Twelve healthy lactating Murrah buffaloes of similar parity (3rd) between 90 and 120 days of lactation, selected from the herd of National Dairy Research Institute (Karnal, India) and maintained at managemental practices as followed at the Institute they were included in this experiment. The animals were divided into two groups based on their production level in previous lactation. The average milk production level of group 1 and II was 9.3 and 6？lit/day, respectively. Blood was collected from these buffaloes on three occasions 10 days apart. The lymphocytes were separated and cultured in RPMI 1640 medium with PHA-P for 24？h at 37°C in a humidified CO2 incubator (95% air and 5%？？CO2). The lymphocyte responsiveness was also evaluated in response to the in vivo heat stress and in vitro cortisol. Mitogen-induced stimulation index was not affected by production level ( ). Stimulation index was significantly reduced ( ) in both the groups when cortisol was added at 2.0？ng level in the culture. However, in heat-stressed buffaloes stimulation index did not vary despite increasing levels of cortisol, thus indicating that lymphocyte may become cortisol resistant during periods of acute heat stress. The results showed that lymphocyte proliferation response can be effectively used to study buffalo cell-mediated immunity in vitro. 1. Introduction Induction of lymphocyte proliferative response induced by antigen/mitogen in vitro has been shown to be representative of cellular immunocompetence. This measure can potentially be used as an indicator of an individual’s ability to mount an immune response to specific pathogen or immunomodulators. A series of in vitro studies in dairy cattle have demonstrated that exposure of bovine peripheral blood mononuclear cells to short and severe heat shock reduced their responsiveness to mitogens or decreased the number of viable cells [1, 2]. These authors suggested that exposure to high temperature in vitro can depress responses of lymphocytes; apparent adaptive mechanisms induced by in vivo heat stress provide protection from effects of high temperature seen in vitro. But so far no studies on validation have been conducted on the in vitro immune competence of buffaloes in response to heat stress. Therefore the present study has been designed to evaluate the potential of using in vitro lymphocyte proliferation assay for determining the cellular immunocompetence of lactating buffaloes in response to in vivo heat stress and in vitro cortisol. 2. Materials and Methods Twelve lactating Murrah buffaloes, apparently healthy and of
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