Effects of dietary n-3-PUFA supplementation, post-insemination plane of nutrition and pregnancy status on the endometrial transcriptome of beef heifers

Reproductive performance in cattle is influenced by many management factors, including diet. Supplementing diets with the n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been shown to enhance traits in cattle and may improve reproductive status. Profitable beef systems require compact calving, which depends on successful establishment and maintenance of pregnancy. The greatest increment of reproductive wastage arises from early embryonic loss, with approximately 80% occurring prior to Day 14–16 post-insemination, coinciding with maternal recognition of pregnancy in ruminants. n-3 PUFA supplementation has been postulated to aid maternal recognition and implantation by suppressing uterine secretion of luteolytic prostaglandin F2α (PGF2α), with evidence for inhibition by EPA/DHA in vitro and in vivo. Differences between dairy and beef cows suggest dietary manipulation may have distinct effects in beef systems, yet prior work often failed to detect effects of omega-3 supplementation on embryo viability or pregnancy rate in beef cattle, potentially due to overall positive energy status. Fluctuations in post-AI dietary energy can reduce conception rates, and endometrial transcriptomic remodeling up to maternal recognition occurs in both cyclic and pregnant cattle, with divergence around Day 16. The aim of this study was to examine the individual and interactive effects of plane of nutrition, dietary n-3 PUFA supplementation, and pregnancy status on the global endometrial transcriptome during maternal recognition of pregnancy.

Previous studies in dairy cows examined fatty acid-based supplements and reproductive processes, but fewer data exist for beef cattle. Physiological differences and energy status between dairy and beef cows may alter responses. Several studies indicate n-3 PUFA can suppress uterine PGF2α secretion and may aid conceptus implantation, yet some work (including by the present group) did not find improvements in embryo viability or pregnancy rates in beef cattle. Nutritional restriction post-AI can markedly reduce conception rates in heifers. Endometrial transcriptome changes preparing for implantation occur in both cyclic and pregnant cows prior to Day 16; significant differences emerge around maternal recognition. Prior transcriptomic work suggests n-3 PUFA can alter uterine transcription factors and genes involved in prostaglandin biosynthesis, indicating potential to influence uterine receptivity. The role of post-insemination plane of nutrition on uterine function, particularly in the context of n-3 PUFA supplementation, is less well characterized.

Design: 2×2 factorial with pre-breeding diet (Control vs n-3 PUFA) and post-insemination plane of nutrition (High vs Low). Heifers: Reproductively normal nulliparous crossbred beef heifers. Estrous synchronization: Two IM injections of 500 µg PGF2α analogue 11 days apart; AI performed ~12 h after estrus detection using semen from a single high fertility sire. Pre-insemination diet (30 days): Control diet included a rumen-protected palmitic acid source (Palmit80) or n-3 PUFA diet included 275 g partially rumen-protected fish oil-derived EPA:DHA (1.5:1), achieving 4.15% fish oil of total DM; diets isonitrogenous and isolipidic (8% added lipid DM). Post-insemination diets (Day 0 to Day 16): High (remain on pre-insemination high plane) or Low (0.6× maintenance energy; 2 kg DM concentrate with same lipid supplement plus 0.85 kg DM barley straw). Group sizes for the broader study: CON_Low n=14, CON_High n=14, PUFA_Low n=15, PUFA_High n=17. Sampling: On Day 16 post-AI, heifers were slaughtered; reproductive tracts flushed with PBS + 5% FCS to confirm pregnancy by conceptus recovery. Pregnancy outcomes within groups: Control High: 11 pregnant/3 non-pregnant; Control Low: 11/3; PUFA High: 10/5; PUFA Low: 13/4. Endometrium from antimesometrial border of horn ipsilateral to CL was dissected, snap frozen. RNA extraction: Qiagen RNeasy; quality assessed by Nanodrop and Agilent Bioanalyzer; RIN 8–10 required. Library prep and sequencing: Illumina TruSeq; 3 µg RNA per sample; libraries validated (~260 bp fragments, >30 ng/µl), sequenced on Illumina HiSeq 2000, 100 bp single-end reads. RNAseq analysis: Quality check with FASTQC v0.10.0. Alignment with TopHat v2.0.9 to bovine UMD3.1 genome. Gene counts with HTSeq v0.5.4p5. Differential expression with edgeR (R v2.14.1; edgeR v3.4.1); thresholds: FDR < 0.1% (Benjamini-Hochberg) and |fold change| > 1.5. Comparisons conducted to assess effects of n-3 PUFA, post-insemination plane of nutrition, and pregnancy status; Venn diagrams via Venny 2.1. Pathway analysis: Bovine-to-human ortholog mapping via Ensembl BioMart; KEGG pathway enrichment; Ingenuity Pathway Analysis (IPA v8.8) for networks, canonical pathways, and biological functions; significance by right-tailed Fisher’s exact test.

- n-3 PUFA supplementation effects: - Non-pregnant heifers: Control vs n-3 PUFA showed no DEG (low plane context) and 335 DEG under high plane (257 lower, 78 higher in Control vs PUFA non-pregnant). - Pregnant heifers: Control vs n-3 PUFA on Low plane yielded 429 DEG (225 lower, 204 higher in Control); enriched pathways included eIF2 signaling, mTOR signaling, and embryonic development. Under High plane, only 1 DEG (GJB2; higher in Control). - Specific genes: Cathepsin V (CTSV) higher with PUFA; UPK3BL ~3× higher with PUFA; GBP2 >5× higher in Control vs PUFA. - Post-insemination plane of nutrition effects: - Within Control group: Low vs High led to 13 DEG in non-pregnant (all lower on Low) and 2 DEG in pregnant (both lower on Low; one was OXTR ~10× lower on Low). Several ISGs (MX1, MX2, IFIT1, IFIT3, RSAD2) were reduced on Low. - Within n-3 PUFA group: Low vs High resulted in 1123 DEG in non-pregnant (762 decreased, 361 increased on Low) and 844 DEG in pregnant (502 decreased, 342 increased on Low). Genes associated with implantation (e.g., CA1, SLC13A5, TNR) were reduced on Low; many ribosomal genes were upregulated on Low (potential increased protein synthesis); multiple zinc finger genes reduced on Low. - Interaction (n-3 PUFA × post-insemination diet) in pregnant animals: 1919 DEG; enriched networks included cellular development, cell signaling, cellular movement, embryonic development; KEGG oxytocin signaling enriched (P<0.01). Biological functions: embryonic development among top affected categories. - Pregnancy status effects on endometrium: - Control Low: 81 DEG (76 higher, 5 lower in pregnant); enriched antimicrobial/inflammatory (interferon) networks; canonical interferon signaling enriched. - Control High: 0 DEG. - PUFA Low: 828 DEG (620 lower, 208 higher in non-pregnant vs pregnant); top canonical pathway interferon signaling; multiple ISGs (e.g., ISG20 ~44× lower and ISG15 >28× lower in non-pregnant). - PUFA High: 493 DEG (90 higher, 403 lower in pregnant vs non-pregnant); networks included embryonic/organismal development; genes involved in estrogen pathways (e.g., EBAG9, ESRRA) higher in non-pregnant. Overall, n-3 PUFA supplementation modulated fertility-related pathways (e.g., mTOR, interferon, oxytocin) and embryonic development networks, with pronounced effects under nutritional restriction. Adequate post-insemination nutrition attenuated many transcriptomic differences.

The study addressed whether dietary n-3 PUFA and post-insemination nutrient supply alter the uterine endometrial transcriptome around maternal recognition of pregnancy. Results show that n-3 PUFA supplementation modulates key fertility-related genes and pathways (mTOR, eIF2, embryonic development) particularly when heifers experience a reduced post-insemination plane of nutrition. In contrast, when animals remained on a higher nutritional plane, n-3 PUFA had minimal impact on the endometrial transcriptome in pregnant heifers (only 1 DEG). Post-insemination nutritional restriction broadly altered gene expression in n-3 PUFA-supplemented heifers (hundreds to thousands of DEG), including reductions in genes linked to implantation (e.g., CA1, TNR, SLC13A5) and zinc finger factors, suggesting potential detriments to uterine receptivity when energy intake is curtailed, even with PUFA supplementation. Pregnancy status robustly influenced interferon-stimulated gene expression, consistent with conceptus-derived IFN signaling, particularly evident under Low plane diets. The observed enrichment of oxytocin signaling and modulation of OXTR aligns with mechanisms of luteolysis prevention during early pregnancy. Collectively, these findings suggest n-3 PUFA can beneficially influence uterine molecular environments conducive to pregnancy establishment, with effects most apparent under nutritional stress, whereas sufficient post-insemination nutrition may buffer or mask such effects.

Dietary n-3 PUFA supplementation and post-insemination plane of nutrition interact to shape the bovine endometrial transcriptome during maternal recognition of pregnancy. n-3 PUFA altered expression of genes and pathways related to embryonic development and nutrient sensing (e.g., mTOR), with the strongest effects evident in animals under a restricted post-insemination diet. Adequate nutrition post-AI minimized transcriptomic differences in pregnant heifers. Pregnancy status drove expected interferon-related signatures. These results provide novel insights into diet–uterus interactions that may influence embryo survival in beef heifers. Future work should validate these transcriptomic signatures with functional assays and proteomics, and further delineate the physiological consequences for implantation and pregnancy maintenance under varying nutritional contexts.

The study assessed transcriptomic changes at a single time point (Day 16) and did not find corresponding clear effects on embryo survival in the precursor study, complicating functional interpretation. Some gene expression changes (e.g., OXTR under Low nutrition) were not fully explained. Findings are based on RNA-level data without proteomic or functional validation; authors note the need for follow-up functional assays and global proteomics to confirm molecular effects. The RNAseq subset included 60 heifers, which may limit power for some comparisons.