RESUMO
The objective of this study was to determine the effect of time of rumen fluid collection relative to feeding on gas production kinetics for in vitro rumen fermentation. Three ruminally cannulated Holstein heifers were rumen fluid donors. Feed was removed from heifers 12 h prior to feeding, rumen fluid was collected from each heifer before feeding (0 h), and at 2, 4, and 6 h after feeding, repeated on three separate incubation days. Buffered rumen fluid (100 mL) was incubated in 250-mL bottles containing 1.4 g of dried TMR, in duplicate for each heifer at each collection time. All bottles were incubated for 24 h at 39°C and constant agitation (60 rpm), and capped with monitors to capture temperature and pressure every 15 min (RF1, Ankom Technology, Macedon, NY). At the end of incubation, final pH was recorded. Gas data were fit with nonlinear regression comparison of fit in GraphPad Prism 7 to find best fit regression. The formula with best fit was as follows: y = Vm(1 - (e(-Kd(x - lag)))), where y is gas produced at time X (mmol), Vm is the asymptotic gas production (mmol), Kd indicates the fractional rate of gas production (mmol/h), X is time (h), and lag refers to the lag time before the start of fermentation (h) as indicated by positive gas production (R 2 = 0.98). Data were analyzed using PROC GLIMMIX of SAS, with donor as the experimental unit and day as the random blocking factor; significance is defined as P ≤ 0.05. Time of rumen fluid collection significantly affected gas production kinetics (lag P = 0.01, Vm P = 0.03, Kd P <0.0001). Gas production was highest in fermenter units fed with rumen fluid collected 2-h post-feeding. Fractional rate of fermentation (Kd) was fastest in rumen fluid collected at 0 h. Lag time was longest in rumen fluid collected at 4-h post-feeding and slowest in rumen fluid collected at 0 h. Time of rumen fluid collection did not alter final pH. Our findings suggest that gas production is maximized when rumen fluid is collected between 2 and 4 h after feeding.
RESUMO
The objective of this study was to determine the effect of time of rumen fluid collection relative to feeding on volatile fatty acid (VFA) production for in vitro rumen fermentation. Three ruminally cannulated Holstein heifers were used as rumen fluid donors. Feed was removed from heifers 12 h prior to feeding, rumen fluid was collected from each heifer before feeding (0 h), and at 2, 4, and 6 h after feeding, repeated on three separate incubation days. Buffered rumen fluid (100 mL) was incubated in 250-mL bottles containing 1.4 g of dried TMR, in duplicate for each heifer at each collection time. All bottles were incubated for 24 h at 39°C and constant agitation (60 rpm), and capped with monitors to capture temperature and pressure every 15 min (RF1, Ankom Technology, Macedon, NY). At the end of incubation, final pH and a sample of rumen fluid were collected for VFA and ammonia nitrogen. Data were analyzed using PROC GLIMMIX of SAS, with donor as the experimental unit and day as the random blocking factor; significance is defined as P ≤ 0.05. Time of rumen fluid collection significantly affected acetate (mmol/liter; P = 0.0004), propionate (mmol/liter; P = 0.02), isobutyrate (mmol/liter; P < 0.0001), valerate (mmol/liter; P = 0.004), isovalerate (mmol/liter; P < 0.00001), and total VFA concentrations (mmol/liter; P = 0.004). All VFA relative proportions were altered due to time of rumen fluid collection (P < 0.02). VFA production was highest when rumen fluid was collected 4-h post-feeding. There was little to no effect on pH. Our findings suggest that VFA production is maximized when rumen fluid is collected between 2 and 4 h after feeding.
RESUMO
Many candidate gene association studies have evaluated incomplete, unrepresentative sets of single nucleotide polymorphisms (SNPs), producing non-significant results that are difficult to interpret. Using a rapid, efficient strategy designed to investigate all common SNPs, we tested associations between schizophrenia and two positional candidate genes: ACSL6 (Acyl-Coenzyme A synthetase long-chain family member 6) and SIRT5 (silent mating type information regulation 2 homologue 5). We initially evaluated the utility of DNA sequencing traces to estimate SNP allele frequencies in pooled DNA samples. The mean variances for the DNA sequencing estimates were acceptable and were comparable to other published methods (mean variance: 0.0008, range 0-0.0119). Using pooled DNA samples from cases with schizophrenia/schizoaffective disorder (Diagnostic and Statistical Manual of Mental Disorders edition IV criteria) and controls (n=200, each group), we next sequenced all exons, introns and flanking upstream/downstream sequences for ACSL6 and SIRT5. Among 69 identified SNPs, case-control allele frequency comparisons revealed nine suggestive associations (P<0.2). Each of these SNPs was next genotyped in the individual samples composing the pools. A suggestive association with rs 11743803 at ACSL6 remained (allele-wise P=0.02), with diminished evidence in an extended sample (448 cases, 554 controls, P=0.062). In conclusion, we propose a multi-stage method for comprehensive, rapid, efficient and economical genetic association analysis that enables simultaneous SNP detection and allele frequency estimation in large samples. This strategy may be particularly useful for research groups lacking access to high throughput genotyping facilities. Our analyses did not yield convincing evidence for associations of schizophrenia with ACSL6 or SIRT5.
