Protein composition affects variation in coagulation properties of buffalo milk.

The aim of this study was to investigate the effects exerted by the content of casein and whey protein fractions on variation of pH, rennet-coagulation time (RCT), curd-firming time (K20), and curd firmness of Mediterranean buffalo individual milk. Measures of milk protein composition and assessment of genotypes at CSN1S1 and CSN3 were obtained by reversed-phase HPLC analysis of 621 individual milk samples. Increased content of αS1-casein (CN) was associated with delayed coagulation onset and increased K20, whereas average pH, RCT, and K20 decreased when ß-CN content increased. Milk with low κ-CN content exhibited low pH and RCT relative to milk with high content of κ-CN. Increased content of glycosylated κ-CN was associated with unfavorable effects on RCT. Effects of milk protein composition on curd firmness were less important than those on pH, RCT, and K20. Likely, this occurred as a consequence of the very short RCT of buffalo milk, which guaranteed a complete strengthening of the curd even in the restricted 31 min time of analysis of coagulation properties and for samples initially showing soft curds. Effects of CSN1S1-CSN3 genotypes on coagulation properties were not to be entirely ascribed to existing variation in milk protein composition associated with polymorphisms at CSN1S1 and CSN3 genes. Although the role of detailed milk protein composition in variation of cheese yield needs to be further investigated, findings of this study suggest that modification of the relative content of specific CN fractions can relevantly influence the behavior of buffalo milk during processing.

Short communication: CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes affect detailed milk protein composition of Mediterranean water buffalo.

The aim of the study was to investigate the effect of composite CSN1S1-CSN3 [α(S1)-κ-casein (CN)] genotype on milk protein composition in Mediterranean water buffalo. Content of α(S1)-CN, α(S2)-CN, ß-CN, γ-CN, κ-CN, glycosylated and unglycosylated κ-CN, α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC using 621 individual milk samples. Genotypes at CSN1S1 and CSN3 were also obtained by reversed-phase HPLC. Two alleles were detected at CSN1S1 (corresponding to the A and B variants, O62823: p.Leu193Ser,) and at CSN3 (corresponding to the X1 and X2 variants, CAP12622.1: p.Ile156Thr). Increased proportions of α(S1)-CN in total casein (TCN) were associated with genotypes carrying CSN1S1 A. Genotypes associated with a marked decrease of the proportion of α(S1)-CN in TCN (composite genotypes AB-X1X1 and BB-X1X2) were associated with marked increases in the proportion of α(S2)-CN. In addition, composite genotypes carrying the X1 allele at CSN3 were associated with a greater proportion of α(S2)-CN in TCN relative to those carrying CSN3 X2. Composite genotypes greatly affected also the variability of ratios of κ-CN to TCN, with genotypes carrying the X1 allele at CSN3 being associated with decreased ratios. The decreased content of glycosylated κ-CN associated with CSN3 X1 was responsible for the overall lower content of total κ-CN in milk of X1-carrying animals. Increasing the frequency of specific genotypes might be an effective way to alter milk protein composition, namely the proportion of α(S1)-CN, α(S2)-CN, and κ-CN in TCN, and the degree of glycosylation of κ-CN.

Effect of parity, days in milk, and milk yield on detailed milk protein composition in Mediterranean water buffalo.

The effects of some nongenetic factors on milk protein fraction contents and relative proportions were estimated in 606 individual milk samples of Mediterranean water buffalo. Content of α(S1)-casein (CN), α(S2)-CN, ß-CN, γ-CN, κκ-CN, glycosylated κ-CN (glyco-κ-CN), α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC. Relative contents of α(S1)-CN%, α(S2)-CN%, ß-CN%, and κ-CN% were, respectively, 32.1, 17.1, 34.5, and 15.7%, whereas γ-CN% accounted for 0.6% of total casein content. Increasing total casein content in milk would result in a greater proportion of ß-CN% at the expense of all of the other major casein fractions, especially of κ-CN%. Values of α(S2)-CN%, ß-CN%, and γ-CN% tended to decrease with parity, although their variations were not significant, whereas α(S1)-CN% and glyco-κ-CN% showed the opposite trend. Contents of most protein fractions showed the typical trends observed for milk components as lactation progressed, with high contents in early lactation, a minimum in midlactation, followed by a gradual increase toward the latter part of lactation. Values of α(S1)-CN% increased during lactation, whereas α(S2)-CN% decreased. The proportion of ß-CN% had its maximum value between 60 and 160 d of lactation, followed by a decrease, whereas κ-CN% had its minimum value in early lactation (<60 d) and remained relatively constant in the period of mid and late lactation. Glyco-κ-CN% and ß-lactoglobulin% decreased in the first part of lactation, to reach their minimum values in midlactation, followed by an increase. Milk of top-producing buffaloes, compared with that of low-producing ones, had a significantly greater value of ß-CN% and glyco-κ-CN%, and lower proportion of α(S1)-CN%. The possible effect exerted by protein genetic variants in affecting variation of milk protein fraction contents and relative proportions should be further considered to better get insight into buffalo milk protein composition.

Effect of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotype on milk production traits and milk coagulation properties in Mediterranean water buffalo.

The aim of this study was to estimate effects of CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes on milk production traits and milk coagulation properties (MCP) in Mediterranean water buffalo. Genotypes at CSN1S1 and CSN3 and coagulation properties [rennet clotting time (RCT), curd firming time (K20), and curd firmness (A30)] were assessed by reversed-phase HPLC and computerized renneting meter analysis, respectively, using single test-day milk samples of 536 animals. Alternative protein variants of α(S1)-CN and κ-CN were detected by HPLC, and identification of the corresponding genetic variants was carried out by DNA analysis. Two genetic variants were detected at CSN1S1 (A and B variants) and 2 at CSN3 (X1 and X2 variants). Statistical inference was based on a linear model including the CSN1S1-CSN3 composite genotype effect (7 genotypes), the effects of herd-test-day (8 levels), and a combined days in milk (DIM)-parity class. Composite genotype AB-X2X2 was associated with decreased test-day milk yield [-0.21 standard deviation (SD) units of the trait] relative to genotype BB-X2X2. Genotypes did not affect milk protein content, but genotype AB-X1X1 was associated with increased fat content compared with genotype BB-X2X2 (+0.28 SD units of the trait) and AB-X1X1 (+0.43 SD units of the trait). For RCT, the largest difference (+1.91 min; i.e., 0.61 SD units of the trait) was observed between genotype AA-X1X2 and AB-X1X1. Direction of genotype effects on K(20) was consistent with that for RCT. The maximum variation in K20 due to genotype effects (between AA-X1X2 and AB-X1X1 genotypes) was almost 0.9 SD units of the trait. Magnitude of genotype effects was smaller for A30 than for RCT and K20, with a maximum difference of 0.5 SD units of the trait between genotype AA-X1X2 and AA-X1X1. The B allele at CSN1S1 was associated with increased RCT and K20 and with weaker curds compared with allele A. Allele X2 at CSN3 exerted opposite effects on MCP relative to CSN1S1 B. Because of linkage disequilibrium, allele B at CSN1S1 and allele X2 at CSN3 tend to be associated and this likely makes their effects cancel each other. This study indicates a role for casein genes in variation of MCP of buffalo milk. Further studies are necessary to estimate the effects of casein genetic variants on variation of cheese yield.

Breed assignment test in four Italian beef cattle breeds.

The assessment of a method able to assign individuals to the breed of origin is needed to certify origin and quality of livestock products. A set of 21 microsatellites was tested for breed identification in four native Italian beef breeds: Chianina, Marchigiana, Romagnola, and Piemontese. Two statistical approaches, based on maximum likelihood and on a Bayesian method, were evaluated. Different marker sets, chosen in order of the highest gene diversity and F(ST) estimates were also tested. The Bayesian method performed better, achieving a correct assignment rate of about 90% even with six microsatellites. The marker sets with the highest gene diversity were shown to perform best. Considering a threshold probability of 90%, only 52.5% of the genotypes were correctly allocated. Such results are mainly due to the low genetic differentiation estimates among breeds (F(ST)=0.049). These findings suggest that markers with high gene diversity and the presence of private alleles should be investigated and the Bayesian method used.

An ultrastructural and cytochemical study of the mesonephros of Rana esculenta during activity and hibernation.

The biological cycle of most amphibians undergoes seasonal variations. In this study, we investigated the mesonephros of Rana esculenta during active life and the natural hibernation period. The ultrastructural morphology of the different tracts constituting the nephron was analysed. Moreover, to evaluate the effect of seasonal temperature variations on the mesonephros function, the activity of some enzymes linked to membrane transport and playing regulatory roles in various metabolic pathways was investigated in different tracts of the frog nephron. During hibernation the glomerular filtration barrier appeared thicker than in the active life, lysosomes and paraplasmatic material, mostly glycogen, being accumulated in the proximal and distal tubule cells respectively. Cytoplasmic organelles, i.e., mitochondria, endoplasmic reticulum were observed in segregated areas. At the same time, changes in some enzyme activities were noted. The activity of some membrane-transport enzymes (5' nucleotidase and K+-p-nitrophenyl phosphatase) and of energetic metabolism (succinic dehydrogenase) was reduced. Nevertheless the alkaline phosphatase activity was not changed significantly, and this suggests that some metabolic activities were preserved in the hibernating samples. These results indicate morpho-functional adaptations of the kidney cells that preserve their role in osmoregulation and some metabolic processes, even during unfavourable seasons.

Cytochemical characterization of the hemocytes of Leucophaea maderae (Dictyoptera: Blaberoidea).

A correlated morphological and cytochemical approach to the hemocytes in Leucophaea maderae characterizes the granule contents of blood cells with May Grunwald-Giemsa (MGG), periodic acid-Schiff (PAS), Alcian blue (AB), and Sudan black B stainings, and after fluorochroming with acridine orange (AO). The cell nucleus and nuclear DNA is demonstrated with the Feulgen reaction. The activities of α-naphtyl butyrate esterase (ANBE) and acid phosphatase (AcPase) have been investigated in hemocytes, by both light and electron microscopy. The study discloses five cells types: prohemocyts, plasmatocytes, granulocytes, spherulocytes, and cytocytes. Occasional Sudan black-positive cells could represent an adipohemocyte category. The different patterns of cytochemical staining suggest a variety of functional roles for hemocytes. The most selective reaction pattern in terms of cytological differentiation among hemocytes occurred with the ANBE reaction. Ultrastructural analysis of intercellular granule contents, and the demonstration of cells with intermediate features, suggests that the hemolymph of Leucophaea maderae contains a developmental series from prohemocyte to plasmatocyte to granulocyte and then to spherulocyte. © 1993 Wiley-Liss, Inc.

Cytofluorometric study of the erythrocyte glycoconjugates content in different species of vertebrates.

The PAS-positive material was measured by cytofluorometry in peripheral circulating erythrocytes of 22 species from different classes of Vertebrates. The following parameters were considered: total fluorescence intensity per cell, concentration index, fluorescence fading. The highest variability of concentration indexes was found in Fishes with a minimum in the more active swimmers (Scylliorhinus and Salmo); this fact may be related to a more intense erythropoiesis. The values were more homogeneous in the Amphibians, Reptiles, and Birds, the difference never exceeding 1/2 between the maximum and minimum values. The lowest concentration indexes were found in Mammals, a class with very specialized and enucleated erythrocytes: the data appear rather heterogeneous with a minimum in Macaca. The patterns of fluorescence fading suggest a biochemical homogeneity of the PAS-positive erythrocyte material in the species considered. This fact might indicate the presence of a fundamental biochemical component possibly linked to different structures of the red cells.

Biochemical and histochemical features of human cultured cells (EUE) adapted to hypertonic medium.

EUE cells from a human heteroploid line cultured in hypertonic medium (0.274 M NaCl) modify their lipid pattern: sulfolipid concentration reaches 86 to 90 microgram/mg protein whilst it ranges between 19 to 32 microgram/mg in cells cultured in isotonic medium. Ganglioside concentration reaches 2.6 nmoles of sialic acid/mg protein (after 75 days) and 13 (after 85 days) in hypertonic saline medium. Whilst it is 0.5 in isotonic medium. Phospholipid concentration does not show any similar change. Cytoenzymatic analysis reveals that dehydrogenases (lactate, G-6-P dehydrogenases, tetrahydrofolate reductase and NADH diaphorase) appear strongly enhanced in cells grown on hypertonic medium. On the contrary higher acid phosphatase and ATPase activity was demonstrable in cells grown on isotonic medium. These results are similar (except for ATPase activity) to those observed in salt secreting glands involved in strong osmotic work. The results are discussed in relation to the problem of energy supply in cells performing osmotic work.