Comparative Protein Profiling of Blood and Milk of Early Pregnant Buffaloes Using SDS-PAGE

Abstract

Conventional methods for detecting pregnancy in buffalo are inefficient before 30-days after artificial insemination. A proteomic approach for high-resolution analysis of blood plasma and milk proteins by SDS polyacrylamide gel electrophoresis (PAGE) is of great importance. Blood and milk samples were collected on days 0 (before insemination) and 20 and 25 after insemination from retrospective pregnant (n = 6) and non-pregnant (n = 6) animals. An equal volume of samples was loaded in (12%-4%) SDS gel to resolve major plasma and milk proteins. Plasma proteins showed three distinct zones – high, medium, and low molecular weight (MW). In the high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the moderate MW zone (60 -75 kDa), 5-6 proteins like albumin (~70 kDa), 75 kDa, and 63 kDa consisting of pregnancy-associated glycoproteins, ~73 kDa being dominant. In the low MW zone (25 – 35 kDa), a clear band of 27 kDa was visible. Proteins in milk supernatants showed four distinct zones of proteins – very high, high, medium, and low molecular weight (MW) zones. In the very high MW zone (135 - 250 kDa), ~208 kDa and ~190 kDa proteins were visible. In the high MW zone (60 -75 kDa), proteins like lactoferrin (~78.2 kDa), 73 kDa, albumen (~66 kDa), and a heavy chain of immunoglobulin (IgG) (~54 kDa) were detected. In the medium MW zone (25 – 35 kDa), clear bands of milk caseins, namely thick alpha S2 casein (29 kDa), and other caseins. In the low MW zone (11 – 20 kDa), two thick bands of 18 kDa and 12 kDa were visible. The mean gray values (sum of the gray values of all the pixels in the peak divided by the number of pixels) of the 73 kDa protein peak were analyzed in two-way ANOVA using GraphPad (ver 8.0). Repeated measures of ANOVA were selected where pregnancy status (pregnant and non-pregnant) defined column factor and time (0-, 20- and 25-day) defined row factor. The expression of 73 kDa protein in blood plasma was higher (P = 0.04) in pregnant women than in non-pregnant buffaloes at 20- and 25-days after insemination. However, in milk, there was no difference in the expression of the 73 kDa protein between pregnant and non-pregnant buffaloes across time. Variation in animals within the groups was significantly high. The differential expression of ~73 kDa protein in plasma and precise identification of the protein can be a diagnostic marker for early pregnancy detection in buffaloes.