Omega-3 fatty acids supplementation from late pregnancy to early lactation attenuates the endocannabinoid system and immune proteome in preovulatory follicles and endometrium of Holstein dairy cows

Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Omega-3 (n-3) fatty acid (FA) supplementation lowers ECS activation and has anti-inflammatory effects. Thus, we hypothesized that supplementing cows with n-3 FA will downregulate components of the ECS and immune system in preovulatory follicles and in the endometrium. Twenty-four multiparous Holstein dairy cows were supplemented from d 256 of pregnancy to d 70 postpartum as follows: (i) control (CTL; n = 12) prepartum with 250 g/d/cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); (ii) FLX (n = 12) prepartum with 700 g/d/cow of extruded flaxseed supplement rich in α-linolenic acid (ALA, C18:3n-3), and postpartum at 6.4% of diet (DM basis) of the same supplement. Ovaries were monitored at 30 DIM, and following estrous cycle synchronization we aspirated the follicular fluids (FF) of follicles ≥7 mm, separated the granulosa cells (GC), and performed endometrium biopsies at 58 ± 5 DIM. The FF were analyzed for concentrations of estradiol (E₂) and progesterone (P₄), and E₂-active follicles were declared when E₂/P₄ was >1. The FA and endocannabinoid (eCB) profiles were determined in plasma and in the reproductive tissues. Proteomic analyses and mRNA abundances were determined in GC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the omega-6 (n-6) to n-3 FA ratio in plasma, FF and GC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (AA; C20:4n-6), and increased the abundance of the n-3 FA-derived eCB eicosapentaenoyl ethanolamide compared with CTL. In the endometrium, n-3 FA supplementation reduced the abundance of the n-6 FA-derived eCB 2-arachidonoylglycerol (2-AG) compared with CTL. Proteomic analysis of GC showed that n-3 FA supplementation increased the abundance of FA-binding-protein-5, which is involved in intracellular transport of eCB, as well as the abundances of the cytokine-receptor-like-factor-2 and glutathione-S-transferase-LANCL1, whereas it reduced the abundances of several complement proteins: complement factors I, D, H, complement components C7 chain and C8 β chain, and complement component 1 Q subcomponent-binding protein, mitochondrial (C1QBP). In addition, the abundance of superoxide-dismutase (SOD3) was lower in FLX GC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GC, n-3 FA supplementation reduced the relative mRNA abundances of type 1 and type 2 cannabinoids receptors compared with CTL. Across treatments, a positive correlation was found between the relative abundance in FF of the eCB anandamide with C7, C1QBP and SOD3 in GC; while FF 2-AG had a negative correlation with them. Overall, in line with our premise, dietary n-3 FA supplementation attenuated the levels of some eCB and reduced the expression of several proteins and genes related to the ECS and immune system in the preovulatory follicle and in the endometrium, which may be part of the etiology of the positive effects of n-3 FA on the reproductive system in dairy cows.