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

P. dos; Y. Butenko; G. Kra; S. Malitsky; M. Itkin; Y. Levin; U. Moallem; M. Zachut

doi:10.3168/jds.2024-25409

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

P. dos, Y. Butenko, G. Kra, S. Malitsky, M. Itkin, Y. Levin, U. Moallem, M. Zachut^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

1 Citation (Scopus)

Abstract

Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Supplementation with n-3 fatty acid (FA) 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: (1) control (CTL; n = 12), prepartum with 250 g/d per cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); or (2) extruded flaxseed (FLX; n = 12) supplement rich in α-linolenic acid (C18:3n-3), prepartum with 700 g/d per cow and postpartum at 6.4% of diet (DM basis). 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 (GRC), 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 GRC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the ratio of n-6 to n-3 ratio in plasma, FF and GRC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (ARA; 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 GRC 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 GRC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GRC, n-3 FA supplementation reduced the relative mRNA abundances of cannabinoid receptors 1 and 2 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 GRC, whereas 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.

Original language	English
Pages (from-to)	4299-4317
Number of pages	19
Journal	Journal of Dairy Science
Volume	108
Issue number	4
Early online date	15 Jan 2025
DOIs	https://doi.org/10.3168/jds.2024-25409
Publication status	Published - Apr 2025

Funding

Funding This research was financially supported by the Chief Scientist of the Ministry of Agriculture, grant number 20-04-0015, Rishon Lezion, Israel. This research was financially supported by the Chief Scientist of the Ministry of Agriculture, grant number 20-04-0015, Rishon LeZion, Israel. The authors thank the staff of the Dairy Cattle Experimental Farm of the Volcani Institute (Rishon LeZion, Israel) for their assistance with animal care while conducting this experiment. The authors also wish to thank Valorex Company (France) for partly donating the fat supplements. Supplemental material for this article is available athttps://data.mendeley.com/datasets/n7v3mrmkch/1 (Supplemental File S1),https://data.mendeley.com/datasets/29fbd2nn4x/1 (Supplemental File S2), andhttps://data.mendeley.com/datasets/p9j6fpdxhy/1 (Supplemental File S3). Author contributions were as follows. Priscila Dos Santos Silva: methodology, project administration, data curation, visualization, formal analysis, validation, writing\u2013original draft, writing\u2013review and editing; Gitit Kra: methodology, project administration, data curation; Yana Butenko: formal analysis, writing\u2013review and editing; Sergey Malitsky: data curation, formal analysis, methodology, validation; Maxim Itkin: data curation, formal analysis, methodology, validation; Yishai Levin: data curation, formal analysis, methodology, validation; Uzi Moallem: data curation; writing\u2013review and editing; Maya Zachut: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing\u2013review and editing. The experimental protocol for this study was approved by the Volcani Institute Animal Care Committee (approval number IL 882/20), and it was performed in accordance with the relevant guidelines and regulations. The authors have not stated any conflicts of interest. Nonstandard abbreviations used: 1AdG = 1-docosatetraenoyl glycerol; 2AdG= 2-docosatetraenoyl glycerol; 2-AG = 2-arachidonoylglycerol; 1-HG = 1-dihomo-gamma-lineoyl glycerol; 2-HG = 2-dihomo-gamma-lineoyl glycerol; 1-LG = 1-linoleoyl glycerol; 2-LG = 2-linoleoyl glycerol; 1-PG = 1-palmitoyl glycerol; 2-PG = 2-palmitoyl glycerol; 1-POG = 1-palmitoleoyl glycerol; 2-POG = 2-palmitoleoyl glycerol; 1-SG = 1-stearoyl-rac-glycerol; 2-SG = 2-stearoyl-rac-glycerol; ACN = acetonitrile; AEA = anandamide; ALA = \u03B1-linolenic acid; ARA = arachidonic acid; ARA-Tau = N-arachidonoyl taurine; ARAG = arachidonoyl glycine; AU = arbitrary unit; CL = corpus luteum; Ct = cycle threshold; CTL = control; DAP = differentially abundant proteins; DEA = docosatetraenoyl ethanolamide; DEEA = docosaenoyl ethanolamide; DHA = docosahexaenoic acid; DHEA = docosahexaenoyl ethanolamide; DSEA = docosanoyl ethanolamide; DTEA = docosatrienoyl ethanolamide; E2 = estradiol; eCB = endocannabinoid; ECS = endocannabinoid system; EPA = eicosapentaenoic acid; EPEA = eicosapentaenoyl ethanolamide; ESI = electrospray ionization; ETEA = 5(Z),8(Z),11(Z)-eicosatrienoic acid ethanolamide; FA = fatty acid; FC = fold change; FF = follicular fluid; FLX = extruded flaxseed; GRC = granulosa cells; HEA = dihomo-gamma-linolenoyl ethanolamide; LA = linoleic acid; Lauroyl_EA = lauroyl ethanolamide; LCEA = lignoceroyl ethanolamide; LEA = linoleoyl ethanolamide; MEA = myristoyl ethanolamide; MTBE = methyl-tert-butyl-ether; N-OT = N-oleoyl taurine; N-P-glycine = N-palmitoyl glycine; O_Ala = oleoyl alanine; OEA = N-oleoylethanolamide; OG = oleoyl glycine; OS = oleoyl serine; P4 = progesterone; PBMC = peripheral blood mononuclear cells; PEA = N-palmitoylethanolamide; ROS = reactive oxygen species; RT-qPCR = reverse-transcription quantitative PCR; SEA = stearoyl ethanolamide; ULC = ultra liquid chromatography; UPLC = ultra-performance liquid chromatography; VEA = vaccenoyl ethanolamide; VS = vaccenoyl serine.

All Science Journal Classification (ASJC) codes

Food Science
Animal Science and Zoology
Genetics

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10.3168/jds.2024-25409Licence: CC BY

Cite this

dos, P., Butenko, Y., Kra, G., Malitsky, S., Itkin, M., Levin, Y., Moallem, U., & Zachut, M. (2025). Omega-3 fatty acid supplementation from late pregnancy to early lactation attenuates the endocannabinoid system and immune proteome in preovulatory follicles and endometrium of Holstein dairy cows. Journal of Dairy Science, 108(4), 4299-4317. https://doi.org/10.3168/jds.2024-25409

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

abstract = "Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Supplementation with n-3 fatty acid (FA) 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: (1) control (CTL; n = 12), prepartum with 250 g/d per cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); or (2) extruded flaxseed (FLX; n = 12) supplement rich in α-linolenic acid (C18:3n-3), prepartum with 700 g/d per cow and postpartum at 6.4% of diet (DM basis). 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 (GRC), 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 GRC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the ratio of n-6 to n-3 ratio in plasma, FF and GRC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (ARA; 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 GRC 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 GRC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GRC, n-3 FA supplementation reduced the relative mRNA abundances of cannabinoid receptors 1 and 2 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 GRC, whereas 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.",

author = "P. dos and Y. Butenko and G. Kra and S. Malitsky and M. Itkin and Y. Levin and U. Moallem and M. Zachut",

note = "Publisher Copyright: {\textcopyright} 2025 American Dairy Science Association",

year = "2025",

month = apr,

doi = "10.3168/jds.2024-25409",

language = "English",

volume = "108",

pages = "4299--4317",

journal = "Journal of Dairy Science",

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dos, P, Butenko, Y, Kra, G, Malitsky, S , Itkin, M , Levin, Y, Moallem, U & Zachut, M 2025, 'Omega-3 fatty acid supplementation from late pregnancy to early lactation attenuates the endocannabinoid system and immune proteome in preovulatory follicles and endometrium of Holstein dairy cows', Journal of Dairy Science, vol. 108, no. 4, pp. 4299-4317. https://doi.org/10.3168/jds.2024-25409

Omega-3 fatty acid supplementation from late pregnancy to early lactation attenuates the endocannabinoid system and immune proteome in preovulatory follicles and endometrium of Holstein dairy cows. / dos, P.; Butenko, Y.; Kra, G. et al.
In: Journal of Dairy Science, Vol. 108, No. 4, 04.2025, p. 4299-4317.

Research output: Contribution to journal › Article › peer-review

TY - JOUR

T1 - Omega-3 fatty acid supplementation from late pregnancy to early lactation attenuates the endocannabinoid system and immune proteome in preovulatory follicles and endometrium of Holstein dairy cows

AU - dos, P.

AU - Butenko, Y.

AU - Kra, G.

AU - Malitsky, S.

AU - Itkin, M.

AU - Levin, Y.

AU - Moallem, U.

AU - Zachut, M.

PY - 2025/4

Y1 - 2025/4

N2 - Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Supplementation with n-3 fatty acid (FA) 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: (1) control (CTL; n = 12), prepartum with 250 g/d per cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); or (2) extruded flaxseed (FLX; n = 12) supplement rich in α-linolenic acid (C18:3n-3), prepartum with 700 g/d per cow and postpartum at 6.4% of diet (DM basis). 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 (GRC), 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 GRC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the ratio of n-6 to n-3 ratio in plasma, FF and GRC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (ARA; 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 GRC 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 GRC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GRC, n-3 FA supplementation reduced the relative mRNA abundances of cannabinoid receptors 1 and 2 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 GRC, whereas 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.

AB - Activation of the endocannabinoid system (ECS) elicits negative effects on the reproductive system in mammals. Supplementation with n-3 fatty acid (FA) 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: (1) control (CTL; n = 12), prepartum with 250 g/d per cow calcium salts of FA and postpartum at 1.6% of the diet (DM basis); or (2) extruded flaxseed (FLX; n = 12) supplement rich in α-linolenic acid (C18:3n-3), prepartum with 700 g/d per cow and postpartum at 6.4% of diet (DM basis). 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 (GRC), 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 GRC and endometrium. Supplementation of n-3 FA increased the proportion of total n-3 FA and decreased the ratio of n-6 to n-3 ratio in plasma, FF and GRC compared with CTL. In plasma and FF, n-3 FA supplementation decreased the proportion of the n-6 FA eCB precursor arachidonic acid (ARA; 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 GRC 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 GRC than in CTL. In the endometrium, n-3 FA supplementation decreased the abundance of a few immune-related proteins. In the GRC, n-3 FA supplementation reduced the relative mRNA abundances of cannabinoid receptors 1 and 2 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 GRC, whereas 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.

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

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