Dietary beauvericin and enniatin B exposure cause different adverse health effects in farmed Atlantic salmon.

The extensive use of plant ingredients in novel aquafeeds have introduced mycotoxins to the farming of seafood. The emerging enniatin B (ENNB) and beauvericin (BEA) mycotoxins have been found in the novel aquafeeds and farmed fish. Little is known about the potential toxicity of ENNs and BEA in farmed fish and their feed-to-organ transfer. Atlantic salmon (Salmo salar) pre-smolt (75.3 ± 8.10 g) were fed four graded levels of spiked chemical pure ENNB or BEA feeds for three months, in triplicate tanks. Organismal adverse health end-point assessment included intestinal function (protein digestibility), disturbed hematology (red blood cell formation), bone formation (spinal deformity), overall energy use (feed utilization), and lipid oxidative status (vitamin E). Both dietary BEA and ENNB had a low (<∼0.01%) transfer to organs (kidney > liver > brain > muscle), with a higher transfer for ENNB compared to BEA. BEA caused a growth reduction combined with a decreased protein digestion and feed conversion rate- ENNB caused a stunted growth, unrelated to feed utilization capacity. In addition, ENNB caused anemia while BEA gave an oxidative stress response. Lower bench-mark dose regression assessment showed that high background levels of ENNB in commercial salmon feed could pose a risk for animal health, but not in the case of BEA.

Effect of combinations of feed-grade urea and slow-release urea in a finishing beef diet on fermentation in an artificial rumen system.

This study evaluated the effect of combinations of feed-grade urea and slow-release urea (SRU) on fermentation and microbial protein synthesis within two artificial rumens (Rusitec) fed a finishing concentrate diet. The experiment was a completely randomized, dose-response design with SRU substituted at levels of 0% (control), 0.5%, 1%, or 1.75% of dry matter (DM) in place of feed-grade urea, with four replicate fermenters per dosage. The diet consisted of 90% concentrate and 10% forage (DM basis). The experiment was conducted over 15 d, with 8 d of adaptation and 7 d of sampling. Dry matter and organic matter disappearances were determined after 48 h of incubation from day 9 to 12, and daily ammonia (NH3) and volatile fatty acid (VFA) production were measured from day 9 to 12. Microbial protein synthesis was determined on days 13-15. Increasing the level of SRU quadratically affected total VFA (Q, P = 0.031) and ammonia (Q, P = 0.034), with a linear increment in acetate (L, P = 0.01) and isovalerate (L, P = 0.05) and reduction in butyrate (L, P = 0.05). Disappearance of neutral detergent fiber (NDF) and acid detergent fiber (ADF) was quadratically affected by levels of SRU, plateauing at 1% SRU. Inclusion of 1% SRU resulted in the highest amount of microbial nitrogen associated with feed particles (Q, P = 0.037). Responses in the efficiency of microbial protein synthesis fluctuated (L, P = 0.002; Q, P = 0.001) and were the highest for 1% SRU. In general, the result of this study showed that 1% SRU in combination with 0.6% urea increased NDF and ADF digestibility and total volatile fatty acid (TVFA) production.

Effect of exogenous fibrolytic enzymes and ammonia fiber expansion on the fermentation of wheat straw in an artificial rumen system (RUSITEC)1.

This study investigated the effect of treatment of wheat straw using ammonia fiber expansion (AFEX) and exogenous fibrolytic enzymes (Viscozyme) on fiber digestibility, rumen fermentation, microbial protein synthesis, and microbial populations in an artificial rumen system [Rumen Simulation Technique (RUSITEC)]. Four treatments were assigned to 16 vessels (4 per treatment) in 2 RUSITEC apparatuses in a randomized block design. Treatments were arranged as a 2 × 2 factorial using untreated or AFEX-treated wheat straw with or without exogenous fibrolytic enzymes [0 or 500 µg of protein/g straw dry matter (DM)]. Fibrolytic enzymes were applied to straw, prior to sealing in nylon bags. The concentrate mixture was provided in a separate bag within each fermentation vessel. The RUSITECs were adapted for 8 d and disappearance of DM, neutral detergent fiber (NDF), acid detergent fiber (ADF), and crude protein (CP) was measured after 48 h of incubation. Ammonia fiber expansion increased (P < 0.01) the disappearance of wheat straw DM (69.6 vs. 38.3%), NDF (65.6 vs. 36.8%), ADF (61.4 vs. 36.0%), and CP (68.3 vs. 24.0%). Total dietary DM, organic matter (OM), and NDF disappearance was also increased (P ≤ 0.05) by enzymes. Total microbial protein production was greater (P < 0.01) for AFEX-treated (72.9 mg/d) than untreated straw (63.1 mg/d). Total gas and methane (CH4) production (P < 0.01) were also greater for AFEX-treated wheat straw than untreated straw, with a tendency for total gas to increase (P = 0.06) with enzymes. Ammonia fiber expansion increased (P < 0.01) total volatile fatty acid (VFA) production and the molar proportion of propionate, while it decreased (P < 0.01) acetate and the acetate-to-propionate ratio. The AFEX-treated straw had lower relative quantities of fungi, methanogens, and Fibrobacter succinogenes (P < 0.01) and fewer protozoa (P < 0.01) compared to untreated straw. The pH of fermenters fed AFEX-treated straw was lower (P < 0.01) than those fed untreated straw. Both AFEX (P < 0.01) and enzymes (P = 0.02) decreased xylanase activity. There was an enzyme × straw interaction (P = 0.02) for endoglucanase activity. Enzymes increased endoglucanase activity of AFEX-treated wheat straw, but had no effect on untreated straw. The addition of enzymes lowered the relative abundance of Ruminococcus flavefaciens, but increased F. succinogenes. These results indicate that AFEX increased the ruminal disappearance of wheat straw and improved fermentation and microbial protein synthesis in the RUSITEC.

Intake, digestibility, and growth performance of Girolando bulls supplemented on pasture in Bahia, Brazil.

The objective of this trial was to evaluate intake, digestibility, and growth performance of Girolando bulls submitted to two nutritional planes while grazing on Brachiaria brizantha cv. Marandu pasture. Twenty-two animals, with average initial body weight = 209.1 ± 8.2 kg, were used in this trial. The experimental design was repeated measurements, in a 2 × 3 factorial arrangement, with two nutritional planes (NP1 and NP2) and three seasons of the year, with 11 replicates per treatment. The animals of the NP1 received mineral mixture ad libitum during rainy season 1 (15 February through 5 July 2014), energy protein supplement in the amount of 1 g d kg BW-1 during the dry season (from 6 July through 22 November 2014), and again mineral mixture ad libitum during rainy season 2 (from 23 November 2014 through 9 March 2015). The NP2 animals received 2 g d kg BW-1, 2 g d kg BW-1, and 1 g d kg BW-1 of energy-protein supplement in the respective seasons of the year. Forage intakes were similar between nutritional planes, 6.8 and 7.6 kg DM day-1 and 2.1 and 2.22% BW for NP1 and NP2, respectively. There was no statistical difference (level) between the intakes of neutral detergent fiber corrected for ash and protein (4.1 and 4.3 kg day-1 and 1.2 and 1.3% BW, respectively for nutritional planes 1 and 2). For the other nutrients, NP2 showed greater values. The highest intakes and digestibilities of dry matter, organic matter, and non-fiber carbohydrate were in rainy season 2. Performance and feed conversion were similar among NPs. This study showed that lower levels of supplementation could be done in order to reduce feeding costs with no impact on performance.

Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperature.

Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low temperature. Those identified ten different genes, three of which had previously been reported to affect cold-tolerance. Four genes were identified in both the salt and the cold-sensitivity screen. We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype. Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, http://ygacmed.yale.edu/triples/triples.htm) only marginally overlap. We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified.

Existence of a tightly regulated water channel in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae strain Sigma1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.

Polymorphism of Saccharomyces cerevisiae aquaporins.

Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyces cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the sigma 1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from sigma 1278b is highly homologous to functional aquaporins, we did not observe Aqy2p-mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.

Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain.

The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the Sigma1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DeltaCp), mediated an approximately 3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in Sigma1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.

Purification and functional reconstitution of the human CHIP28 water channel expressed in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used for heterologous expression of the human CHIP28 water Aquaporin-1 channel (Aquaporin-1). A nine-amino-acid epitope of the influenza hemagglutinin protein (HA epitope), recognized by the monoclonal antibody 12CA5, was chosen to tag CHIP28 at its N-terminus. Epitope-tagged CHIP28 was purified from yeast extracts by immunochromatography on protein A/ 12CA5-coupled beads, after KI extraction and detergent solubilization, then concentrated by anion exchange chromatography. Purified protein was reconstituted in proteoliposomes and was shown to function as a water channel by stopped-flow spectrophotometry. This study demonstrates that the yeast has the capacity to produce functional aquaporins at levels sufficient for biochemical and biophysical analyses.

Functional expression of the human CHIP28 water channel in a yeast secretory mutant.

The temperature-sensitive Saccharomyces cerevisiae mutant strain NY17, deficient in the secretory pathway (sec6-4 mutation), is used for the heterologous expression of the human CHIP28 water channel. After a heat-shock, the protein is present in partially purified post-golgi secretory vesicles. Immunodetection and water transport studies, directly made on the vesicles, showed that CHIP28 is highly expressed and active in the yeast membranes.