Long-term effects of prior diets, dietary transition and pregnancy on adipose gene expression in dairy heifers.

Adipose tissue is highly involved in whole-body metabolism and is the main site for lipid synthesis, storage and mobilization in ruminants. Therefore, knowledge about adipose tissue responses to different diets is important, especially in growing heifers as the feeding regimes of replacement heifers affect their future success as dairy cows. However, at gene expression level such knowledge is limited. As part of a larger feed trial, adipose tissue biopsies from 24 Norwegian Red heifers were collected at 12 months of age (12MO) and at month seven of gestation (PREG) and analyzed by next-generation mRNA sequencing. Between these two sampling points, all heifers had gone through a successful conception and a feed change from four dietary treatments of high or low energy (HE/LE) and protein (HP/LP) content (treatments LPHE, HPHE, LPLE and HPLE) to a low-energy, low-protein pregnancy feed given to all animals. Gene expression differences between different feed treatments at 12MO are described in an earlier publication from our group. The main objectives of this study were to investigate the long-term effects of diets differing in protein and energy density level on gene expression in adipose tissue of growing replacement dairy heifers. To achieve this, we examined the post-treatment effects between the treatment groups at month seven of gestation; 6 months after the termination of experimental feeding, and the long-term gene expression changes occurring in the adipose tissue between 12MO and PREG. Post-treatment group comparisons showed evidence of long-term effects of dietary treatment on adipose gene expression. Differences between protein treatments were smaller than between energy treatments. Adipose gene expression changes from 12MO to PREG were much larger for the HE than the LE treatments and seemed to mostly be explained by the characteristics of the diet change. 97 genes displayed a unidirectional expression change for all groups from 12MO to PREG, and are considered to be treatment-independent, possibly caused by pregnancy or increased age. This study provides candidate genes and key regulators for further studies on pregnancy preservation (TGFB1, CFD) and metabolic regulation and efficiency (PI3K, RICTOR, MAP4K4,) in dairy cattle.

RNA-seq analysis of bovine adipose tissue in heifers fed diets differing in energy and protein content.

Adipose tissue is no longer considered a mere energy reserve, but a metabolically and hormonally active organ strongly associated with the regulation of whole-body metabolism. Knowledge of adipose metabolic regulatory function is of great importance in cattle management, as it affects the efficiency and manner with which an animal converts feedstuff to milk, meat and fat. However, the molecular mechanisms regulating metabolism in bovine adipose tissue are still not fully elucidated. The emergence of next-generation sequencing technologies has facilitated the analysis of metabolic function and regulation at the global gene expression level. The aim of this study was to investigate the effect of diets differing in protein and energy density level on gene expression in adipose tissue of growing replacement dairy heifers using next-generation RNA sequencing (RNAseq). Norwegian Red heifers were fed either a high- or low-protein concentrate (HP/LP) and a high- or low-energy roughage (HE/LE) diet from 3 months of age until confirmed pregnancy to give four treatments (viz, HPHE, HPLE, LPHE, LPLE) with different growth profiles. Subcutaneous adipose tissue sampled at 12 months of age was analyzed for gene expression differences using RNAseq. The largest difference in gene expression was found between LPHE and LPLE heifers, for which 1092 genes were significantly differentially expressed, representing an up-regulation of mitochondrial function, lipid, carbohydrate and amino acid metabolism as well as changes in the antioxidant system in adipose tissue of LPHE heifers. Differences between HPHE and HPLE heifers were much smaller, and dominated by genes representing NAD biosynthesis, as was the significantly differentially expressed genes (DEG) common to both HE-LE contrasts. Differences between HP and LP groups within each energy treatment were minimal. This study emphasizes the importance of transcriptional regulation of adipose tissue energy metabolism, and identifies candidate genes for further studies on early-stage obesity and glucose load in dairy cattle.

Condensed tannins from Sesbania sesban and Desmodium intortum as a means of Haemonchus contortus control in goats.

Two experiments were conducted to determine whether the proanthocyanidin (condensed tannin)-containing forage legumes Desmodium intortum cv Greenleaf and Sesbania sesban (accession 15019) could be integrated into a feeding management strategy as a means of Haemonchus contortus control in goats. The anthelmintic effects of condensed tannin extracts from the two legumes on H. contortus L(3) larvae were studied in an in vitro larval migration inhibition system. The extracts inhibited larval migration in a dose-dependent manner, and at concentrations from 1,000 µg/ml condensed tannin, the extract from D. intortum caused a significantly higher inhibition of larval migration than did the corresponding concentrations of the S. sesban extract (P < 0.01). Prolonged feeding of tanniniferous forage legumes showed that animals receiving D. intortum had the lowest total worm burden, the lowest female to male parasite ratio, the lowest number of eggs in the uterus of each female worm and the lowest per capita fecundity (P < 0.01). However, there was no change in the performance (weight gain) of parasite-infected goats probably due to incomplete removal of the parasite or prolonged confinement of goats in small pens, which calls for further investigation. However, since there is no single efficient method in control of parasites, based on the obtained data from this experiment, integrated feeding of D. intortum with other suitable method of parasite control is thus suggested.

A novel electroporation device for gene delivery in large animals and humans.

Intramuscular injection of plasmid DNA followed by electrical stimulation (electroporation) is an efficient method for achieving therapeutic levels of encoded proteins or eliciting efficient immune responses in smaller animals such as mice and rats. Electroporation in larger animals and humans poses new technical challenges, the main difficulty being to maintain efficacy while limiting invasiveness and pain. Here we present data using a new device for combined injection and electroporation in large animals and humans. The device injects DNA through two needles during insertion into the muscle and thus distributes the injection volume along the needles which also serve as electrodes. Since the electrical field is strongest close to the needle-electrode, a near perfect match between the DNA and the electric field is achieved. We show that using moderate amounts of DNA: (1) muscle tissue is transfected along the entire length of the needle path, (2) the efficacy is higher compared to when the DNA is injected between the electrodes, (3) level of protein expression can be tightly controlled by the number of treatments, and (4) efficient immunization is achieved.