Effect of inoculum and pepsin–pancreatin hydrolysis on fibre fermentation measured by the gas production technique in pigs

Two experiments were undertaken to adapt the in vitro gas production technique in syringes, used for ruminants, to fibre fermentation studies in the large intestine of pigs.In a first experiment, two inocula (faeces and large intestine content) were compared at four dilution levels in a buffer solution (0.025, 0.05, 0.1 and 0.2 g ml⁻¹) with two substrates: wheat bran and sugar–beet pulp. The accumulated gas produced over 72 h was modelled and the kinetics parameters compared. The time to half asymptote was lower for the intestinal inoculum (5.5 versus 8.0 h, P<0.02), but the 2 inocula yielded similar fractional rates of degradation (0.16 h⁻¹) and gave equal final gas production (252 ml g⁻¹ substrate). No interaction (P>0.05) was observed between inocula and substrates. The dilution of the samples in the buffer solution increased (P<0.001) the lag time (from 0.9 to 2.1 h for dilution rates ranging from 0.2 to 0.025 g ml⁻¹, respectively) and decreased (P<0.001) the rates of substrate degradation (from 0.18 to 0.13 h⁻¹).A second experiment aimed to study the effect of an in vitro pepsin–pancreatin hydrolysis of the sample prior to the gas test. Six substrates were tested: maize, wheat bran, sugar–beet pulp, lupins, peas and soybean meal. The enzymatic hydrolysis affected (P<0.001) the kinetics parameters and the ranking order of the fermented substrates. The lag times also increased for all ingredients. The rate of degradation decreased when peas, lupins, maize and wheat bran were hydrolysed (P<0.001) but it increased with soybean meal (P=0.014) and sugar–beet pulp (P<0.001). Final gas production increased with peas and soybean meal (P<0.001), remained unchanged for lupins and decreased for the other substrates (P<0.001).In conclusion, the method using faeces as a source of microbial inoculum is reliable to characterise the fermentation kinetics of ingredients in the large intestine of pigs. However, it is important to hydrolyse the substrates with pepsin and pancreatin before the gas tests.     

Effect of elemental nano-selenium on feed digestibility,rumen fermentation,and purine derivatives in sheep

The objective of this experiment was to study the effect of elemental nano-selenium (NS) on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Eight male ruminally cannulated sheep (42.5 ± 3.2 kg of body weight, BW) were used in a replicated 4×4 Latin square experiment in four 20 day periods. Depending on treatment designation, sheep were fed the basal diet supplemented with 0 (control), 0.3, 3 and 6 g of nano-Se/kg dry matter (DM). Ruminal pH (range of 6.68–6.80) and ammonia N concentration (range of 9.95–12.49 mg/100 mL) was decreased (P<0.01), and total VFA concentration (range of 73.63–77.72 mM) was increased linearly (P<0.01) and quadratically (P<0.01) with increasing nano-Se supplementation. The ratio of acetate to propionate was linearly (P<0.01) and quadratically (P<0.01) decreased due to the increasing of propionate concentration. In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis and crude protein (CP) of soybean meal were linearly (P<0.01) and quadratically (P<0.01) improved by feeding nano-Se. Similarly, nutrients digestibility in the total tract and urinary excretion of purine derivatives were also quadratically (P<0.01) changed by increasing nano-Se supplementation. The present results indicated that nano-Se supplementation in basal diet improved rumen fermentation and feed utilization. Nano-Se could also stimulate rumen microbial activity, digestive microorganisms or enzyme activity. The optimum dose of nano-Se was about 3.0 g/kg dietary DM in sheep.     

Nutritional characterization of Mucuna pruriens: 3. Effect of replacing soybean meal with Mucuna on intake,digestibility, N balance and microbial protein synthesis in sheep

Mucuna pruriens seeds have relatively high crude protein (CP) concentrations, but little is known about their potential to replace commonly used CP supplements in ruminant rations. The aim of this experiment was to determine effects of replacing soybean meal (SB) with Mucuna on the performance of lambs. Forty Rambouillet lambs (33.2 ± 5.73 kg) fed a basal diet of maize grain, cottonseed hulls and urea were randomly assigned to one of four supplements formulated by substituting 0 (SB), 330 (Lo), 670 (Med) or 1000 g/kg (Hi) of soybean meal with rolled Mucuna seeds. Lambs were housed individually in metabolic crates and allowed ad libitum access to isocaloric (metabolizable energy=11.7 MJ/kg dry matter, DM) and isonitrogenous (CP = 146 g/kg, DM) diets for 14 d of adaptation and 7 d of total fecal collection. Fecal egg counts and coccidian oocyst scores were determined on d 14. Dry matter intake (1.7 kg/d versus 1.5 kg/d; P<0.05), CP digestibility (774 g/kg versus 714 g/kg DM; P<0.05) and N retention (28.0 g/d versus 20.4 g/d; P<0.01) were higher and amylase-pretreated neutral detergent fiber digestibility (617 g/kg versus 686 g/kg DM) was lower (P<0.05) in sheep fed SB versus Mucuna diets. However, supplementary protein source did not affect rumen pH, blood urea N or glucose concentration, or fecal egg counts. Increasing the level of Mucuna supplementation increased (P<0.05) level and efficiency of microbial protein synthesis, ruminal fluid acidity, total volatile fatty acid concentration, decreased (P<0.05) coccidian oocyst scores, and tended (P<0.10) to increase N retention. Therefore, SB is a better supplement than Mucuna to support performance of lambs. Nevertheless, Mucuna seeds are a promising CP supplement for situations where cost or availability precludes use of SB in ruminant rations.     

The interaction between lactose level and crude protein concentration on piglet post-weaning performance,nitrogen metabolism,selected faecal microbial populations and faecal volatile fatty acid concentrations

A performance study and a nitrogen balance study (2×3 factorial) were conducted to investigate the interaction between lactose level (215 and 125 g/kg) (lactofeed 70; 860 g whey permeate/kg, 140 g soya bean meal/kg, Volac International, UK) and crude protein (CP) concentration (160, 185 and 210 g/kg) on post-weaning piglet performance, nitrogen metabolism, faecal microbiology and faecal volatile fatty acid concentrations. In the performance trial, 252 piglets (7.6 kg; 33 days of age) were assigned to one of six dietary treatments following a 12-day period on a commercial creep diet (17 MJ/kg DE, 16 g lysine/kg). The experimental diets were fed for 28 days (days 12–40) and were formulated to have identical digestible energy (15 MJ/kg) and total lysine (14.5 g/kg) contents. In the N balance experiment, 24 boars (20 kg live weight) were offered the same diets as in the performance trial. Faecal samples were collected for selected microbial populations. There was an interaction (P<0.05) between lactose and CP concentration in daily gain (ADG) and daily feed intake (ADFI) (P<0.01) during the weaner period (days 12–40). At the high lactose level there was a linear increase in ADG and ADFI with increasing CP. However, at the low lactose level there was no increase in ADG or ADFI above the medium CP. Pigs offered 215 g lactose/kg had a higher dry matter (P<0.001), organic matter (P<0.001), energy (P<0.001), nitrogen (P<0.01) and neutral detergent fibre (P<0.05) coefficient of total tract apparent digestibility compared to pigs offered 125 g lactose/kg. There was an interaction between lactose and CP concentration for nitrogen intake (NI) (P<0.05), urine pH (P<0.05) and selected faecal microbial populations. At the high CP level, pigs offered diets containing 215 g lactose/kg had a higher NI and a lower urine pH than pigs offered 125 g lactose/kg (P<0.05). However, the inclusion of lactose had no significant effect on either NI or urine pH at the low or medium CP concentration. At the low lactose level there was a linear increase in faecal E. coli population and a linear decrease in faecal Lactobacilli population with increasing CP. However at high lactose levels CP concentration had no effect on either E. coli or Lactobacilli populations. Pigs offered 215 g lactose/kg had a significantly higher Bifidobacteria population compared to pigs offered 125 g lactose/kg. There was a linear decrease in Bifidobacteria population as CP increased. In conclusion, at the high lactose level there was a linear increase in ADG and ADFI with increasing CP concentrations. There was no increase in these parameters above 185 g CP/kg at the low lactose level.     

Extracellular expression of a thermostable phytase (phyA) in Kluyveromyces lactis

Functional expression of a thermostable phytase from A. niger was achieved in Kluyveromyces lactis GG799 cells. Effective secretion of recombinant enzyme (198 U ml⁻¹) in the fermentation broth at 72 h incubation at 22 °C was obtained. Purified enzyme showed a specific activity of 72 U mg⁻¹) and was detected on SDS-PAGE as a heavily glycosylated protein with a molecular weight of ≥140 kDa. Optimum temperature of the enzyme was at 55 °C and it showed a characteristic bi-hump pH profile with two pH optima (at pH 2.5 and 5.5). Enzyme showed considerable pepsin resistance with 60% activity retention after incubation with pepsin at the ratio of 1:1000. Enzyme was thermostable retaining 69 and 37% activity at 90 and 100 °C for 10 min respectively and remained active at these temperatures till 1 h. Deglycosylation studies demonstrated negligible effect of N-linked glycans on thermal properties. Multiple sequence alignment data revealed a conserved Asn at position 345 of this phytase which might contribute to its thermal properties. This thermostable phytase coupled with its noticeable protease resistance could be a better alternative to current commercial phytases.     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Diet selection and eating behaviour of lactating goats subjected to time restricted feeding in choice and single feeding system

This study was carried out to investigate diet selection and eating behaviour of lactating German Fawn × Hair Crossbred goats in different feeding methods and levels. Twenty German Fawn × Hair first backcross does (B₁) were allocated into 4 treatment groups (2 feeding methods single (TMR) and choice feeding × 2 feeding levels ad libitum and restricted) with 5 replicates. Restricted feeding was applied only 4 h feed allocation during day. Barley, corn, soybean meal, corn gluten meal, wheat bran and alfalfa hay were feed ingredients for single and choice feeding. Eating patterns, milk yield and composition were determined for 8 weeks. The following results were obtained: (1) the meal criteria for goats restricted single and choice-fed, ad libitum single and choice-fed were determined as 1.00 and 0.63, 12.88 and 10.23 min, respectively. (2) Ad libitum feeding increased meal size, meal length, intermeal interval, total eating duration and decreased eating rate and meal number, compared to restricted feeding (P < 0.01). Choice feeding decreased meal size (P < 0.05), meal length (P < 0.01) and increased eating rate and meal number (P < 0.01), compared to single feeding. Restricted fed goats decreased intermeal interval in single feeding compared to choice feeding (P < 0.01), but increased meal number in choice feeding (P < 0.01). (3) Ad libitum choice-fed does made a diet containing 12.79% corn, 35.41% barley, 13.21% wheat bran, 5.35% soybean meal, 1.28% corn gluten meal and 29.80% alfalfa meal while restricted choice-fed does made a diet having more corn (27.69%), corn gluten meal (5.62%) and wheat bran (16.17%) and less barley (14.37%) and soybean meal (4.51%). (4) Choice feeding decreased RUP intake (P < 0.05) without affecting milk protein, irrespective to feeding levels, while having a tendency to increase in milk yield (14.2%) and 4% FCM (8.8%). (5) Restricted feeding decreased DM, ME, ADF and NDF intakes (P < 0.05) with concomitant decreases in 4% FCM, total milk solid, ash and fat compositions (P < 0.05), irrespective to feeding methods. (6) Choice-fed goats changed their preferences for a possible synchronized nutrient intake during a daytime, as sorted barley, soybean meal and alfalfa hay from early morning to late afternoon.It could be concluded that choice-fed goats have the ability to make their diet to meet nutrient requirements and had a tendency to increase in milk yield. Restriction in feeding time resulted in lower feed intake and milk yield, although the animal changed their feed preference in favour of high quality ingredients and eating pattern with lower meal criterion and intermeal interval.     

Does dietary supplementation with phytases affect the thermoregulatory and behavioral responses of pullets in a tropical environment?

Dietary supplementation of two types of phytases (fungal and bacterial) with different dosages (300 and 900 FTUs) was evaluated in the thermoregulatory and behavioral responses of replacement pullets in a tropical environment. 288 Hy-Line White laying birds with a mean weight of 639.60 ± 6.05 g, clinically healthy, and eight weeks old were used in the study. Respiratory rate (RR, breaths. min⁻¹), Cloacal temperature (CT, °C), Surface temperature with feathers (STWF, °C), and Surface temperature featherless (STF, °C) were measured in the morning and afternoon. Behavioral data were observed through the following activities: sitting, eating, drinking, exploring feathers (EF), non-aggressive pecking (NAP), and object pecking (OP) recorded every 10 min from 6 a.m. to 5 p.m. Environmental variables were measured along with thermoregulatory and behavioral responses. There was an interaction for RR between phytase and period of the day (P < 0.05). The lowest RR (morning) was observed in fungal phytase. STF and STWF were higher (P < 0.05) in the afternoon. Birds supplemented with fungal phytase showed lower STWF (P < 0.05). The variables that contributed to explain physiological and behavioral responses are shown in order of importance for (i) periods of day: morning (sitting, STWF, drinking, eating, and CT) and afternoon (STF, STWF, OP, drinking, eating, RR and sitting); (ii) phytases: fungal (STF, STWF, RR, sitting, eating and drinking); and bacterial (RR, STF, STWF, CT and sitting). Thermoregulatory and behavioral responses were similar between dosages, but different between types of phytases. Birds supplemented with fungal phytase used sensible heat dissipation mechanisms and exhibited thermal comfort behaviors. The 300 and 900 FTUs phytase doses did not influence the thermoregulatory and behavioral responses of birds, while they showed natural heat dissipation and heat stress behaviors in the afternoon. We recommend a dietary supplementation of 300 FTUs fungal phytases.     

Biophysical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Molecular Size, Glycosylation Pattern, and Engineering of Proteolytic Resistance

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.     

Effects of exogenous enzymes on apparent nutrient digestibility in rainbow trout (Oncorhynchus mykiss) fed diets with high inclusion of plant-based protein

Plant-based protein ingredients are increasingly used in feed for carnivorous fish as replacement for fish meal. This study investigated if supplementing diets with high inclusion levels of plant-based protein with three different enzymes could improve the apparent nutrient digestibility in juvenile rainbow trout (Oncorhynchus mykiss). Three diets with high inclusion levels of either de-hulled, solvent extracted soybean meal (344 g/kg), sunflower meal (246 g/kg), or rapeseed meal (264 g/kg) were produced and feed batches were coated post-extrusion with the different enzymes: β-glucanase at 67 mg/kg, xylanase at 208 mg/kg, protease at 228 mg/kg, or a combination of the three enzymes at the same doses. Three consecutive digestibility trials were carried out using a flow-through, modified Guelph System. Each trial was designed to include five dietary treatment groups: a non-supplemented control diet and 4 diets supplemented with either β-glucanase, xylanase, protease or a combination of the three enzymes. Diets were fed to triplicate tanks and two faecal sampling periods for digestibility measurements for each tank were scheduled. Each experiment comprised a 10-day digestibility trial succeeded by a water sampling period for measuring the dissolved nitrogen (N) output. Each experiment lasted 17–19 days in total.Apparent digestibility coefficients (ADCs) of protein, fat, ash, phosphorus and dry matter (DM) were derived from the three digestibility trials, along with calculations of the specific growth rate (SGR, %/d) and feed conversion ratio (FCR). Nitrogen mass-balance and energy retention were evaluated for each dietary treatment group to elucidate on the utilization of digested nutrients and energy.Enzyme supplementation had only moderate effect on apparent nutrient digestibility in the sunflower and rapeseed experiments, while β-glucanase and protease improved the apparent digestibility of all dietary nutrients in the soybean experiment (P<0.05). The effect was more pronounced for lipid than for other nutrients. β-Glucanase had a positive effect on energy retention in the soybean experiment (P<0.05), while there were no effects on nitrogen retention or fish performance in any of the three experiments (P>0.05) during the short feeding periods. The study thus provides preliminary results on the potential of β-glucanase and protease to increase apparent nutrient digestibility of soybean meal in fish feed.     

Rumen microbial responses in fermentation characteristics and production of CLA and methane to linoleic acid in associated with malate or fumarate

An in vitro incubation in batch was conducted to investigate the effect of propionate precursor (malate or fumarate) on fermentation characteristics, and production of CLA and methane by rumen microbes when incubated with linoleic acid (C_18:2). Sixty milligrams of C_18:2 alone (LA), 60 mg C_18:2 with 24 mM malic acid (M-LA), or 60 mg C_18:2 with 24 mM fumaric acid (F-LA) was added to 150 ml culture solution consisting of 75 ml strained rumen fluid and 75 ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate, and C_18:2 (control). Two grams of feed consisting of 1.4 g concentrate and 0.6 g ground alfalfa (DM basis) was also added to the culture solution of each treatment. An in vitro incubation in batch was made anaerobically in a shaking incubator for up to 12 h at 39 °C.The pH of the culture solution was increased (P<0.0001) in M-LA or F-LA treatments from 3 h to 12 h compared with the control and LA treatments. At 12 h incubation, the concentration of total VFA in the culture solution was higher (P<0.01) in M-LA and F-LA than in control and LA treatments. Concentration of C₃ by M-LA and F-LA was increased at 3 h (P<0.01), 6 h (P<0.01) and 12 h (P<0.01) compared with control and LA. However, no difference in C₃ concentration was observed between control and LA, or between M-LA and F-LA. Accumulated total gas produced for up to 12 h incubation was increased (P<0.01) by M-LA or F-LA compared with the control. Accumulated total methane produced for up to 12 h incubation, however, was greatly reduced (P<0.01) by all the supplements compared with control, and its production from M-LA or F-LA was smaller than the LA. The M-LA or F-LA also increased (P<0.05–<0.001) the concentrations of cis9, trans11-CLA for all incubation times and trans10, cis12-CLA at 1 h (P<0.01), 3 h (P<0.05), and 12 h (P<0.05) incubation times compared with LA.It can be concluded that malate and fumarate, as propionate precursors, act as alternative electron sinks and may compete with CH₄ generation and bio-hydrogenation of C_18:2 in the utilization of metabolic H₂. The highest CLA concentration at the early incubation stage (1 h) was accompanied by reduced propionate proportion. Linoleic acid is also considered one of the potential alternatives to suppress CH₄ generation.     

Effect of dietary β-glucan on growth performance,fecal microbial shedding and immunological responses after lipopolysaccharide challenge in weaned pigs

Two experiments (Exp.) were conducted to evaluate the effects of β-glucan inclusion in the diet on growth performance and immune function after lipopolysaccharide (LPS) challenge. In Exp. 1, a total of 40 weaned pigs (progeny of Landrace×Yorkshire sows by Duroc) with an initial body weight (BW) of 7.89 ± 0.84 kg (21 ± 2 d) of age) were used in a 28-day (d) experiment to determine the effects of dietary β-glucan on growth performance. Pigs were allotted randomly to two treatments consisting of addition of 0 or 0.1 g β-glucan/kg diet with four replicate pens per treatment and five pigs per pen. Growth performance was not affected by β-glucan supplementation throughout the experiment. However, dietary β-glucan reduced (P<0.05) the number of fecal Escherichia coli. In Exp. 2, a total of 20 weaned barrows (6.22 ± 0.25 kg of BW and 21 ± 2 d of age) individually raised in metabolic cages were used to evaluate immunological responses following LPS challenge. Pigs were fed 0 or 0.1 g β-glucan/kg diet for 42 d. At the end of the trial, half of the pigs (n = 5) from each treatment were injected intraperitoneal with E. coli LPS at a concentration of 100 μg/kg BW and the other half were injected with sterile saline solution. Treatments were arranged as a 2×2 factorial, with the main effect of LPS challenge (saline vs. LPS) and β-glucan supplementation (0 g/kg vs. 0.1 g/kg). After LPS injection, blood was taken at 0, 2, 4, 6, 8 and 12 hours (h) for the blood cell counts and blood inflammatory response. Dietary β-glucan increased (P<0.05) leukocytes counts at 4, 6 and 8 h, and blood lymphocyte concentrations at 2, 4 and 6 h and LPS challenge increased (P<0.05) counts of leukocytes at 2, 4, 6 and 8 h and blood lymphocyte at 2 and 4 h post-challenge. The rectal temperature was increased (P<0.05) at 2, 4, 6 and 8 h after LPS challenge. Dietary β-glucan reduced (P<0.05) and LPS challenge increased (P<0.05) blood plasma tumor necrosis factor-α (TNF-α) concentration at 2 and 4 h post-challenge. Dietary β-glucan increased (P<0.05) the concentration of the cluster of differentiation antigens 4 cells (CD4⁺) at 2, 4 and 6 h, and of 8 (CD8⁺) at 4 and 6 h post-challenge, respectively. The LPS challenge increased (P<0.05) CD4⁺ and CD8⁺ cell concentrations at 2, 4 and 6 h post-challenge. The CD4⁺:CD8⁺ ratio was reduced (P<0.05) by LPS challenge but was increased (P<0.05) by dietary β-glucan at 2, 4, 6 and 8 h post-challenge. In conclusion, dietary β-glucan decreased E. coli numbers but did not affect growth performance in weaned pigs and may offer benefits on immune function in weaned pigs challenged with LPS.     

Characterization of a HAP–phytase from a thermophilic mould Sporotrichum thermophile

The phytase of Sporotrichum thermophile was purified to homogeneity using acetone precipitation followed by ion-exchange and gel-filtration column chromatography. The purified phytase is a homopentamer with a molecular mass of ～456 kDa and pI of 4.9. It is a glycoprotein with about 14% carbohydrate, and optimally active at pH 5.0 and 60 °C with a T_1/2 of 16 h at 60 °C and 1.5 h at 80 °C. The activation energy of the enzyme reaction is 48.6 KJ mol^?1 with a temperature quotient of 1.66, and it displayed broad substrate specificity. Mg²⁺ exhibited a slight stimulatory effect on the enzyme activity, while it was markedly inhibited by 2,3-butanedione suggesting a possible role of arginine in its catalysis. The chaotropic agents such as guanidinium hydrochloride, urea and potassium iodide strongly inhibited phytase activity. Inorganic phosphate inhibited enzyme activity beyond 3 mM. The maximum hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) for sodium phytate were 83 nmol mg^?1 s^?1 and 0.156 mM, respectively. The catalytic turnover number (K_cat) and catalytic efficiency (K_cat/K_m) of phytase were 37.8 s^?1 and 2.4 × 10⁵ M^?1 s^?1, respectively. Based on the N-terminal and MALDI–LC–MS/MS identified amino acid sequences of the peptides, the enzyme did not show a significant homology with the known phytases.     

Supplementation of isonitrogenous oil seed cakes in cactus (Opuntia ficus-indica)–tef straw (Eragrostis tef) based feeding of Tigray Highland sheep

The experiment was conducted at Maichew Agricultural Technical Vocational Education and Training College, Ethiopia. Twenty four male yearling Tigray Highland sheep with mean body weight (BW) of 21 ± 2.6 kg (mean ± S.D.) were used to investigate the effect of different protein sources on feed intake, nutrient digestibility, BW change and carcass parameters in a study comprising of 90 days feeding trial, followed by 7 days of digestibility trial and evaluation of carcass parameters. Six individually fed animals were used per treatment in a randomized complete block design. The treatments consisted of ad libitum feeding of tef (Eragrostis tef) straw plus 172 g dry matter (DM) of cactus (Opuntia ficus-indica) pear (T1, control) and supplementation with 145 g DM cotton seed cake (CSC) (T2), 195 g DM noug seed cake (NSC) (T3) or 149 g DM peanut cake (PNC) (T4) per head per day. The quantity of the supplements was set to supply 62.5 g crude protein (CP). Tef straw DM intake was depressed (P<0.01) as the result of NSC supplementation. Sheep supplemented with CSC and PNC had higher (P<0.001) total DM intake than the control and NSC supplemented ones. Supplementation with NSC and PNC also resulted in higher (P<0.01) apparent digestibility of DM and OM compared to the control treatment. Supplementation with CSC and PNC resulted in better daily BW gain (P<0.001), feed conversion efficiency (FCE) and dressed carcass weight (P<0.01) compared to the non-supplemented diet. Dressing percentage on slaughter weight base was higher (P<0.01) in supplemented sheep than in the non-supplemented ones. Supplementation with PNC also promoted higher (P<0.05) rib-eye muscle area than in the non-supplemented ones. It was concluded that supplementation with 145 g DM CSC and 149 g DM PNC resulted in better feed intake, BW gain and carcass traits in cactus–tef straw based feeding of sheep.     

Effect of the dietary oregano (Origanum vulgare) on Cu and Zn balance in weaned piglets

A 4-week study conducted on 20 weaned piglets (average initial weight 15 kg) evaluated the effects of dietary oregano (Origanum vulgare) used in the presence/absence of phytase on the Cu and Zn balance, while reducing/eliminating their inclusion in the diet as inorganic salts. Oregano was harvested from the wild flora. The Cu and Zn concentrations that were taken into consideration (9.85 ppm and 53.31 pmm, respectively) were the consensus values obtained in an interlaboratory study. The piglets were assigned to 4 groups (C, E1, E2, E3), housed in individual metabolic cages and fed on corn–soybean meal-based diets. The diet of the control group (C) with addition of 1% inorganic mineral premix (MP), contained: 40.92 ppm Cu, 144.96 ppm Zn. The experimental diets differed from the C diet as follows: E1 – 3% oregano, 0% phytase (5000 PU/g), 0% MP; E2 – 3% oregano, 0.01% phytase, 0% MP; E3 – 3% oregano, 0% phytase, 0.5% MP, E4 – 3% oregano, 0.01% phytase, 0,5% premix. For groups E1, E2, E3 and E4, 0.5% Zn of the MP were included in the diet, because the dietary oregano amount did not meet the requirements (NRC) for piglets. The mineral balance was determined during 3 periods of 5 days each. The levels of Cu and Zn were measured by FAAS in the samples (weekly samples/piglet) of ingesta, faeces and urine. It was noticed that although the dietary Cu ingested by the groups without MP was 75% (10.08 ppm) lower than C, the absorption coefficients were only 47% (28.83) lower than for group C (54.22%), while in the groups with 0.5% MP, the absorption was just 10% (48.86%) lower than for group C. For Zn, where the amount ingested by the experimental groups was 33% (97.62 ppm) lower than for group C, the absorption coefficients were just 20% (46.3%) lower than for group C (57.64%). No significant differences were noticed for Cu and Zn in terms of apparent absorption, between the groups with/without phytase. The deposits of Cu and Zn in the main organs and serum (from slaughtered piglets) were also evaluated.     

The effect of feeding expeller-pressed canola meal on growth performance and diet nutrient digestibility in weaned pigs

The effects of feeding increasing levels of expeller-pressed (EP) canola meal in substitution for soybean meal as an energy and amino acid source were evaluated in 240 weaned pigs with an initial body weight of 7.3 ± 0.6 kg. Five pelleted wheat-based diets containing 0, 50, 100, 150 or 200 g EP canola meal/kg were formulated to contain 10.0 MJ net energy (NE)/kg and 1.18 g standardised ileal digestible (SID) lysine/MJ NE and were fed for 4 wk starting 1 wk after weaning at 19 days of age. Expeller-pressed canola meal was added at the expense of soybean meal and the diets were balanced for NE using canola oil and for amino acids using crystalline lysine, methionine, threonine and tryptophan. Increasing inclusion of EP canola meal linearly reduced (P<0.001) the apparent total tract digestibility of energy, dry matter and crude protein and the digestible energy content of diets. From 0 to 28 days on trial, increasing inclusion of EP canola meal did not affect body weight gain, feed intake and feed efficiency. In conclusion, up to 200 g EP canola meal/kg can replace soybean meal in diets formulated to equal NE and SID amino acid content and fed to nursery pigs starting 1 wk after weaning without reducing growth performance.     

Combined effect of probiotics and specific immunoglobulin Y directed against Escherichia coli on growth performance,diarrhea incidence,and immune system in calves

Enterotoxigenic Escherichia coli (ETEC) K99 is one of the major pathogens associated with calf diarrhea. The induction of passive immunity in animals by immunoglobulin Y and using probiotics are inexpensive alternatives to antibiotics for the prevention and treatment of a number of bacterial infections, including diarrhea. Hence, the aim of this research was to evaluate the impact of dietary probiotics and ETEC K99-specific egg yolk antibody supplements, alone and in combination with each other, on health and growth parameters, diarrhea incidence and immune stimulation in newborn Holstein calves. One hundred and twenty neonatal calves were allocated randomly into 4 dietary groups (n = 30 per group) received colostrum/milk without any additives (control group), or supplemented with egg yolk powder contained E. coli K99-specific antibody (Ab group; 1 g/day), a commercial probiotic, Hypro-calves (Pro group; 3 g/day), and their combination (Ab + Pro group), from day (d) 1 to d28 of age. Analyses of the growth parameters, feed efficiency, fecal score, and microbiota and immune function were carried out on d0, 14, 21, and 28 of the experiment. Calves in Ab or Ab + Pro group had higher (P < 0.05) average daily gain compared to control and Pro groups during 0–14d. Feed efficiency of calves in Ab and Ab + Pro groups was significantly higher than that in control group during the period of 0–14d; however, no significant differences were observed in 0–28d period. Diarrhea prevalence and fecal score in Ab + Pro group were lower than control group (P < 0.05). Calves in Ab + Pro group had the lowest number of fecal E. coli in comparison to other groups on d28 (P < 0.05). Feeding Ab + Pro supplement increased (P < 0.05) concentrations of blood IgA and serum CD4 compared to the control group. Likewise, calves in Pro group had higher CD4 levels as compared to the control calves (P < 0.05). Serum concentration of interferon-gamma in control group was lower than other groups (P < 0.05). Overall, these data suggest that feeding a combination of probiotic and specific antibody against ETEC to neonate Holstein calves enhances feed efficiency, boosts immunity, and reduces diarrhea prevalence.     

Feeding soybean meal increases the blood level of isoflavones and reduces the steroidogenic capacity in bovine corpora lutea,without affecting peripheral progesterone concentrations

Thirty-three Holstein-Friesian cows were followed from 14 days pre partum until the fourth ovulation post partum. Housing conditions and basic ration were identical for all animals. Concentrates were individually supplemented according to the daily milk production level, using two different types of protein rich concentrates: soybean meal and rapeseed meal. Soybean and rapeseed meal are known to be respectively high and low in isoflavones. Cows were randomly divided into three groups and blocked for parity. Group I (n = 11) was supplemented with soybean meal and acted as control group. Groups II (n = 11) and III (n = 11) were respectively supplemented with soybean and rapeseed meal and were subjected to a biopsy sampling of the corpus luteum at day 9 of the first three postpartal estrous cycles.Soybean meal supplementation to lactating dairy cows (1.72 kg on average) induced an increase in the blood concentration of equol, dihydrodaidzein, o-desmethylangolensin in both soy groups and resulted in a reduced area occupied by steroidogenic (P = 0.012) and endothelial cells (P = 0.0007) in the luteal biopsies. Blood concentrations of equol and glycitein were negatively correlated with the areas occupied by steroidogenic (r = −0.410 with P = 0.0002, respectively r = −0.351 with P = 0.008) and endothelial cells (r = −0.337 with P = 0.01, respectively r = −0.233 with P = 0.085) in the 3 first estrous cycles. The latter however did not affect the diestrous peripheral blood progesterone concentration.     

In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus)

Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20–25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p < 0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3 mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.     

The modification of glucose levels and N source in the Hungate's medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp. YQ3 grown on corn stalks

Hungate's method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate's medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G⁺, glucose in the Hungate's medium as a glucose control; G^?, glucose removed in a modified Hungate's medium) and four N sources (N1: yeast extract + tryptone + (NH₄)₂SO₄ in Hungate's medium (control); N2: yeast extract + (NH₄)₂SO₄; N3: tryptone + (NH₄)₂SO₄; and N4: tryptone + yeast extract). In the G^? media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G⁺ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G^? medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r > 0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate's media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate's medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate's medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals.