Global population growth poses a threat to sustainable development. Meanwhile, the use of plant proteins as healthy and sustainable alternatives to animal proteins needs further research. Therefore, this investigation was designed to study the nutritive, structural, and thermal properties of isolated protein fractions from different legumes, i.e., faba bean (FPI), soybean (SPI), and lupine (LPI). As a prospective plant-based protein powder, an equal mixture (MPI) of the three prior legume samples was formulated to study its properties compared to each sole sample. The alkaline extraction and isoelectric precipitation (AE-IP) technique was used for protein isolation. Results showed that all protein isolates had reasonable levels of protein with maximum protein content in SPI (96.15%). The MPI sample, however, came out on top in terms of amino acid profile followed by FBI. Compared to SPI and LPI, it had the highest isoleucine content and higher methionine, valine, leucine, phenylalanine, and lysine. Moreover, MPI showed a median particle charge (−37.1 mV) compared to FPI, SPI, and LPI samples. MPI sample peak showed resistance to heat denaturation at a temperature greater than 200 °C when the DSC test
was conducted. With respect to its rheological characteristics, it outperformed the other three protein isolates and exhibited the highest values of storage modulus G’ and loss modulus G”. Consequently, our study suggests that pulse-derived protein isolate mixture can be used as a unique type of nutritious dietary protein supplement. It could be a good nutritional alternative to proteins derived from animals.

Fusarium graminearum produces zearalenone (ZEA), a mycotoxin that is widely found in
food and feed products and is toxic to humans and livestock. Piper sarmentosum extract (PSE) inhibits
F. graminearum, and Oroxylin A appears to be a major antifungal compound in PSE. The aim of this
study is to quantify the Oroxylin A content in PSE using UPLC-QTOF-MS/MS, and to investigate the
antagonistic activity of Oroxylin A against F. graminearum and its inhibitory effect on ZEA production.
The results indicate that Oroxylin A inhibits both fungal growth and ZEA production in a dosedependent
manner. Oroxylin A treatment downregulated the mRNA expression of zearalenone
biosynthesis protein 1 (ZEB1) and zearalenone biosynthesis protein 2 (ZEB2). The metabolomics
analysis of F. graminearum mycelia indicated that the level of ribose 5-phosphate (R5P) deceased
(p < 0.05) after Oroxylin A treatment (64–128 ng/mL). Moreover, as the Oroxylin A treatment content
increased from 64 to 128 ng/mL, the levels of cis-aconitate (p < 0.05) and fumarate (p < 0.01) were
upregulated successively. A correlation analysis further showed that the decreased R5P level was
positively correlated with ZEB1 and ZEB2 expression, while the increased cis-aconitate and fumarate
levels were negatively correlated with ZEB1 and ZEB2 expression. These findings demonstrate the
potential of Oroxylin A as a natural agent to control toxigenic fungi and their mycotoxin.

In this study, we performed a quantitative analysis of 12 compounds derived from
Piper sarmentosum extract (PSE) and guava leaf extract (GE). In addition, we
investigated the effects of mixed extract (ME) of PSE and GE (1:1) on piglets' gut
microbiome and metabolome. A total of 200 piglets (Duroc × Landrace × Large
Yorkshire, 21‐day‐old) were randomly assigned into two groups with five replicates
of 20 piglets/pen having the same initial body weight. Piglets were fed a basal diet
supplemented with ME at 0 (T0) or 200 mg/kg (T1) for 3 weeks. The quantitation
results by ultraperformance liquid chromatography linked to triple‐quadrupole
tandem mass spectrometry showed that vitexin 2‐O‐rhamnoside and pellitorine
were the greatest abundant among six compounds detected in the PSE. In addition,
quercetin, isoquercitrin and avicularin were found to be the richest of all detected
compounds in the GE. Findings on experimental animals indicated that three
differential metabolites, comprising L‐alanine, sarcosine and dihydrofolic acid, in T1
compared with T0 groups, have exactly opposite levels trends in serum and faeces.
Moreover, two metabolic pathways (i.e., urea cycle and glutamate metabolism)
differed significantly in the serum and faeces of piglets betweenT0 and T1 (p < 0.05).
At the same time, T1 had significantly higher relative abundances of Agathobacter
and Alloprevotella than T0 at genus level (p < 0.05). Correlation analysis revealed that
the genus Agathobacter correlated positively with carbamoyl phosphate (p < 0.01)
and oxoglutaric acid (p < 0.05), and negatively with succinic acid (p < 0.01) and
ornithine (p < 0.05). These four differential metabolites were also involved in the
urea cycle and/or glutamate metabolism pathways. The results here indicated that
the tested plant extract mixture represents a worthy feed additive with obvious
antioxidative properties.

The search for a natural antimicrobial agent is ongoing and critical because of the rise and rapid proliferation of antibiotic-resistant pathogenic bacteria. The current study aims to examine the effect of Paenibacillus polymyxa AM20 as an alternative antibiotic and feed additive on Indian river broiler performance, digestive enzymes, thyroid hormones, lipid profile, hepatosomatic index, immunological response, gut bacteria, and antioxidant parameters. The bacterial isolate AM20 was identified at the gene level by isolating DNA and using PCR to detect genes. Based on 16S rRNA gene sequence analysis, the bacterial isolate was identified as Paenibacillus polymyxa. One hundred twenty Indian river broilers (1-day old) were randomly divided into 4 groups of 10 chicks each, with 3 replicates. The control group was fed a basal diet only, while the other 3 were administered control diets supplemented with P. polymyxa at 3 concentrations: 0.5, 1, and 1.5 mg/kg. The findings revealed that all groups that received graded amounts of P. polymyxa increased all growth parameters throughout the study. P. polymyxa treatment at 1.5 mg/kg increased body gain by 9% compared to the control due to increased feed intake (P = 0.0001), growth rate (P = 0.0001), and decreased feed conversion ratio. Compared to the control group, P. polymyxa (1.5 mg/kg) enhanced kidney functions in chickens by reducing uric acid and creatinine levels (P = 0.0451). Compared to the control group, alanine aminotransferase and aspartate transaminase levels in the liver were significantly reduced at all P. polymyxa doses. Liver function values were highest for P. polymyxa at 1.5 mg/kg. Compared to the control group, those whose diets included P. polymyxa had significantly better blood cholesterol levels, high-density lipoprotein, low-density lipoprotein, immunological response, thyroid function, and gut microbiota. In general, broiler chickens' economic efficiency was improved by including P. polymyxa in their diet, which also improved their growth performance, carcass dressing, specific blood biochemical levels and enzymes, and the composition of the gut microbiota.

Optimal dietary non-phytate phosphorus (NPP) is essential in poultry to maximise productive and reproductive
performance, along with indices of egg and bone quality. This study aimed to establish the NPP
requirements of egg-type duck breeders aged from 54 to 80 weeks on the following traits: egg production,
egg incubation, egg quality, tibial characteristics, reproductive organ, plasma indices, and the
expression of genes related to phosphorus absorption. Longyan duck breeders aged 54 weeks (n = 300)
were randomly allotted to five treatments, each containing six replicates of 10 individually caged birds.
Birds were fed corn-soybean meal-based diets containing 0.18, 0.25, 0.32, 0.38, and 0.45% NPP/kg for
27 weeks. The tested dietary NPP levels did not affect egg production or egg quality indices. The hatchling
weight of ducklings increased (quadratic, P < 0.01) as dietary NPP level increased, and the highest value
occurred with 0.25% NPP. The number of large yellow follicles (LYF), and the relative weights of LYF and
ovary showed linear and quadratic responses to dietary NPP levels; the lowest number and relative
weight of LYF occurred with 0.38% NPP, and the lowest ovarian weight was obtained with 0.25% NPP.
There were no differences in tibial length, breaking strength, and mineral density in response to dietary
NPP levels. In contrast, tibial content of Ca increased (linear, P < 0.01) with dietary NPP levels increasing
from 0.18 to 0.45%, and the tibial content of P increased at 0.32% NPP and the higher dietary NPP levels.
Plasma concentration of P showed a quadratic (P < 0.05) response to the dietary NPP levels, where the
highest value was seen at 0.38% NPP. In conclusion, dietary NPP levels from 0.18 to 0.45% had no effects
on egg production, and egg and tibial quality of duck breeders. The duck breeders fed a diet with 0.25%
NPP showed the highest hatchling weight of their offspring, while those fed 0.38% NPP had the lowest
number and relative weight of LYF. These results indicated that the diet with 0.25% NPP can be used
in egg-type duck breeders to improve the hatchling weight of their offspring, without adverse effects
on their productivity. The regression model indicated that the maximal hatchling weight of ducklings
was obtained from duck breeders fed the diet with 0.30% NPP.

Thepresentstudyinvestigatedtheinflu-enceofexposingquaileggstolow-dosegammaradiation(GR)andinovofeedingwith2sourcesofamixtureoftraceelements(Zn,Fe,andCu),includingsulfate(TES)andloadedwithmontmorillonite(TEM),onembryonicdevelopmentactivitiesandprehatchquality.Atotalof960eggsontheseventhdayofincubationwererandomlydividedinto6groups(160eggs/group)with4replicateof40eggsineach.A3£2factorialarrangementexperimentwasperformedandincluded3sourcesinovofeedingwithamixtureoftraceelements(Zn,Fe,andCu),including0mg/egg,50mgTES/egg,and50mgTEM/eggwitheggirradiationusing0and0.2GyfromGR.Eggsinjectedwith50mgTEM/eggandexposedto0.2GyfromGR(TEM/GR)wassignificantly(P≤0.05and0.01)higherinhatchability,hatchbodyweight,andrelativeorganweight(liver,gizzard,proventriculus,heart,andintes-tine).Theobtainedresultsindicatedsignificant(P≤0.05)decreasedintheserumconcentrationofmalondialdehyde(MDA)inTEM/GRgroup.Therewassignificant(P≤0.05)increasedofcatalase(CAT)activityandthe concentrationsofgrowthhormone(GH)andinsulin-likegrowthfactor-1(IGF-1)inTEM/GRgroup;however;totalantioxidantcapacity(T-AOC)wassignificant(P≤0.05)increasedinCT/GRgroup.SerumconcentrationsofimmunoglobulinM(IgM)(P≤0.05)andtumornecrosisfactor-alpha(TNF-a)wereincreasedintheTEM/CRgroup;theconcentrationoftransforminggrowthfactorbeta(TGF-b)significant(P≤0.05)increasedintheTEM/GRgroup;andinterleukins(IL6andIL10)showednosignificantdifferencesamongthegroups.Ourresultsshowedincreaseinthyroxineandmyostatincon-centrationswithTES/CRandCT/GRofourstudygroups,respectively.TherelativemRNAexpressionlevelsoftheGH,IGF-1,andFascellsurfacedeathreceptor(FAS)genesweresignificantly(P≤0.05and0.01)upre-gulatedinthelivertissueoftheTEM/GRgroupcom-paredwiththeothergroups.Inconclusion,TEM/GRwasthebesttreatmentforimprovingprehatchquality,increasingserumantioxidantenzymeactivities,andpro-motingtheexpressionofgrowthandimmunegenesinfer-tilizedquaileggs.

Intestinal oxidative stress in broilers is produced by chronic heat stress (HS) and has a negative
impact on poultry performance as it induces intestinal inflammation and promotes the invasion of
gram-negative bacteria, such as bacterial lipopolysaccharide (LPS). Therefore, dietary inclusion
of the antioxidant compound, ethoxyquin (EQ), could improve enteric antioxidant capacity,
immune responses, and the epithelial barrier, and maintain the symbiotic gut microbiota
community. To investigate the effects of EQ supplementation on alleviating enteric oxidative
stress in heat-stressed broilers, 200 one-day-old male Ross 308 broilers were randomly assigned
to four groups (n = 50 chicks/group; n = 10 chicks/replicate) and fed a basal diet supplemented
with 0 (CT), 50 (EQ-50), 100 (EQ-100), and 200 (EQ-200) mg EQ/ kg-1 for 5 weeks. The chicks
were raised in floor pens inside the broiler farm at a temperature and humidity index (THI) of 29
from day 21 to day 35. Growth performance traits, relative organ index, hepatic antioxidant
enzymes, serum immunity, total adenylate, and cytokine activities were improved in the EQ-50
group (linear or quadratic P < 0.05), promoting the relative mRNA expression of cytokine generelated
anti-inflammatory and growth factors. A distinct microbial community colonised the gut
microbiota in the EQ-50 group, with a high relative abundance of Lactobacillus,
Ligilactobacillus, Limosilactobacillus, Pediococcus, Blautia, and Faecalibacterium compared to
the other groups. Dietary supplementation with 50 mg EQ/ kg-1 for 5 wk attenuates enteric
oxidative stress and intestinal inflammation by enhancing serum immune and cytokine content
(IgG, IL-6, and TGF-β,) and symbiotic microbiota in heat-stressed broilers. EQ promotes the
expression of Hsp70, SOD2, GPx 4, IL-6, and IGF-1 cytokine gene-related anti-inflammatory and
growth factors in heat-stressed hepatic broilers. Collectively, EQ-50 could be a suitable feed
supplement for attenuating enteric oxidative stress and intestinal inflammation, thereby promoting
the productivity of heat-stressed broilers.

Abstract: Fusarium graminearum produces zearalenone (ZEA), a mycotoxin that is widely found in
food and feed products and is toxic to humans and livestock. Piper sarmentosum extract (PSE) inhibits
F. graminearum, and Oroxylin A appears to be a major antifungal compound in PSE. The aim of this
study is to quantify the Oroxylin A content in PSE using UPLC-QTOF-MS/MS, and to investigate the
antagonistic activity of Oroxylin A against F. graminearum and its inhibitory effect on ZEA production.
The results indicate that Oroxylin A inhibits both fungal growth and ZEA production in a dosedependent
manner. Oroxylin A treatment downregulated the mRNA expression of zearalenone
biosynthesis protein 1 (ZEB1) and zearalenone biosynthesis protein 2 (ZEB2). The metabolomics
analysis of F. graminearum mycelia indicated that the level of ribose 5-phosphate (R5P) deceased
(p < 0.05) after Oroxylin A treatment (64–128 ng/mL). Moreover, as the Oroxylin A treatment content
increased from 64 to 128 ng/mL, the levels of cis-aconitate (p < 0.05) and fumarate (p < 0.01) were
upregulated successively. A correlation analysis further showed that the decreased R5P level was
positively correlated with ZEB1 and ZEB2 expression, while the increased cis-aconitate and fumarate
levels were negatively correlated with ZEB1 and ZEB2 expression. These findings demonstrate the
potential of Oroxylin A as a natural agent to control toxigenic fungi and their mycotoxi

There is scarce information on the effects of feed storage period on poultry performance, which has not been investigated thus far. The aim of this study was to evaluate the effect of feeding broilers on feeds stored for different periods, with or without supplemental amino acids and vitamins. This study followed a factorial design (3 x 3), i.e. three dietary treatments and three feed storage periods. The three dietary treatments consisted of 1) a basal diet, 2) a basal diet including additional 5g methionine and 13 g lysine/kg, and 3) a basal diet including 3 g vitamin premix/kg. The 3 dietary treatments were tested with feeds stored for three different periods (Fresh, 4-months, and 6-months). A total of 450 one-day old broiler chicks were randomly assigned to the 9 treatments, each treatment contained 5 replicates of 10 chicks each (n= 50/group). The experiment lasted for 42 days. The results indicated that the final BWs of the birds received 4- or 6-months stored feed were greater than that of those fed fresh diets. The best total BWG was seen in the 4-month-stored feed group. There was no significant effect of storage period on total FC and FCR during period from 0 to 6 weeks of age, but the FCR tended to be better with the feed stored for 4 months. Haemoglobin was significantly higher in the birds fed fresh or 4-month stored feed. The percent of liver and spleen of the birds received 6-month-stored feed were the highest (P ˂ 0.01). Plasma content of albumin was higher in the birds received 4-or 6-month stored feed than the fresh diet. The use of additional amino acids increased the final BW and total BWG than those of the control and vitamin-supplemented treatments. The main effect of dietary treatments on percentages of immune organ weight was insignificant. The control (basal diet) recorded higher lymphocytes percentage, while T2 (BD + double level of amino acids) recorded higher eosinophil, heterophil and H/L ratio, whereas T3 (BD + double level of vitamins) recorded higher levels of haemoglobin, eosinophil, and monocytes. The main effect of dietary treatments on blood plasma proteins was insignificant. The tested treatments did not show a significant effect on broilers mortality. The detected values of AF1, AF2, AG1, and AG2 were very low in all 9 diets. In conclusion, enrichment of broiler feeds with higher levels of lysine and methionine (double level of NRC, 1994) together with antioxidants supplement could prolong their shelf-life to 6 months without any deleterious impact on productive performance of broiler chickens.