A New Rapid and Quantitative Assay to Determine the Phytase Activity of Feed.

The fortification of animal feed with enzymes in order to optimize feed utilization has become a standard for the meat production industry. A method for measuring levels of active enzymes that can be carried out quickly would ensure that feed has been supplemented with the appropriate amount of enzyme. Phytase is the most widely used feed enzyme and is routinely quantified with an activity assay in a limited number of specialized laboratories. As an alternative, we report here the development of a rapid and easy method to perform a quantitative assay for the phytase from Citrobacter braakii. The method is suitable for use at local sites with a minimum lab setup and will reduce delays and potential interferences due to improper sample storage and shipment. The new assay is based on a lateral flow immunoassay that utilizes magnetic immune-chromatographic test (MICT) technology to quantify the phytase content of a feed extract. After extraction of the phytase from the feed, the sample is simply diluted and added to a reaction tube containing a specific anti-phytase antibody coupled to superparamagnetic particles. The mixture is then applied on an assay cassette, where the formed particle-antibody-phytase complexes are captured by immobilized antibodies on a nitro-cellulose strip housed in a cassette. The cassette is placed in the MICT reader that measures the magnetic signal of the captured particles. Using the calibration information stored in the cassette barcode, the signal is converted to a phytase concentration, given as phytase activity (FYT) per kilogram of feed. The accuracy and robustness of the assay compared to the ISO phytase activity assay were demonstrated through a large validation study including real feed samples from different compositions and origins. The MICT assay is the first quantitative assay for feed enzymes that is fast, reliable, and simple to use outside of a specialized reference laboratory and that is suitable for use in place of the current ISO assay.

Supplementation of a lacto-fermented rapeseed-seaweed blend promotes gut microbial- and gut immune-modulation in weaner piglets.

BACKGROUND: The direct use of medical zinc oxide in feed will be abandoned after 2022 in Europe, leaving an urgent need for substitutes to prevent post-weaning disorders. RESULTS: This study investigated the effect of using rapeseed-seaweed blend (rapeseed meal added two brown macroalgae species Ascophylum nodosum and Saccharina latissima) fermented by lactobacilli (FRS) as feed ingredients in piglet weaning. From d 28 of life to d 85, the piglets were fed one of three different feeding regimens (n = 230 each) with inclusion of 0%, 2.5% and 5% FRS. In this period, no significant difference of piglet performance was found among the three groups. From a subset of piglets (n = 10 from each treatment), blood samples for hematology, biochemistry and immunoglobulin analysis, colon digesta for microbiome analysis, and jejunum and colon tissues for histopathological analyses were collected. The piglets fed with 2.5% FRS manifested alleviated intraepithelial and stromal lymphocytes infiltration in the gut, enhanced colon mucosa barrier relative to the 0% FRS group. The colon microbiota composition was determined using V3 and V1-V8 region 16S rRNA gene amplicon sequencing by Illumina NextSeq and Oxford Nanopore MinION, respectively. The two amplicon sequencing strategies showed high consistency between the detected bacteria. Both sequencing strategies indicated that inclusion of FRS reshaped the colon microbiome of weaned piglets with increased Shannon diversity. Prevotella stercorea was verified by both methods to be more abundant in the piglets supplied with FRS feed, and its abundance was positively correlated with colonic mucosa thickness but negatively correlated with blood concentrations of leucocytes and IgG. CONCLUSIONS: FRS supplementation relieved the gut lymphocyte infiltration of the weaned piglets, improved the colon mucosa barrier with altered microbiota composition. Increasing the dietary inclusion of FRS from 2.5% to 5% did not lead to further improvements.

Effects of Increasing Doses of Lactobacillus Pre-Fermented Rapeseed Product with or without Inclusion of Macroalgae Product on Weaner Piglet Performance and Intestinal Development.

This study evaluated the effects of increasing doses of pre-fermented rapeseed meal (FRM) without or with inclusion of the brown macroalgae Ascophyllum nodosum (AN) on weaner piglets' performance and gut development. Ten days pre-weaning, standardized litters were randomly assigned to one of nine isoenergetic and isoproteic diets comprising (on DM basis): no supplement (negative control, NC), 2500 ppm ZnO (positive control, PC), 8, 10, 12, 15 or 25% FRM, and 10% FRM plus 0.6 or 1.0% AN. Fifty piglets receiving the same pre-weaning diets were weaned at 28 days of age and transferred to one pen, where they continued on the pre-weaning diet until day 92. At 41 days, six piglets per treatment were sacrificed for blood and intestinal samplings. The average daily gain was at least sustained at any dose of FRM (increased at 8% FRM, 28-41 days) from 18-41 days similar to PC but unaffected by inclusion of AN. The percentage of piglets that completed the experiment was increased by FRM compared to NC, despite detection of diarrhea symptoms. FRM showed quadratic dose-response effects on colon and mid-jejunum crypts depth, and enterocyte and mid-jejunum villus heights with optimum development at 8% or 10% FRM, respectively, but this was abolished when AN was also added. In conclusion, FRM sustained piglet growth performance and intestinal development similar to ZnO with an optimum inclusion level of 8-10% of dietary DM.

Impact of Dietary Supplementation of Lactic Acid Bacteria Fermented Rapeseed with or without Macroalgae on Performance and Health of Piglets Following Omission of Medicinal Zinc from Weaner Diets.

The feeding of medicinal zinc oxide (ZnO) to weaner piglets will be phased out after 2022 in Europe, leaving pig producers without options to manage post-weaning disorders. This study assessed whether rapeseed meal, fermented alone (FRM) or co-fermented with a single (Ascophylum nodosum; FRMA), or two (A. nodossum and Saccharina latissima; FRMAS) brown macroalagae species, could improve weaner piglet performance and stimulate intestinal development as well as maturation of gut microbiota in the absence of in-feed zinc. Weaned piglets (n = 1240) were fed, during 28-85 days of age, a basal diet with no additives (negative control; NC), 2500 ppm in-feed ZnO (positive control; PC), FRM, FRMA or FRMAS. Piglets fed FRM and FRMA had a similar or numerically improved, respectively, production performance compared to PC piglets. Jejunal villus development was stimulated over NC in PC, FRM and FRMAS (gender-specific). FRM enhanced colon mucosal development and reduced signs of intestinal inflammation. All fermented feeds and PC induced similar changes in the composition and diversity of colon microbiota compared to NC. In conclusion, piglet performance, intestinal development and health indicators were sustained or numerically improved when in-feed zinc was replaced by FRM.