TECHNICAL NOTE: Analysis of volatile fatty acids in rumen fluid by gas chromatography mass spectrometry using a dimethyl carbonate extraction.

Analysis of rumen fluid volatile fatty acids (VFA) is typically conducted by injecting acidified aqueous rumen fluid into a gas chromatograph (GC) with a flame ionization detector (FID). Aqueous samples are highly problematic because of the large vapor volume that can lead to poor peak shape and contamination of inlets, potentially causing sample carryover. Methods using aqueous samples are not well suited for use in a mass spectrometer (MS) detector system. The objective of this project was to validate a dimethyl carbonate (DMC) extraction process and GCMS method for rumen VFA analysis. To perform the extraction, 100 µL of sample, KHSO4 (500 g/L), and 2-ethylbutyrate (internal standard; 8.5 mM) were added to a microcentrifuge tube (in order) followed by 1 mL of DMC. The mixture was thoroughly vortexed and centrifuged. The organic layer (top) was removed and placed in a GC vial. The DMC extract was injected (0.5 µL) into an Agilent 5977B GCMS (8:1 split injection) with a polar DB-FFAP column. The column was held at 105 °C for 5 min, increased at 10 °C/min to 150 °C, then 65 °C/min to 240 °C, and held constant for 10 min. The peak area of acetate relative to the internal standard is linear from approximately 2 mM to at least 130 mM and encompasses the expected values of rumen concentrations for the other VFA. Recovery of VFA from spiked rumen fluid was tested at three concentrations in rumen fluid from steers fed a finishing diet or grazing wheat pasture. Recovery was not affected by the diet of the animals (P > 0.10) or the amount of VFA spiked (P > 0.19) for acetate, propionate, isobutyrate, or butyrate. There was an interaction of amount of VFA spiked and the diet of the animal (P = 0.021) for valerate and a tendency for an interaction (P = 0.051) for isovalerate, due to the recovery of the VFA being lower in the medium spike amount in rumen fluid from cattle on wheat pasture. Overall, recovery was greatest for propionate (101.9 ± 1.67%) and lowest for valerate (95.7 ± 1.95%). Including the 10-min hold at 240 °C at the end of each run prevented carryover from sample to sample. This method appears to perform well in a GCMS system and accurately and precisely quantifies rumen fluid VFA.

Scientists need to measure the end-products of microbial digestion called volatile fatty acids in the gastrointestinal tract of ruminants for many reasons. The methods currently available for the analysis of volatile fatty acids have evolved over time along with new technology. Many of the methods available are either not compatible with a mass spectrometer detector or require hazardous chemicals to prepare the samples. The method described here uses a less-hazardous chemical to prepare the samples for analysis with a mass spectrometer detector. The method tested in this manuscript was adequate in preparing samples for volatile fatty acid analysis.

Energy balance affects pulsatile secretion of luteinizing hormone from the adenohypophesis and expression of neurokinin B in the hypothalamus of ovariectomized gilts.

The pubertal transition of gonadotropin secretion in pigs is metabolically gated. Kisspeptin (KISS1) and neurokinin B (NKB) are coexpressed in neurons within the arcuate nucleus of the hypothalamus (ARC) and are thought to play an important role in the integration of nutrition and metabolic state with the reproductive neuroendocrine axis. The hypothesis that circulating concentrations of luteinizing hormone (LH) and expression of KISS1 and tachykinin 3(TAC3, encodes NKB) in the ARC of female pigs are reduced with negative energy balance was tested using ovariectomized, prepubertal gilts fed to either gain or lose body weight. Restricted feeding of ovariectomized gilts caused a rapid and sustained metabolic response characterized by reduced concentrations of plasma urea nitrogen, insulin, leptin, and insulin-like growth factor-1 and elevated concentrations of free fatty acids. The secretory pattern of LH shifted from one of low amplitude to one of high amplitude, which caused overall circulating concentrations of LH to be greater in restricted gilts. Nutrient-restricted gilts had greater expression of follicle-stimulating hormone and gonadotropin-releasing hormone receptor, but not LH in the anterior pituitary gland. Expression of KISS1 in the ARC was not affected by dietary treatment, but expression of TAC3 was greater in restricted gilts. These data are consistent with the idea that hypothalamic expression of KISS1 is correlated with the number of LH pulse in pig, and further indicate that amplitude of LH pulses may be regulated by NKB in the gilt.

The effects of Capn1 gene inactivation on the differential expression of genes in skeletal muscle.

Protein turnover is required for muscle growth and regeneration and several proteolytic enzymes, including the calpains, degrade myofibrillar proteins during this process. In a previous experiment, phenotypic differences were observed between µ-calpain knockout (KO) and wild type (WT) mice, including nutrient accretion and fiber type differences. These changes were particularly evident as the animals aged. Thus, we utilized 18 mice (9 KO and 9 WT) to compare transcript abundance to identify differentially expressed genes (DEGs) at 52 wk of age. A total of 55 genes were differentially expressed, including adiponectin, phosphoenolpyruvate carboxykinase 1, uncoupling protein 1, and lysine deficient protein kinase 2. These genes were analyzed for over- and underrepresented gene ontology (GO) terms. Several GO terms, including response to cytokine, response to interferon-beta, regulation of protein phosphorylation, and hydrolase activity, were identified as overrepresented. Pathways related to taurine biosynthesis, nitric oxide synthase signaling, amyloid processing, and L-cysteine degradation were also identified. Our results are consistent with previous experiments, in that identified DEGs may explain, at least in part, some of the phenotypic differences between µ-calpain KO and WT mice. Clearly muscle growth and maintenance are complex, multifaceted processes. Genes affected by the silencing of the µ-calpain gene have been identified, but the relationship between µ-calpain and these pathways requires further investigation.