The embryo affects day 14 uterine transcriptome depending on the nutritional status in sheep

Transcriptomics and functional bioinformatics were used to investigate the potential interactions of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on uterine indicators of metabolism and reproduction. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition during 28 days: maintenance energy intake (control; 5 cyclic, 6 pregnant ewes) providing 7.8 MJ of metabolisable energy per ewe and 0.5 maintenance intake (undernourished; 6 cyclic, 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. RNA from uterine tissue was harvested at slaughter on day 14 of estrus or pregnancy, and hybridized to the Agilent 15K Sheep Microarray chip. Functional bioinformatics analyses were performed using the Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis. Among metabolic pathways, citrate cycle, oxidative phosphorylation, pentose and glucuronate interconversions and biosynthesis of unsaturated fatty acids were upregulated in control pregnant compared with control cyclic ewes. However, these pathways were not altered in undernourished ewes. The presence of an embryo in undernourished ewes upregulated fatty acid and glycogenic amino acid metabolism. RIG-I and Toll like receptors and chemokine signaling pathways were upregulated by the presence of the embryo in both control and undernourished ewes, but in the latter group the impact was lower. Undernutrition alone upregulated carbohydrate metabolism, but different pathways were altered in cyclic versus pregnant ewes. Citrate cycle, glycolysis/gluconeogenesis, and pentose and glucuronate interconversions were upregulated in undernourished cyclic compared with control cyclic ewes. Glycolysis/gluconeogenesis, pyruvate and propanoate metabolism, beta-alanine and phenylalanine metabolism were upregulated in undernourished pregnant compared with control pregnant ewes, whereas biosynthesis of unsaturated fatty acids and replication and repair were downregulated. Undernutrition alone led to an overall weak activation of immune system pathways both in cyclic and pregnant ewes. However, undernourished cyclic compared with control cyclic ewes had a high activation of NOD-like and RIG-1 like receptor signaling pathways, whereas undernourished pregnant compared with control pregnant ewes only had a weak upregulation of T cell receptor signaling pathways. Overall, data revealed that metabolic and immune adaptations of the uterus to nutrient restriction are dependent on the presence of the conceptus. 46 adult multiparous Rasa Aragonesa ewes were housed in individual pens and offered a diet (once daily) that provided 1 x live weight maintenance requirements for 1 month prior to the beginning of the experimental procedures [12]. The estrus cycles were synchronized using intravaginal sponges, which were inserted for 14 days. At the time of sponge insertion, ewes were allocated to one of two planes of nutrition: a control (n= 21; 7.8 MJ of metabolisable energy per ewe), which continued to receive the same diet for 28 days, and a low plane of nutrition (n = 25; 3.9 MJ of metabolisable energy per ewe), which was offered at 50% of estimated daily requirements (pellet and straw) until the end of the experiment. At the time of sponge withdrawal, ewes were injected with 300 IU, i.v., equine chorionic gonadotropin (Intervet, Salamanca, Spain), and the occurrence of estrus (Day 0) was monitored every 8 h. Thirteen control ewes and 18 ewes in the low plane of nutrition were mated to vasectomized or intact rams to establish a cyclic and pregnant group, respectively, within each plane of nutrition