Supplementary Material for: Involvement of Plasma Endocannabinoids and the Hypothalamic Endocannabinoid System in Increasing Feed Intake after Parturition of Dairy Cows

The endocannabinoids (ECs) N-arachidonylethanolamide (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) participate in the control of feed intake and energy metabolism. Most mammals increase their feed intake after parturition to cope with the increased energy and nutrient requirements for milk synthesis, thereby increasing their metabolic rate. Here we investigated in experiment 1 the regulation of plasma AEA and 2-AG concentrations during the transition from late pregnancy to early lactation in dairy cows, and analyzed in experiment 2 the expression of the EC system in the paraventricular nucleus (PVN) and the arcuate nucleus (ARC) of the hypothalamus of late and early lactating cows using immunohistochemistry. Cows in experiment 1 were retrospectively grouped based on peak plasma fatty acid concentrations to a high (H) or low (L) group. Feed intake was not different between groups before parturition, but was lower in H than L cows during early lactation. Plasma AEA and 2-AG concentrations increased 2.2- to 2.4-fold during early lactation, in which time plasma AEA concentrations rose faster in H cows than in L cows postpartum. Upregulation of N-acyl phosphatidylethanolamine-specific phospholipase D together with tending increased cannabinoid receptor 1 (CB1) expression, and downregulation of fatty acid amide hydrolase in early lactating cows suggested an increased PVN AEA tone. The abundance of CB1 in the ARC and diacylglycerol lipase-alpha was not different between late and early lactating cows, but PVN monoacylglycerol lipase expression was 30% higher in early lactating cows, indicating diminished PVN 2-AG concentrations. The results show a potential involvement of AEA in stimulating feed intake and of 2-AG in regulating energy metabolism of early lactating cows.