Methods for rapid prototyping novel labware: using CAD and desktop 3D printing in the microbiology laboratory.

Although the microbiology laboratory paradigm has increasingly changed from manual to automated procedures, and from functional to molecular methods, traditional culture methods remain vital. Using inexpensive desktop fused filament fabrication 3D printing, we designed, produced and tested rapid prototypes of customised labware for microbial culture namely frames to make dip slides, inoculation loops, multi-pin replicators, and multi-well culture plates for solid medium. These customised components were used to plate out samples onto solid media in various formats, and we illustrate how they can be suitable for many microbiological methods such as minimum inhibitory concentration tests, or for directly detecting pathogens from mastitis samples, illustrating the flexibility of rapid-prototyped culture consumable parts for streamlining microbiological methods. We describe the methodology needed for microbiologists to develop their own novel and unique tools, or to fabricate and customise existing consumables. A workflow is presented for designing and 3D printing labware and quickly producing easy-to-sterilise and re-useable plastic parts of great utility in the microbiology laboratory.

Phosphorus feeding practices, barriers to and motivators for minimising phosphorus feeding to dairy cows in diverse dairy farming systems.

Minimising phosphorus (P) feeding to dairy cows can reduce feed costs and minimise water pollution without impairing animal performance. This study aimed to determine current P feeding practices and identify the barriers to and motivators for minimising P feeding on dairy farms, using Great Britain (GB) dairy farming as an example of diverse systems. Farmers (n = 139) and feed advisers (n = 31) were involved simultaneously in independent questionnaire surveys on P feeding in dairy farms. Data on the herd size, milk yield and concentrate fed were analysed using ANOVA to investigate the effect of farm classification, region, and feed professional advice. Chi-square tests were used to investigate associations between farm characteristics and implemented P feeding and management practices. Most farmers (72%) did not know the P concentration in their lactating cow's diet and did not commonly adopt precision P feeding practices, indicating that cows might have been offered dietary P in excess of recommended P requirement. Farmers' tendency to feed P in excess of recommendations increased with herd size, but so did their awareness of P pollution issues and likeliness of testing manure P. However, 68% of farmers did not analyse manure P, indicating that mineral P fertiliser application rates were not adjusted accordingly, highlighting the risk of P being applied beyond crops' requirement. Almost all farmers (96%) were willing to lower dietary P concentration but the uncertainty of P availability in feed ingredients (30%) and concerns over reduced cow fertility (22%) were primary barriers. The willingness to reduce dietary P concentrations was driven by the prospect of reducing environmental damage (28%) and feed costs (27%) and advice from their feed professionals (25%). Most farmers (70%) relied on a feed professional, and these farmers had a higher tendency to analyse their forage P. However, farmers of pasture-based systems relied less on feed professionals. Both farmers (73%) and feed advisers (68%) were unsatisfied with the amount of training on P management available. Therefore, the training on P management needs to be more available and the influence that feed professionals have over P feeding should be better utilised. Study findings demonstrate the importance of considering type of dairy farming systems when developing precision P feeding strategies and highlight the increasing importance of feed professionals in minimising P feeding.

Short communication: Effect of abomasal inorganic phosphorus infusion on phosphorus absorption in large intestine, milk production, and phosphorus excretion of dairy cattle.

The objective of the study was to evaluate the effect of inorganic phosphorus (Pi) infusion on P absorption in large intestine, milk production, and phosphorus excretion. Four ruminally and ileally cannulated crossbred cows were used in a 4 × 4 Latin square with 21-d periods. Cows were fed a total mixed ration containing 0.21% P, providing 50% of the cows' P requirement. Cobalt-EDTA was used as marker to measure large intestine digesta flow. On d 13 to 21 of each period, each cow was infused daily with 0, 20.1, 40.2, or 60.3 g of Pi into the abomasum and total collection was conducted on d 18 to 21. Ileal samples were collected every 9 h on d 18 to 21. Feed, digesta, and fecal samples were analyzed for total P and Pi using the molybdovanadate yellow method and blue method, respectively. All data were analyzed using PROC GLIMMIX in SAS 9.3 (SAS Institute Inc., Cary, NC) using contrasts to evaluate linear, quadratic, and cubic effects of Pi infusion dose. Dry matter intake, apparent dry matter digestibility, milk yield, and milk total P were unaffected by Pi infusion. Ileal flow and fecal excretion of total P and Pi increased linearly with increasing infused Pi. In the large intestine, net absorption of TP and Pi was increased linearly with increasing infused Pi. The magnitude of absorption from the large intestine was greater than reflected in current models, raising questions that could be evaluated with longer infusion periods or dietary alteration.

The effects of forage particle length and exogenous phytase inclusion on phosphorus digestion and absorption in lactating cows.

Accurate estimates of phosphorus (P) availability from feed are needed to allow P requirements to be met with reduced P intake, thus reducing P excretion by livestock. Exogenous phytase supplementation in poultry and swine diets improves bioavailability of P, and limited research suggests that this strategy may have some application in dairy cattle rations. The effects of exogenous phytase and forage particle length on site and extent of P digestion were evaluated with 5 ruminally and ileally cannulated lactating cows (188 ± 35 d in milk). Cows were assigned in a 2 × 2 factorial arrangement of treatments in 2 incomplete Latin squares with four 21-d periods. Diets contained P slightly in excess of National Research Council requirements with all P from feed sources. During the last 4d of each period, total mixed ration, refusals, omasal, ileal, and fecal samples were collected and analyzed for total P, inorganic P (Pi), and phytate (Pp). Total P intake was not influenced by dietary treatments but Pp intake decreased and Pi intake increased with supplemental phytase, suggesting rapid action of the enzyme in the total mixed ration after mixing. Omasal flow of Pi decreased with phytase supplementation, but we observed no effect of diet in ileal flow or small intestinal digestibility of any P fraction. Fecal excretion of total P was slightly higher and Pp excretion was lower for cows receiving diets supplemented with phytase. Milk yield and composition were unaffected by diets. When phytase was added to the mixed ration, dietary Pp was rapidly degraded before intake and total-tract Pp digestion was increased. The lack of effect of phytase supplementation on dietary P utilization was probably because these late-lactation cows had a low P requirement and were fed P-adequate diets.

Effect of dietary phytate on phosphorus digestibility in dairy cows.

The objective was to evaluate the effect of dietary phytate P (Pp) supply on ruminal and postruminal Pp digestion and net disappearance of P from the lower digestive tract of lactating cows. Six ruminally and ileally cannulated crossbred lactating cows were used in 2 incomplete Latin squares with four 21-d periods (17d of diet adaptation, 4d of total collection). Dietary treatments were low Pp, medium Pp, and high Pp, and a high inorganic P (Pi) diet with the same total P content as the highest Pp diet but with P mostly from inorganic sources. The diets contained 0.10, 0.18, 0.29, and 0.11% Pp and 0.43, 0.48, 0.54, and 0.53% total P on a dry matter basis, with cottonseed meal used to increase Pp content. Ytterbium-labeled corn silage and Co-EDTA were used as particulate and liquid phase markers to measure omasal and ileal digesta flow. Omasal and ileal digesta were collected every 6h on d 20 and 21 and rumen contents were collected on d 21. Samples were analyzed for total P (molybdovanadate yellow method), Pi (blue method), and Pp (high performance ion chromatography). Phytate P and total P intake increased linearly with increasing dietary Pp. Ruminal Pp disappearance also increased linearly with dietary Pp but the magnitude of change was small. Small intestinal net disappearance of Pi was not affected by dietary Pp. Phytate P was hydrolyzed in the large intestine but its hydrolysis was not influenced by dietary Pp. Net disappearance of Pi form the large intestine did not vary with dietary Pp. Dry matter digestibility decreased linearly with increasing dietary Pp, as did apparent digestion of P, and fecal P increased linearly. Dry matter digestibility was higher for high Pi than for high Pp, likely due to the effect of cottonseed meal in the latter diet. Replacing a portion of Pp with Pi resulted in decreased P excretion but this effect was confounded with increased fecal dry matter for the high-Pp (high-cottonseed meal) diet. In lactating cows Pp digestibility was not negatively influenced by dietary Pp and fecal P excretion was regulated by dietary total P rather than by form of dietary P.

Disappearance of infused phytate from the large intestine of dairy heifers.

The objective of this study was to investigate the disappearance of phytate from the large intestine of dairy heifers. Uncertainty about the availability of phosphorus (P) in different feeds may limit implementation of dietary strategies to reduce fecal P excretion by dairy cows. Increased understanding of the dynamics of phytate degradation and disappearance of P in the large intestine may improve prediction of intestinal P digestion and absorption. Eight ruminally- and ileally-cannulated crossbred dairy heifers were used in two 4×4 Latin square designs with 9-d periods, including 3d of washout. All heifers were fed a high-forage diet containing 0.14% P throughout the study. Ytterbium-labeled corn silage and Co-EDTA were dosed to the rumen 4 times daily as particulate and liquid phase markers, respectively, to measure ileal digesta flow. Ond 4 to 7 of each period, each heifer was infused ileally with 0, 5, 15, or 25 g/d of phytate (phytic acid) in solution and total fecal collection was conducted. When infusion ceased (d 8 and 9) ileal digesta was sampled to measure P flow to the ileum from the basal diet. Feed, digesta, and feces were dried, ground, and analyzed for phytate P, inorganic P, and total P using high performance ion chromatography, inductively coupled plasma atomic emission spectroscopy, and the molybdovanadate yellow method, respectively. Phytate degradation in the large intestine was observed but was not complete, and the amount of infused phytate did not influence the degradability of phytate. Fecal excretion of total P increased with increasing total P infused. The slope coefficient for ileal P flow (dietary only) to feces was 0.56 ± 0.26 (mean ± SE), whereas the slope coefficient for infused P was 0.75 ± 0.13. These indicate net absorption of P from the large intestine and greater disappearance of P from dietary P flowing to the ileum than from the infused pure phytate (44 vs. 25%). This data will support mechanistic modeling efforts to improve prediction of P digestion, allowing more accurate estimation of P bioavailability in feeds.

Quantifying phytate in dairy digesta and feces: alkaline extraction and high-performance ion chromatography.

Development of an analytical method with appropriate combination of extraction and quantification approaches for undigested phytate in ruminant feces and digesta will advance knowledge of phytate degradation in ruminants and help to reduce phosphorus excretion. Established quantification methods give satisfactory results for feedstuffs and nonruminant manure but recovery of phytate is incomplete for ruminant feces and digesta because of their complex sample matrix and low ratio of phytate to inorganic P. The objective was to develop a robust, accurate, sensitive, and inexpensive method to extract and quantify phytate in feeds, ruminant feces, and digesta. Diets varying in phytate content were fed to dairy heifers, dry cows, and lactating cows to generate digesta and fecal samples of varying composition to challenge extraction and quantification methods. Samples were extracted with 0.5 M HCl or 0.25 M NaOH + 0.05 M EDTA. Acid extracts were mixed with 20% NaCl, alkaline extracts were acidified to final pH < 2, and then both extracts were clarified with C18 cartridges and 0.2-µm filters. High-performance ion chromatography (HPIC) was used to quantify phytate. In feed samples, the measured phytate was comparable in alkaline and acid extracts (2,965 vs. 3,085 µg/g of DM). In digesta and fecal samples, alkaline extraction yielded greater estimates of phytate content than did acid extraction (40.7 vs. 33.6 and 202.9 vs. 144.4 µg/g of DM for digesta and fecal samples, respectively). Analysis of alkaline extracts by HPIC is usually not possible because of sample matrix interferences; acidification and C(18)-cartridge elution of alkaline extracts prevented this interference. Pure phytate added to dry samples before extraction was almost completely recovered (88 to 105%), indicating high extraction efficiency, no adverse effect of extract clean-up procedures, and accurate quantification of phytate. The proposed method is rapid, inexpensive, robust, and combines the extraction power of NaOH-EDTA with the precision and sensitivity of HPIC quantification, allowing accurate quantification of phytate in feeds, ruminant digesta, and fecal samples.

Adrenomedullary hormonal and glycemic responses to high ambient temperature in the soft-shelled turtle, Lissemys punctata punctata.

The aim of the investigation was to study the influence of high ambient temperature on adrenomedullary activity and blood glucose levels in adult female soft-shelled turtles (Lissemys punctata punctata). Experiments were carried out at 25 degrees, 35 degrees, and 38 degrees, and one group was exposed to 38 degrees for 15 days and then maintained at 25 degrees for another 15 days. Exposure to a low ambient temperature of 25 degrees had no clear effect on adrenomedullary function with respect to histology (nuclear diameter), epinephrine and norepinephrine concentrations, and blood glucose level of turtles, but higher temperatures of 35 degrees and 38 degrees stimulated adrenomedullary activity as well as blood glucose level in turtles compared with controls (30 degrees ). The extent of these changes was greater at 38 degrees than that at 35 degrees, and withdrawal from high ambient temperature reversed the effect in turtles.