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

Reproductive efficiency in beef cattle is crucial for economic viability, and early embryonic loss represents a significant portion of reproductive wastage. Improving pregnancy establishment is a key goal for enhancing profitability. Dietary manipulation, specifically supplementation with n-3 PUFAs like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has shown promise in improving reproductive outcomes in cattle. The proposed mechanism involves the suppression of luteolytic prostaglandin F2α (PGF2α) secretion, promoting successful maternal recognition of pregnancy and implantation. However, the impact of n-3 PUFA supplementation in beef cattle remains less explored than in dairy cattle, particularly when considering the interplay with fluctuations in dietary energy intake during early pregnancy. Short-term changes in feed availability, commonly experienced in seasonal grazing systems, can dramatically reduce conception rates. This study aimed to investigate the combined effects of n-3 PUFA supplementation and post-insemination plane of nutrition on the endometrial transcriptome, focusing on the period of maternal recognition of pregnancy (Day 16 post-insemination) and considering the influence of pregnancy status on gene expression.

Several studies have demonstrated the positive effects of n-3 PUFA supplementation on various aspects of reproductive performance in dairy cows. These effects include improved pregnancy rates and reduced embryonic loss. The mechanisms are often linked to altered prostaglandin production and improved uterine environment. However, research on n-3 PUFA supplementation in beef cattle is limited, and there's a need to investigate the effects specifically in this population. Furthermore, few studies have investigated how post-insemination plane of nutrition influences endometrial transcriptome, specifically in combination with n-3 PUFA supplementation. In ruminants, the endometrial transcriptome undergoes significant orchestrated changes in preparation for implantation. Progesterone plays a key role in this process, regulating endometrial function crucial for conceptus growth, implantation, placentation, and fetal development. By Day 16 in cattle, noticeable differences in endometrial transcriptome emerge between pregnant and cyclic animals, emphasizing the critical role of this period in pregnancy establishment.

This study employed a 2x2 factorial design. 179 nulliparous crossbred beef heifers had their estrous cycles synchronized using PGF2α. Heifers were randomly assigned to one of two pre-breeding dietary treatments: a control diet or a diet supplemented with a high level of n-3 PUFA (4.15% fish oil). Both diets were isonitrogenous and isolipidic. The pre-breeding diet was administered for 30 days prior to induced estrus and artificial insemination (AI). After AI (Day 0), heifers were allocated to either a high or low post-insemination plane of nutrition. The high plane maintained the pre-insemination diet, while the low plane restricted energy intake to 0.6 times estimated maintenance requirements. This post-insemination diet was maintained for 16 days, until Day 16 when heifers were slaughtered. Pregnancy status was confirmed by flushing uterine horns and examining for a conceptus. Endometrial tissue samples from the antimesometrial border of the uterine horn ipsilateral to the corpus luteum were collected, snap frozen, and stored at -80°C. RNA was isolated, purified, and subjected to RNA sequencing. Differential gene expression analysis was performed using EdgeR, with a false discovery rate (FDR) <0.1% and fold change >1.5 defining differentially expressed genes (DEGs). Pathway analysis was conducted using BioMart, KEGG, and Ingenuity Pathway Analysis (IPA) to identify enriched pathways and networks related to the identified DEGs.

The study identified significant differences in endometrial gene expression based on post-insemination dietary treatments and pregnancy status. In non-pregnant heifers, n-3 PUFA supplementation did not alter gene expression. However, in pregnant heifers, n-3 PUFA supplementation led to 429 DEGs (225 downregulated and 204 upregulated compared to the control group) when heifers were on a low post-insemination plane of nutrition. Pathway analysis revealed that several networks including embryonic development, nervous system development and organ development were associated with these DEGs. Specifically, the mTOR signaling pathway, crucial for cell growth and proliferation during implantation, was enriched. Analysis of the effect of post-insemination nutritional plane within each dietary group revealed a substantial number of DEGs in n-3 PUFA-supplemented pregnant heifers (1123 DEGs) with the lower dietary allowance, while only a small number of changes were observed in control heifers. Networks associated with cellular development, growth and proliferation, and embryonic development were significantly enriched. The interaction between n-3 PUFA supplementation and post-insemination diet in pregnant heifers resulted in 1919 DEGs. Enriched networks and pathways in this interaction included oxytocin signaling and embryonic development. In pregnant compared to non-pregnant heifers, differential gene expression analyses revealed variations in the expression of interferon-stimulated genes (ISGs) associated with maternal immune response. This variation was observed in both control and n-3 PUFA-supplemented groups and was more pronounced in heifers on the low post-insemination plane of nutrition.

The findings suggest a beneficial role for n-3 PUFA supplementation, particularly when coupled with a low post-insemination plane of nutrition, for pregnant heifers. The observed changes in gene expression profiles, especially those related to embryonic development and mTOR signaling, support the hypothesis that n-3 PUFA supplementation might enhance pregnancy establishment even under nutrient-restricted conditions. The enrichment of mTOR signaling pathway in n-3 PUFA-supplemented pregnant heifers on low post-insemination diets is noteworthy because mTOR is known to be crucial in nutrient sensing and regulation of cell growth and proliferation during implantation. Furthermore, the impact of ISGs on uterine immune response underscores the complex interaction between nutrition, immune function, and pregnancy outcomes. The contrasting gene expression patterns in high vs. low post-insemination nutrition in n-3 PUFA-supplemented heifers suggest that nutritional consistency is vital during the peri-implantation period. The results highlight the potential of n-3 PUFA to modulate various aspects of uterine function, leading to improved reproductive outcomes, especially when facing challenges such as short-term nutritional fluctuations.

This study provides novel insights into the intricate interplay between dietary n-3 PUFA supplementation, post-insemination plane of nutrition, and the endometrial transcriptome in beef heifers. The results suggest a positive effect of n-3 PUFA supplementation on pregnancy establishment, particularly under nutrient-restricted conditions, likely via modulation of key pathways like mTOR signaling. Future research should focus on validating these findings through functional assays and proteomics studies, and exploring the long-term effects of n-3 PUFA supplementation on pregnancy maintenance and calf health. Further investigation into the specific mechanisms by which n-3 PUFAs influence gene expression and uterine function is warranted.

The study's limitations include the relatively small sample size for each treatment group. Although the study used a large number of heifers in the experiment, the sample size for each of the experimental group used for RNAseq was relatively small which would limit the statistical power and ability to identify subtle changes in gene expression. The crossbred nature of the heifers might restrict the generalizability of the findings to other breeds. The study was conducted under controlled conditions, and the results may not fully reflect the complex environmental factors influencing reproductive performance under field conditions. Additional research exploring alternative pathways and specific protein analysis will further enhance our understanding of the complex molecular interactions involved.