0.89 for αS2-CN-12P. Furthermore, the high intra-herd heritabilities of αS1-CN PD and αS2-CN PD imply a strong genetic control of the phosphorylation process, which is independent of casein production. The genetic correlations between αS2-CN phosphorylation isoforms are positive and moderate to high (0.33–0.90). Furthermore, the strong positive genetic correlation (0.94) between αS1-CN PD and αS2-CN PD suggests that the phosphorylation processes of αS1-CN and αS2-CN are related. This study shows the possibility of breeding for specific αS1-CN and αS2-CN phosphorylation isoforms, and relations between the phosphorylation degrees of αS1-CN and αS2-CN and technological properties of milk need to be further investigated to identify potential benefits for the dairy industry. [ABSTRACT FROM AUTHOR]

Local zebu cattle breeds in Indian villages are crossbred with exotic breeds at an unprecedented rate without utilising the full potential of genomic technologies. In addition to agriculture produce, livestock, particularly cattle, constitute a vital source of livelihood for farmers in India. Age-old agricultural practices, errant monsoon, and frequent crop failures have resulted in Maharashtra having the highest number of farmer suicides in the country. Local cattle breeds are considered low-yield breeds and thus are primarily used as beasts of burden. Information on functional gene variants in Indian cattle breeds is scant and limited to PCR-RFLP study in few breeds. In this study, 32 samples from 8 cattle breeds were obtained from remote villages of Maharashtra state. By using the re-sequencing technology, we sequenced 403 bp of the exon IV CSN3 allele and inferred its haplotypes. From 32 samples, 14 genotypes (G1-G14) defined by 7 single-nucleotide polymorphisms (SNP's) were identified. From these 14 genotypes, we reconstructed 3 haplotypes (H01, H02, and H03) and estimated their frequencies. Of the 3 haplotypes, two (H01 and H03) corresponded to CSN3*B4 and CSN3*B and CSN3*H and CSN3*G alleles, respectively. The third haplotype H02 was identified as a new allele and differed from CSN3*B4 and CSN3*B and CSN3*H and CSN3*G alleles by one nonsynonymous mutation at the position C5306T-Ile100Thr. The neighbour-joining tree reconstruction revealed haplotype sharing or hybridisation between B. t. indicus and B. t. taurus because the 3 haplotypes originated in this study clustered with A1, B2, B4, B, G, and H alleles, which were reported previously in both the subspecies of B. taurus. The occurrence of one new allele in a small sample size highlights the urgency to screen local zebu cattle breeds by using genomic tools for circumventing genetic erosion that is widespread in Indian villages in India. [ABSTRACT FROM AUTHOR]

Objective: From a review of published information on genetic association studies, a meta-analysis was conducted to determine the influence of the genes κ-casein (CSN3) and β-lactoglobulin (LGB) on milk yield traits in Holstein, Jersey, Brown Swiss, and Fleckvieh. Methods: The GLIMMIX procedure was used to analyze milk production and percentage of protein and fat in milk. Models included the main effects and all their possible two-way interactions; not estimable effects and non-significant (p>0.05) two-way interactions were dropped from the models. The three traits analyzed used Poisson distribution and a log link function and were determined with the Interactive Data Analysis of SAS software. Least square means and multiple mean comparisons were obtained and performed for significant main effects and their interactions (p<0.0255). Results: Interaction of breed by gene showed that Holstein and Fleckvieh were the breeds on which CSN3 (6.01%±0.19% and 5.98%±0.22%), and LGB (6.02%±0.19% and 5.70%±0.22%) have the greatest influence. Interaction of breed by genotype nested in the analyzed gene indicated that Holstein and Jersey showed greater influence of the CSN3 AA genotype, 6.04%±0.22% and 5.59%±0.31% than the other genotypes, while LGB AA genotype had the largest influence on the traits analyzed, 6.05%±0.20% and 5.60%±0.19%, respectively. Furthermore, interaction of type of statistical model by genotype nested in the analyzed gene indicated that CSN3 and LGB genes had similar behavior, maintaining a difference of more than 7% across analyzed genotypes. These results could indicate that both Holstein and Jersey have had lower substitution allele effect in selection programs that include CSN3 and LGB genes than Brown Swiss and Fleckvieh. Conclusion: Breed determined which genotypes had the greatest association with analyzed traits. The mixed model based in Bayesian or Ridge Regression was the best alternative to analyze CSN3 and LGB gene effects on milk yield and protein and fat percentages. [ABSTRACT FROM AUTHOR]

Casein proteins (αS1-, αS2-, β- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 1015 micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pKa) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pKa = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pKa = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ∼5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ∼65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk. [ABSTRACT FROM AUTHOR]

The genetic variants of kappa-casein gene were found in different frequencies in various cattle breeds. These genetic variants cause differences in quality and composition of the milk. The objective of this work was to identify the genotypes and allelic frequencies of kappa-casein locus in Brown Swiss as an exotic breed and Busha cattle as indigenous cattle breed in Montenegro. The DNA of 21 blood samples of Busha breed cattle and 19 blood samples of Brown Swiss cattle were genotyped for polymorphisms at the kappa-casein gene by a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) essay. A 351 bp fragment of kappa casein was amplified and digested with HinfI restriction endonuclease. Three genotypes were identified (AA, AB and BB) with frequencies of 0.117, 0.450 and 0.433 respectively in Brown Swiss breed and 0.274, 0.499 and 0.227 in Busha breed. The frequencies of alleles were 0.316 (A) and 0.684 (B) in Brown Swiss and 0.524 and 0.476 (respectively) in Busha cattle. The genotyping of kappa-casein alleles (A and B) is of practical importance, since B allele is in positive correlations with commercially valuable parameters of cheese yielding efficiency. The results of very high frequency of B allele in Brown Swiss cattle confirmed good performance of this breed in Montenegrin population. This determination of A and B allele frequencies in population of Busha breed is the first research of this kind in Montenegro. It could be used for possible increasing the frequency of desired alleles and genotypes by including in the programs of selection and preservation of the Busha breed in Montenegro, as an important animal genetic resource. [ABSTRACT FROM AUTHOR]

Transglutaminase (TGase) and succinylation could modify the physicochemical characteristics and the functional properties of proteins. The aim of this work was to cross-link egg white protein (EWP) and succinylated casein with TGase and to assess the feasibility of making a novel composite protein film. The effects of succinylated casein content, reaction time, and TGase concentration on the mechanical properties, physical characteristics (thermal property and degree of crystallinity), and structure properties (secondary structure and surface micrograph structure) of the film were investigated in this study. The results revealed that the susceptibility of EWP to TGase-mediated cross-linking modification was enhanced by succinylated casein. Meanwhile, the films with TGase were more homogeneous and smoother and possessed better water resistance and thermal stability. The content of α-helix, β-turn structures were increased whereas β-sheet structure was decreased. The spatial conformation and degree of crystallinity of composite protein film were also affected by TGase. The increase of the degree of crystallinity of the composite film further proved the improvement of film mechanical properties induced by TGase and succinylated casein. Based on the above results, this study provides relevant data and insights for the TGase/chemical modification of protein-based edible films. [ABSTRACT FROM AUTHOR]

Casein (CN) phosphorylation is an important posttranslational modification and is one of the key factors responsible for constructing and stabilizing casein micelles. Variation in phosphorylation degree of αS-CN is of great interest because it is suggested to affect milk technological properties. This study aimed to investigate the variation in phosphorylation degree of αS-CN among milk of individual cows and to explore relationships among different phosphorylation isoforms of αS-CN. For this purpose, we analyzed morning milk samples from 529 French Montbéliarde cows using liquid chromatography coupled with electrospray ionization mass spectrometry. We detected 3 new phosphorylation isoforms: αS2-CN-9P, αS2-CN-14P, and αS2-CN-15P in bovine milk, in addition to the known isoforms αS1-CN-8P, αS1-CN-9P, αS2-CN-10P, αS2-CN-11P, αS2-CN-12P, and αS2-CN-13P. The relative concentrations of each αS-CN phosphorylation isoform varied considerably among individual cows. Furthermore, the phenotypic correlations and hierarchical clustering suggest at least 2 regulatory systems for phosphorylation of αS-CN: one responsible for isoforms with lower levels of phosphorylation (αS1-CN-8P, αS2-CN-10P, and αS2-CN-11P), and another responsible for isoforms with higher levels of phosphorylation (αS1-CN-9P, αS2-CN-12P, αS2-CN-13P, and αS2-CN-14P). Identifying all phosphorylation sites of αS2-CN and investigating the genetic background of different αS2-CN phosphorylation isoforms may provide further insight into the phosphorylation mechanism of caseins. [ABSTRACT FROM AUTHOR]

The objective of this study was to identify allele and genotype frequencies of the κ-CN and β-LG genes in Anatolian water buffalo. A total of 126 water buffalos from Turkey were genotyped using the PCR-RFLP method. For gene κ-CN, only B allele and BB genotype were observed. And for gene β-LG, two types of alleles (A and B) and three types of genotypes were observed. The genotype frequencies of AA, AB and BB of β-LG in Anatolian water buffalo were 0.254, 0.698 and 0.048, respectively. Surprisingly, the frequency of allele A was higher than that of allele B in contrast to world buffalo breeds. [ABSTRACT FROM AUTHOR]

Genetic analysis of loci affecting production and reproduction traits in livestock animals make possible the evaluation of animal breeding values using molecular markers. The aim of this study was to detect polymorphisms in the leptin and κ-casein genes of a Cuban water buffalo population to provide markers useful for associating studies. Genomic DNA from 68 unrelated Cuban buffaloes, uncontrolled crossbreds of river (Buffalypso) and swamp (Carabao) animals, was analyzed using PCR-RFLP. Fragments of leptin and ê-casein genes were amplified and digested with Bsa AI and Hind III/Taq I, respectively. All the amplified samples were monomorphic BB after digestion with Hind III/Taq I. Genotypic frequencies found in the leptin gene with Bsa AI were 0.47 (AA), 0.42 (AG) and 0.11 (GG). The population was in HWE (p=1.0000), possibly because there was not selection for this locus on it. The FIS estimate (0.0212) showed no inbreeding in the population regarding this locus. He (0.436) and Ho (0.426) values were below 0.5 (50%), indicating low genetic variation in this locus in the population. PCR-RFLP detected the two genetic variants described for the leptin gene A1620G. However, a higher number of animals and SNPs in the leptin and/or other genes should be analyzed for a more accurate estimate of the genetic diversity in this population. DNA sequencing should be assayed by nucleotide sequence analysis to detect the new A and B variants of κ-casein reported in buffalo. [ABSTRACT FROM AUTHOR]

Background: Cows' milk generally contains two types of β-casein, A1 and A2 types. Digestion of A1 type can yield the peptide β-casomorphin-7, which is implicated in adverse gastrointestinal effects of milk consumption, some of which resemble those in lactose intolerance. This study aimed to compare the effects of milk containing A1 β-casein with those of milk containing only A2 β-casein on inflammation, symptoms of post-dairy digestive discomfort (PD3), and cognitive processing in subjects with self-reported lactose intolerance.Methods: Forty-five Han Chinese subjects participated in this double-blind, randomized, 2 × 2 crossover trial and consumed milk containing both β-casein types or milk containing only A2 β-casein. Each treatment period was 14 days with a 14-day washout period at baseline and between treatment periods. Outcomes included PD3, gastrointestinal function (measured by smart pill), Subtle Cognitive Impairment Test (SCIT), serum/fecal laboratory biomarkers, and adverse events.Results: Compared with milk containing only A2 β-casein, the consumption of milk containing both β-casein types was associated with significantly greater PD3 symptoms; higher concentrations of inflammation-related biomarkers and β-casomorphin-7; longer gastrointestinal transit times and lower levels of short-chain fatty acids; and increased response time and error rate on the SCIT. Consumption of milk containing both β-casein types was associated with worsening of PD3 symptoms relative to baseline in lactose tolerant and lactose intolerant subjects. Consumption of milk containing only A2 β-casein did not aggravate PD3 symptoms relative to baseline (i.e., after washout of dairy products) in lactose tolerant and intolerant subjects.Conclusions: Consumption of milk containing A1 β-casein was associated with increased gastrointestinal inflammation, worsening of PD3 symptoms, delayed transit, and decreased cognitive processing speed and accuracy. Because elimination of A1 β-casein attenuated these effects, some symptoms of lactose intolerance may stem from inflammation it triggers, and can be avoided by consuming milk containing only the A2 type of beta casein.Trial Registration: ClinicalTrials.gov/NCT02406469. [ABSTRACT FROM AUTHOR]

Casein molecules are used in food industry as ingredients. They can be used as isolated forms and under micellar form consisting in an association of different casein molecules and calcium phosphate. In this review, after a brief reminder of the main characteristics of casein molecules and casein micelles, the modifications of caseins induced by physical, chemical, and enzymatic actions are reported. The resulting new physicochemical properties (mineral and casein compositions, charge, hydrophobicity, aggregation state, and morphology) and techno-functionalities (heat stability, viscosity, gelation, emulsifying, and foaming properties) are described and discussed with a special attention paid to the results obtained in our laboratory since several decades. [ABSTRACT FROM AUTHOR]

The present study was designed to estimate the relative gene expression of major genes responsible for β-casein and its transcription regulation during early lactation cycle in Surti and Jaffarabadi buffaloes. Ten buffaloes of each breed maintained at Livestock Research Station, Navsari and Cattle Breeding Farm, Junagadh, respectively were selected for the study. The RNA was extracted from noninvasively isolated primary buffalo mammary epithelial cells (pBMEC) obtained using antibody mediated magnetic separation method from milk samples collected at day 15 and 60 postpartum (pp). Primers used for amplification of β-casein gene (CSN2) and its transcription regulatory genes were bovine specific. The mean relative expressions of C/EBPβ, RUNX2 and STAT5A genes were significantly higher in Jaffarabadi buffaloes as compared to Surti buffaloes at day 15 pp. However, between breed differences in the mean relative expressions of C/EBPβ, RUNX2 and STAT5A genes were not observed at 60 days pp. The relative expressions of CSN2 and YY1 genes did not differ significantly within or between breeds. The mean relative expression of KRT8 gene was almost comparable among all the groups under present study. Thus, only β- casein transcription regulatory genes C/EBPβ, RUNX2 and STAT5A amongst all studied genes showed significant between breed variability at day 15 pp, however this variability too got subsided as lactation stage advanced to day 60 pp. [ABSTRACT FROM AUTHOR]

The main objective of the present work was to identify the b - casein gene polymorphism in Tharparkar and Gir breeds of cattle. The beta - casein constitutes up to 45 % of the casein of bovine milk. The majority of beta-casein in dairy cattle breeds are found A1 and A2, while B is veryless common. The β-casein A1 variant was associated with the incidence of diabetes mellitus type -1, coronary heart disease and autism. The A2 variant reduces serum cholesterol. The present study was conducted on 72 cows (Tharparkar - 36 cows, Gir - 36 cows). Bovine genomic DNA was extracted from whole blood by using the method of phenol - Chloroformdeproteinization and ethanol precipitation and used in order to estimate β - casein genotypes by means of the PCR-RFLP method. The studied populations revealed detection of two genotypes -heterozygote genotype (A1A2) and homozygote genotype (A2A2) with frequencies of 0.11 and 0.89 in Tharparkar breed; 0.13, and 0.87 in Gir breed respectively. It is observed that both the breeds Tharparkar and Gir, contained a higher frequency of allele A2 (0.95 and 0.94) with that of allele A1. The present study revealed that A2 variant of beta casein is predominant over the A1 variant (very low frequency) in the studied population of Tharparkar and Gir breeds. [ABSTRACT FROM AUTHOR]

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region. [ABSTRACT FROM AUTHOR]

Quantitative individual differences in the amount of β-casein in goat milk are determined by at least nine alleles. In particular, two alleles ( CSN2 0 and CSN2 01) are associated with an undetectable amount of this protein in milk. The CSN2 01 allele is characterized by a single nucleotide substitution at position 373 of the seventh exon ( AJ011018:g.8915C>T), responsible for the formation of a premature stop codon at the 182 position. Herein, we report the contribution of the SNP g.1311T>C, which demonstrates a linkage with the SNP AJ011018:g.8915C>T, to the promoter transcriptional activity. Particularly, we indicate that the nucleotide C at position 1311 negatively affects the promoter activity of the CSN2 gene. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate diversity of the αS1-casein gene in Maiwa yak. A polymorphism of αS1-casein gene has been identified in Chinese Maiwa yak by PCR single-strand conformation polymorphism protocol. Comparing the X59856 sequence, some non-coding and coding DNA variants of yak CSN1S1 gene were detected in this present study, including KJ397913 (promoter region, 9969A/G, 9984 A/G, 10153 C/T, 10175 A/C, 10261 G/C), KJ397914 (promoter region 10261 G/C), KJ397896 (intron III, 14862 T/C), KJ397897 (intron III, 14862 T/C, and deleted 14944-14946 CTT), KJ397899 (intron IV, 15297 A/C), KJ397901 (intron VIII, 17503 A/C, 17666 A/C), KJ397902 (exon IX, 18901C/A, intron IX, 18970 T/G, 19088 add A, 19120 add A, 19202 add T, 19296 G/T), KJ397903 (intron IX, 18970 T/G, 19088 add A, 19120 add A, 19202 add T, 19296 G/T), KJ397904 (intron IX, 19537 T/G, 19539T/G, 19599G/A, 19638 C/T), KJ397905 (intron XIV, 23402 T/G, 23417 C/T, 23480 T/C, 23511 G/A), and KJ397907 (intron XVI, 25976 G/T; exon XVII, 26181A/G). The polymorphism of αS1-casein gene may be helpful for future studies on genetic variation within and between yak populations or on associated traits. [ABSTRACT FROM AUTHOR]

In selecting cows for higher milk yields and milk quality, it is important to understand how these traits are affected by the bovine genome. The major milk proteins exhibit genetic polymorphism and these genetic variants can serve as markers for milk composition, milk production traits, and technological properties of milk. The aim of this study was to investigate the relationships between casein (CN) genetic variants and detailed protein composition in Swedish and Danish dairy milk. Milk and DNA samples were collected from approximately 400 individual cows each of 3 Scandinavian dairy breeds: Swedish Red (SR), Danish Holstein (DH), and Danish Jersey (DJ). The protein profile with relative concentrations of α-lactalbumin, β-lactoglobulin, and αS1-, αS2-, k-, and β-CN was determined for each milk sample using capillary zone electrophoresis. The genetic variants of the αS1- (CSN1S1), β- (CSN2), and k-CN (CSN3) genes for each cow were determined using TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA). Univariate statistical models were used to evaluate the effects of composite genetic variants, αS1-β-k-CN, on the protein profile. The 3 studied Scandinavian breeds differed from each other regarding CN genotypes, with DH and SR having similar genotype frequencies, whereas the genotype frequencies in DJ differed from the other 2 breeds. The similarities in genotype frequencies of SR and DH and differences compared with DJ were also seen in milk production traits, gross milk composition, and protein profile. Frequencies of the most common composite αS1-β-k-CN genotype BB/A²A²/AA were 30% in DH and 15% in SR, and cows that had this genotype gave milk with lower relative concentrations of k- and β-CN and higher relative concentrations of αS-CN, than the majority of the other composite genotypes in SR and DH. The effect of composite genotypes on relative concentrations of the milk proteins was not as pronounced in DJ. The present work suggests that a higher frequency of BB/ A¹A²/AB, together with a decrease in BB/A²A²/AA, could have positive effects on DH and SR milk regarding, for example, the processing of cheese. [ABSTRACT FROM AUTHOR]

Kappa-casein (κ-casein) gene is known as a gene that plays a role in controlling milk protein and also play a crucial role in the coagulation and curdling of milk. This study was aimed to identify polymorphisms of the κ-Casein gene of local buffaloes in Indonesia. A total number of 40 heads of riverine buffalo and 250 heads of swamp buffalo. This study used PCR-RFLP method, which amplification of the κ-Casein gene resulted an amplicon with length of 157 bp, located in exon 4. The amplified fragment were digested with EcoRV restriction enzyme, which cut the κ-Casein gene in exon 4 at nucleotides of GAT∣ATC, revealed the presence of one polymorphism at the base position of 23 bp that occurs with a substitution of Ile (ATC) of the T genetic variant into Thr (ACC) of the C genetic variant. Genotyping κ-Casein gene in riverine buffalo produced two types of allele, namely C allele (157 bp) and T allele (136 and 21 bp). These two alleles resulted in three types of genotypes, namely CC, CT, and TT. Frequency of the C allele was dominant to T allele. κ-Casein gene in swamp buffalo was monomorphic with one allele, namely C allele. Heterozygosity value of riverine and swamp buffaloes were low. PIC value in riverine and swamp buffalo ranged 0.000-0.288. Fixation index of κ-Casein gene in riverine buffalo was low (Siborong-borong SBBC = -0,0036; Deli Serdang = -0.025), but in swamp buffalo was in fixation. This study showed that κ-Casein∣EcoRV were polymorphic in riverine buffalo and monomorphic in swamp buffalo. [ABSTRACT FROM AUTHOR]

Abstract: Sheep milk is an excellent raw material for the milk processing industry especially in cheese production. The protein content and composition of sheep milk are important in the cheese manufacturing. The casein fraction of ruminant milk proteins consists of four caseins, namely αs1, αs2, β and κ-Casein. Casein genetic polymorphisms are important due to their effects on quantitative traits and technological properties of milk. This study aimed to detect the genetic polymorphism of αs1- and αs2-Casein genes in three native Egyptian sheep breeds; Rahmani, Barki and Ossimi. PCR-SSCP and PCR-RFLP were used to detect the genetic polymorphism of αs1-CN and αs2-CN genes, respectively. A 223-bp fragment of αs1-CN gene was amplified by PCR and SSCP results recorded the presence of three different patterns; TT, TC and CC; in 87 tested sheep animals. The sequence analysis of two homologous patterns showed a single nucleotide polymorphism (SNP) (T→C) at position 170. The frequencies of three patterns in the tested sheep breeds were 43.33%, 50.00%, and 6.67% in Rahmani; 83.33%, 13.33%, and 3.33% in Ossimi and 74.07%, 22.22%, and 3.70% in Barki, respectively. Our nucleotide sequences of αs1-CN T and C alleles were submitted to GenBank with the accession numbers KF018339 and KF018340, respectively. The restriction digestion of αs2-CN PCR product (1300-bp) by Tru1I endonuclease revealed three different genotypes; AA, AG and GG with frequencies of 66.67%, 30.00%, and 3.33% in Rahmani; 96.67%, 3.33%, and 0.00% in Ossimi and 96.15%, 3.85%, and 0.00% in Barki, respectively. The sequence analysis revealed the presence of a single nucleotide polymorphism (A→G) in intron 6 of αs2-CN gene. Our nucleotide sequence of αs2-CN gene was submitted to GenBank with the accession number JX080380. [Copyright &y& Elsevier]

Abstract: Genetic polymorphism k-casein (CSN3) gene was investigated in lactating Egyptian buffalo using nucleotide sequencing. Primer pairs amplified a 453 nucleotide fragment of CSN3 exon IV with an open reading frame of 421 nucleotides encoding 139 amino acids of the mature peptide and 32 nucleotides 3′UTR. Two SNPs (nt-315 C/T and nt-319 C/T) occurred in amplified fragment. These SNPs were reflected at codon 105 (ACC/ATC) and codon 106 (ACC/ACT) which correspond to codon 135 and 136 of the CSN3 mature peptide, respectively. Variation at codon 135 caused a change from ACC (Threonine) versus ATC (Isoleucine) whereas variation at codon 136(Thr/Thr) is a silent mutation. The results show, contrary to previous reports, that Egyptian buffalo has both alleles A (135ThrACC/136ThrACC) and B (135IleATC/136ThrACT) with allelic frequencies of 0.57 and 0.43, respectively. The Egyptian buffalo genotype frequencies were 0.294 (AA), 0.647 (AB) and 0.058 (BB). The polymorphic site at codon 135 (A[C/T]C) and 136 (AC[C/T]) has C bases in allele A and T bases in allele B, resulting in two haplotypes; 135Thr(ACC)/136Thr(ACC) and 135Ile(ATC)/136Thr(ACT). The frequency of the former haplotype was 0.57. In this study we investigated the reason why buffalo samples, analyzed by RFLP technique, using HindIII and HinfI used in cattle, were mistakenly identified as BB monomorphic. We suggest the use of restriction enzymes AcuI or Eco57MI to be used in buffalo CSN3 RFLP analysis. Digestion of buffalo CSN3-exon VI fragment (453bp) with either enzyme will generate two fragments of 339bp and 114bp in allele B. [Copyright &y& Elsevier]

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer× Arsi-Bale and Boer ×Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32 g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05 g/kg) and reference (AA; 7.61 g/kg) genotype animals. The αs2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the sequence corresponded to allele A and presumably derived from it. Therefore, this allele is denoted by A3. All goats from the reference genotype (AA) were homozygous for the allele at nucleotide position 1374 and 1866, whereas all mutations in the 5' UTR existed in a heterozygous form in both heterozygous (CA) and the new mutation (CC) genotype. The newly identified mutation (CC) detected in some of the goat breeds is, therefore, important in selection for genetic improvement and high-quality milk for the emerging goat cheese-producing industries. The finding will also benefit farmers raising these goat breeds due to the increased selling price of goats. Further studies should investigate the effect of this amino acid exchange on the secondary and tertiary structure of the αs1-CN molecule and on the susceptibility of peptide hydrolysis by digestive enzymes. [ABSTRACT FROM AUTHOR]

In cattle, at least 39 variants of the 4 casein proteins (αS1-, β-, αS2- and κ-casein) have been described to date. Many of these variants are known to affect milk-production traits, cheese-processing properties, and the nutritive value of milk. They also provide valuable information for phylogenetic studies. So far, the majority of studies exploring the genetic variability of bovine caseins considered European taurine cattle breeds and were carried out at the protein level by electrophoretic techniques. This only allows the identification of variants that, due to amino acid exchanges, differ in their electric charge, molecular weight, or isoelectric point. In this study, the open reading frames of the casein genes CSN1S1, CSN2, CSN1S2, and CSN3 of 356 animals belonging to 14 taurine and 3 indicine cattle breeds were sequenced. With this approach, we identified 23 alleles, including 5 new DNA sequence variants, with a predicted effect on the protein sequence. The new variants were only found in indicine breeds and in one local Iranian breed, which has been phenotypically classified as a taurine breed. A multidimensional scaling approach based on available SNP chip data, however, revealed an admixture of taurine and indicine populations in this breed as well as in the local Iranian breed Golpayegani. Specific indicine casein alleles were also identified in a few European taurine breeds, indicating the introgression of indicine breeds into these populations. This study shows the existence of substantial undiscovered genetic variability of bovine casein loci, especially in indicine cattle breeds. The identification of new variants is a valuable tool for phylogenetic studies and investigations into the evolution of the milk protein genes. [ABSTRACT FROM AUTHOR]

Milk fat secretion is a complex process that initiates in the endoplasmic reticulum of the mammary epithelial cell by the budding of lipid droplets. Lipid droplets are finally released as fat globules in milk enveloped by the apical plasma membrane of the mammary epithelial cell. The milk fat globule membrane (MFGM) thus comprises membrane-specific proteins and polar lipids (glycerophospholipids and sphingolipids) surrounding a core of neutral lipids (mainly triacylglycerols and cholesterol esters). We have recently described major proteins of the MFGM in the goat and we have highlighted prominent differences between goats and bovine species, especially regarding lactadherin, a major MFGM protein. Here, we show that, in the goat species, the well-documented genetic polymorphism at the as1-casein (CSN1S1) locus affects both structure and composition of milk fat globules. We first evidenced that both milk fat globule size and ζ-potential are related to the as1-casein genotype. At midlactation, goats displaying strong genotypes for as1-casein (A/A goats) produce larger fat globules than goats with a null genotype at the CSN1S1 locus (O/O goats). A linear relationship (R² = 0.75) between fat content (g/ kg) in the milk and diameter of fat globules (µm) was established. Moreover, we found significant differences with regard to MFGM composition (including both polar lipids and MFGM proteins) from goats with extreme genotype at the CSN1S1 locus. At midlactation, the amount of polar lipids is significantly higher in the MFGM from goats with null genotypes for as1-casein (O/O goats; 5.97 ± 0.11 mg/g of fat; mean ± standard deviation) than in the MFGM from goats with strong genotypes for as1-casein (A/A goats; 3.96 ± 0.12 mg/g of fat; mean ± standard deviation). Two MFGM-associated proteins, namely lactadherin and stomatin, are also significantly upregulated in the MFGM from goats with null genotype for as1-casein at early lactation. Our findings are discussed with regard to techno-functional properties and nutritional value of goat milk. In addition, the genetic polymorphism in the goat species appears to be a tool to provide clues to the lipid secretion pathways in the mammary epithelial cell. [ABSTRACT FROM AUTHOR]

Abstract: Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future. [Copyright &y& Elsevier]

A polymorphism of κ-casein gene CSN3 exon IV was identified in Chinese Maiwa Yak by PCR-restriction fragment length polymorphism protocol to reveal the distribution of diversity of κ-casein in Maiwa Yak at the DNA sequence level. The sequences corresponding to the coding region of κ-casein gene exon IV were used for phylogenetic analysis to establish the phylogenetic relationship of CSN3 variants in the Bos genus. Experimental results indicated that most yak milks were with κ-casein BB genotype. A new variant was identified in Maiwa yak breed by analysis and entered into GenBank with accession number GU441771. The phylogenetic analysis presented κ-casein evolution was consistent with species at a degree. [ABSTRACT FROM AUTHOR]

An abstract of the article "Variation at the 3'-UTR of the goat αS2- and β-casein genes is not associated with milk protein and dry matter contents in Murciano-Granadina goats," by A. Zidi and colleagues is presented.