In ovo feeding of α-ketoglutaric acid improves hepatic antioxidant-gene expression, plasma antioxidant activities and decreases body temperature without affecting broiler body weight under cyclic heat stress.

The broiler industry is adversely affected by the rise in global temperature. This study investigated the effects of in ovo feeding of α-ketoglutaric acid (AKG) on growth performance, organ weight, plasma metabolite, plasma oxidative stress, rectal temperature (RT), and hepatic mRNA expression of antioxidant-related genes in Arbor Acres broilers subjected to cyclic heat stress (HS). Three hundred fifty fertile eggs during incubation were divided into 5 groups according to AKG concentrations and temperature conditions. After dissolving AKG in distilled water at 0, 0.5, 1.0, and 1.5, 0% AKG was in ovo administered to 2 of the 5 groups whereas the remaining 3 groups received 0.5, 1.0, and 1.5%, respectively. From d 29 to 34 of age, 4 groups of birds received heat stress (HS) at 31°C ± 1°C for 6 h per day while the other group was kept at room temperature (21°C ± 1°C; NT). So, the 5 treatment groups were: 1) 0AKG-NT, where chicks hatched from eggs receiving 0% AKG were reared under thermoneutral conditions. 2) 0AKG-HS, where chicks hatched from eggs receiving 0% AKG were reared under cyclic HS conditions. 3) 0.5AKG-HS, where chicks hatched from eggs receiving 0.5% AKG were reared under cyclic HS conditions. 4) 1.0AKG-HS, where chicks hatched from eggs receiving 1.0% AKG were reared under cyclic HS conditions. 5) 1.5AKG-HS, where chicks hatched from eggs receiving 1.5% AKG were reared under cyclic HS conditions. HS significantly reduced body weight change (ΔBW %) and average daily gain (ADG) without affecting average daily feed intake (ADFI). Feed conversion ratio (FCR) was significantly increased (P = 0.003) in all HS-treated groups. A significant linear decrease in the final RT (P = 0.005) and a change in RT (P = 0.003) were detected with increasing AKG concentration. Total antioxidant capacity (P = 0.029) and antioxidant balance (P = 0.001) in plasma increased linearly with increasing AKG concentration whereas malondialdehyde concentrations were linearly decreased (P = 0.001). Hepatic gene expression of CAT (P = 0.026) and GPX1 (P = 0.001) were dose-dependently upregulated while nicotinamide adenine dinucleotide phosphate oxidase (NOX)1, NOX4, and heat shock protein (HSP)70 were linearly downregulated (P < 0.05). Hence, in ovo injection of AKG was effective in mitigating HS-induced oxidative stress without attenuating the adverse effects on broiler growth.

Dietary supplementation of solubles from shredded, steam-exploded pine particles modulates cecal microbiome composition in broiler chickens.

This study evaluated the effects of supplementing solubles from shredded, steam-exploded pine particles (SSPP) on growth performances, plasma biochemicals, and microbial composition in broilers. The birds were reared for 28 days and fed basal diets with or without the inclusion of SSPP from 8 days old. There were a total of three dietary treatments supplemented with 0% (0% SSPP), 0.1% (0.1% SSPP) and 0.4% (0.4% SSPP) SSPP in basal diets. Supplementation of SSPP did not significantly affect growth or plasma biochemicals, but there was a clear indication of diet-induced microbial shifts. Beta-diversity analysis revealed SSPP supplementation-related clustering (ANOSIM: r = 0.31, p < 0.01), with an overall lower (PERMDISP: p < 0.05) individual dispersion in comparison to the control group. In addition, the proportions of the Bacteroides were increased, and the relative abundances of the families Vallitaleaceae, Defluviitaleaceae, Clostridiaceae, and the genera Butyricicoccus and Anaerofilum (p < 0.05) were significantly higher in the 0.4% SSPP group than in the control group. Furthermore, the linear discriminant analysis effect size (LEfSe) also showed that beneficial bacteria such as Ruminococcus albus and Butyricicoccus pullicaecorum were identified as microbial biomarkers of dietary SSPP inclusion (p < 0.05; | LDA effect size | > 2.0). Finally, network analysis showed that strong positive correlations were established among microbial species belonging to the class Clostridia, whereas Erysipelotrichia and Bacteroidia were mostly negatively correlated with Clostridia. Taken together, the results suggested that SSPP supplementation modulates the cecal microbial composition of broilers toward a "healthier" profile.

Dietary supplementation of solubles from shredded, steam-exploded pine particles modifies gut length and cecum microbiota in cyclic heat-stressed broilers.

This study was conducted to investigate the effect of supplementing solubles from steam-exploded pine particles (SSPP) on mitigating the adverse effects of cyclic heat stress (CHS) in broilers which were distributed into 3 dietary treatment groups and 2 temperature conditions. Heat stress (HS) exposure for 6 h daily for 7 d adversely affected performance parameters and rectal temperature of chickens. The absolute and relative weights of the liver and bursa of Fabricius decreased in the CHS group while the relative lengths of the jejunum and ileum increased, which was rescued by dietary supplementation with SSPP. The expression of mucin2 (MUC2) and occludin (OCLN) genes was decreased in CHS birds. The expression of heat shock protein -70 and -90 increased in 0% HS compared to that in 0% NT. Birds supplemented with 0.4% SSPP had higher NADPH oxidase -1 expression than birds in the 0% and 0.1% SSPP treatments. Beta diversity of gut microbiota evaluated through unweighted UniFrac distances was significantly different among treatments. Bacteroidetes was among the 2 most abundant phyla in the cecum, which decreased with 0.1% NT and increased with 0.1% HS in comparison to 0% NT. A total of 13 genera were modified by HS, 5 were altered by dose, and nine showed an interaction effect. In conclusion, CHS adversely affects performance and gut health which can be mitigated with dietary SSPP supplementation that modifies the cecal microbiota in broilers.

Effect of in ovo feeding of γ-aminobutyric acid combined with embryonic thermal manipulation on hatchability, growth, and hepatic gene expression in broilers.

OBJECTIVE: This study investigated the effects of in ovo feeding of γ-aminobutyric acid (GABA) and embryonic thermal manipulation (ETM) on growth performance, organ indices, plasma biochemical parameters, hepatic antioxidant levels, and expression of lipid metabolism-related genes in broilers. METHODS: Two hundred and fifty eggs were assigned to one of four treatments: control eggs incubated under standard conditions (CON); eggs that received an in ovo injection of 10% GABA on day 17.5 of incubation (G10); thermally manipulated eggs between days 10 and 18 of incubation at 39.6°C for 6 h daily (TM); and eggs that received both treatments during incubation (G10+TM). After 28 days of rearing, five birds per treatment were selected for blood and organ sampling. RESULTS: No differences were found in hatchability or growth parameters among different treatment groups. Hepatic gene expression of catalase (CAT) and glutathione peroxidase 1 (GPx1) was upregulated (p = 0.046 and p = 0.006, respectively) in the G10+TM group, while that of nuclear factor erythroid 2-related factor 2 (NRF2) was upregulated (p = 0.039) in the G10 group. In addition, the relative gene expression of NADPH oxidase 1 (NOX1) was significantly lower (p = 0.007) in all treatment groups than that in the CON group. Hepatic fatty acid synthase (FAS) levels and average daily feed intake (ADFI) of last week showed a positive correlation (r = 0.50, p = 0.038). In contrast, the relative gene expression of the extracellular fatty acid-binding protein (EXFAB) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were positively correlated (r = 0.48, p = 0.042 and r = 0.50, p = 0.031) with the overall ADFI of birds. CONCLUSION: Taken together, the results of this study suggest that the combination of in ovo feeding of GABA and ETM can enhance hepatic antioxidant function in broilers.

Dietary shredded steam-exploded pine particle supplementation as a strategy to mitigate chronic cyclic heat stress by modulating gut microbiota in broilers.

Improving the availability of underutilized waste for the economic use of livestock feed can be important in countries where feed grain production is scarce. Modulating the gut microbiota through the fibrous content present in these wastes may help mitigate the adverse effects of heat stress (HS). Here, we investigated the effects of dietary steam-exploded pine particle (SPP), a value-added waste product, on the performance, gut health, and cecum microbiota in heat-stressed broilers. Ross 308 broilers (n = 180) at 29 days of age were distributed into three dietary treatment groups (0%, 1%, and 2% SPP) and two temperature conditions (NT: 21 °C; CHS: 31 °C) and grown for seven days. CHS, but not SPP, adversely affected performance parameters, but SPP did not interactively modulate these results. On the contrary, both differently affected other parameters. CHS resulted in increased rectal temperature, total protein in serum, and Nox4 gene expression, whereas 2% SPP increased GLP-2 and the Nox4 gene expression in the duodenum in comparison to 0% and 1% SPP. CHS significantly modified the beta-diversity of cecal microbiota while 1% SPP supplementation in diets increased the abundance of the favorable bacterial genera in chicken. Concludingly, CHS adversely affects growth performances, gut health, stress-related genes, and cecal microbiota while dietary 1% SPP may facilitate the proliferation of beneficial microorganisms in the cecum of broilers.

Effects of In Ovo Injection of α-Ketoglutaric Acid on Hatchability, Growth, Plasma Metabolites, and Antioxidant Status of Broilers.

Recently, α-ketoglutaric acid (AKG) has gained importance as an antioxidant. Its dietary supplementation in animals and humans has proved beneficial. Moreover, an extensive group of studies on in ovo feeding has proved that it produces better day-old chicks and overall performance. Combining the two, we hypothesized that in ovo feeding of AKG could improve the antioxidant status in addition to chick quality and broiler performance. At 17.5 days of incubation, eggs were divided into one of five groups: eggs that received (i) no injection (U-CON), (ii) distilled water (DDW) only (0 AKG), (iii) 0.5% AKG dissolved in DDW (0.5 AKG), (iv) 1.0% AKG dissolved in DDW (1.0 AKG), or (v) 1.5% AKG dissolved in DDW (1.5 AKG). Chicks were raised until 21 days of age. Biological samples were collected on day 0 and day 21. Body weight (p = 0.020), average daily gain (p = 0.025), and average daily feed intake (p = 0.036) were found to quadratically increase with the amount of AKG during the grower phase. At day 0, the absolute (p = 0.040) and relative weight (p = 0.035) of the liver increased linearly with an increasing amount of AKG. The 0.5 AKG group had significantly higher plasma protein (p = 0.025), absolute and relative heart indices at day 0 (p = 0.006). An in ovo feeding of AKG improved the plasma antioxidant capacity of chicks at day 0 as compared to 0 AKG. AKG effect was seen on the plasma antioxidant balance, which increased linearly with the increasing dose of in ovo AKG. Furthermore, 1.0 AKG and 1.5 AKG showed a significant (p = 0.002) upregulation of the hepatic mRNA expression of nuclear factor erythroid 2-related factor (NRF2) in comparison to 0 AKG. The results imply that without negatively affecting hatchability performance, in ovo feeding of AKG has beneficial effects on the antioxidant status of broilers.

Impact of embryonic manipulations on core body temperature dynamics and survival in broilers exposed to cyclic heat stress.

Ambient temperature-associated stress has been shown to affect the normal physiological functions of birds. The recent literature indicated that both, embryonic thermal manipulation (ETM) and in ovo feeding (IOF) of γ-aminobutyric acid (GABA) can mitigate the deleterious effects of heat stress (HS) in young broiler chicks. Therefore, this study intended to assess the effects of cyclic HS (32 ± 1 °C, 4 h/day from day 29 to 35) on rectal temperatures (RTs) and survival in broiler chickens after ETM and in IOF of GABA. A total of 275 RT data points and survival data were collected from chicks assigned to the following five treatments: chicks hatched from control eggs (CON); chicks hatched from control eggs but exposed to HS (CON + HS); chicks hatched from eggs injected at 17.5 days of incubation with 0.6 mL of 10% GABA and exposed to HS (G10 + HS); chicks hatched from thermally manipulated eggs (39.6 °C, 6 h/day from embryonic days 10 to 18) and exposed to HS (TM + HS); chicks hatched from eggs that received both previous treatments during incubation and exposed to HS (G10 + TM + HS). Under thermoneutral conditions, RTs increased quadratically from 39.9 ± 0.2 °C at hatching to 41.4 ± 0.1 °C at 8 days of age. When exposed to cyclic HS during the last week of rearing, the birds' RTs tended to decrease at the end of the heat stress challenge (from 43.0 ± 0.2 °C on day 29 to 42.4 ± 0.1 °C on day 35). A stepwise Cox regression indicated that treatment was predictive of birds' survival. Hazard ratios (HR) and their confidence intervals (CI) were calculated to assess the likelihood of death during the trial. The birds, belonging to the G10 + TM + HS group, were less likely to die under HS (HR 0.11, 95% CI 0.02 to 0.91, P = 0.041) compared to the CON + HS birds. Taken together, the combination of ETM and GABA IOF may help mitigate the drawbacks of cyclic HS by improving the survival of broilers.

Effects of Dietary Supplementation of Solubles from Shredded, Steam-Exploded Pine Particles on the Performance and Cecum Microbiota of Acute Heat-Stressed Broilers.

Heat stress (HS) negatively influences livestock productivity, but it can be, at least in part, mitigated by nutritional interventions. One such intervention is to use byproducts from various sources that are likely to be included in the consumer chain. Thus, the present study investigated the effects of dietary supplementation of solubles from shredded, steam-exploded pine particles (SSPPs) on the performance and cecum microbiota in broilers subjected to acute HS. One-week-old Ross 308 broilers (n = 108) were fed 0%, 0.1%, or 0.4% SSPP in their diets. On the 37th day, forty birds were allocated to one of four groups; namely, a group fed a control diet without SSPPs at thermoneutral temperature (NT) (0% NT) and acute heat-stressed birds with 0% (0% HS), 0.1% (0.1% HS), and 0.4% (0.4% HS) SSPP-supplemented diets. The NT was maintained at 21.0 °C, while the HS room was increased to 31 °C. The final BW, percent difference in body weight (PDBW), and feed intake (FI) were lower in HS birds, but PDBW was reversely associated with dietary SSPP. Similarly, HS birds had a higher rectal temperature (RT) and ΔT in comparison to birds kept at NT. The FI of SSPP-supplemented birds was not significant, indicating lower HS effects. Plasma triglyceride was decreased in HS birds but not affected in 0.1% HS birds in comparison to 0% NT birds. OTUs and Chao1 were increased by 0.1% HS compared to 0% NT. Unweighted Unifrac distances for 0.1% HS were different from 0% NT and 0.4% HS. The favorable bacterial phylum (Tenericutes) and genera (Faecalibacterium and Anaerofustis) were increased, while the pathogenic genus (Enterococcus) was decreased, in SSPP-supplemented birds. In sum, production performances are negatively affected under acute HS. Dietary supplementation of SSPPs is beneficial for improving community richness indices and unweighted Unifrac distances, and it enhanced the advantageous bacterial phyla and reduced virulent genera and triglyceride hydrolysis in acute HS broilers. Our results indicate that dietary SSPPs modulates the microbial profile of the cecum while resulting in relatively less weight loss and lower rectal temperature compared to control.

Embryonic manipulations modulate differential expressions of heat shock protein, fatty acid metabolism, and antioxidant-related genes in the liver of heat-stressed broilers.

In this study, the effects of in ovo feeding of γ-aminobutyric acid (GABA) and embryonic thermal manipulation (TM) on plasma biochemical parameters, organ weights, and hepatic gene expression in broilers exposed to cyclic heat stress (32 ± 1°C for 8 days) (HS) were investigated. A total of 175 chicks were assigned to five treatments: chicks hatched from control eggs (CON); chicks hatched from control eggs but exposed to HS (CON+HS); chicks hatched from eggs injected at 17.5 days of incubation with 0.6mL of 10% GABA and exposed to HS (G10+HS); chicks hatched from thermally manipulated eggs (39.6°C, 6h/d from embryonic days 10 to 18) and exposed to HS (TM+HS); chicks hatched from eggs that received both previous treatments during incubation and exposed to HS (G10+TM+HS). Results revealed that on day 36 post-hatch, hepatic NADPH oxidase 1 (P = 0.034) and 4 (P = 0.021) genes were downregulated in the TM+HS and G10+TM+HS compared to the CON+HS group. In addition, while acetyl-CoA carboxylase gene expression was reduced (P = 0.002) in the G10+TM group, gene expression of extracellular fatty acid-binding protein and peroxisome proliferator-activated receptor-γ was lower (P = 0.045) in the TM+HS group than in the CON+HS group. HS led to higher gene expression of heat shock protein 70 (HSP70) and 90 (HSP90) (P = 0.005, and P = 0.022). On the other hand, the TM+HS group exhibited lower expression of both HSP70 (P = 0.031) and HSP90 (P = 0.043) whereas the G10+TM+HS group had a reduced (P = 0.016) HSP90 expression compared to the CON+HS. MANOVA on different gene sets highlighted an overall lower (P = 0.034) oxidative stress and lower (P = 0.035) heat shock protein expression in the G10+TM+HS group compared to the CON+HS group. Taken together, the current results suggest that the combination of in ovo feeding of GABA with TM can modulate HSPs and antioxidant-related gene expression in heat-stressed broilers.

Dietary Supplementation of Shredded, Steam-Exploded Pine Particles Decreases Pathogenic Microbes in the Cecum of Acute Heat-Stressed Broilers.

The gut microbiome stimulates nutrient metabolism and could effectively generate heat tolerance in chickens. This study investigates the effects of dietary steam-exploded pine particle (SPP) supplementation and subsequent acute heat stress on productive performance and cecum microbiome in broilers. Eight-day Ross 308 broilers were distributed in three groups with 0%, 1%, and 2% SPP in diets. On the 41st day, forty birds were allocated to four groups with ten birds each. The treatments were control diet at thermoneutral temperature (0% NT) and acute heat-stressed (HS) birds fed control (0% HS), 1% (1% HS), and 2% (2% HS) SPP. Parameters recorded were body weight (BW), feed intake (FI), rectal temperature (RT), relative organ weight, and metagenome analysis from cecum samples. Percent difference in BW, FI, and RT was decreased in HS birds. Metagenome analysis revealed similar richness and diversity in microbial communities. The relative abundance of the bacterial genus such as Limosilactobacillus, Drancourtella, and Ihubacter was increased while that of Alistipes, Alkalibacter, Lachnotalea, and Turicibacter was decreased in SPP supplemented HS birds. Concludingly, the production performance of broilers is negatively influenced during HS, and 2% dietary SPP supplementation may reduce the adverse effects of HS by modifying the microbiota in chickens.

In Ovo Injection of GABA Can Help Body Weight Gain at Hatch, Increase Chick Weight to Egg Weight Ratio, and Improve Broiler Heat Resistance.

The aim of this study was to explore the outcomes of an in ovo GABA injection in broilers challenged with HS. In Experiment 1, 210 Arbor Acres eggs were allocated to five treatments: no-injection, and in ovo injection of 0.6 mL of 0%, 5%, 10%, or 20% of GABA. Hatchling weight and CWEWR were significantly increased in the 5% GABA group. In ovo, injection of 10% GABA solution caused a significant decrease in plasma cholesterol and increased plasma total antioxidant capacity of hatchlings. Experiment 2 was conducted with 126 fertile Arbor Acres eggs distributed into one of two groups. At 17.5 days of incubation, one received no injection, and the other was fed 0.6 mL of 10% GABA. On day 10, one subgroup (4 replicates * 3 birds) from each treatment was submitted to HS (38 ± 1 °C for 3 h) while the other was kept at a thermoneutral temperature (29 ± 1 °C). An in ovo injection of GABA significantly increased total antioxidant capacity, but reduced malondialdehyde levels, hepatic mRNA levels of HSP70, FAS, and L-FABP with HS. In conclusion, an in ovo GABA injection improves CWEWR and antioxidant status at hatch, and enhances antioxidant status while downregulating the expression of HSP70 and fatty acid metabolism-related genes in young chicks under HS.

Embryonic Thermal Manipulation and in ovo Gamma-Aminobutyric Acid Supplementation Regulating the Chick Weight and Stress-Related Genes at Hatch.

Chickens are exposed to numerous types of stress from hatching to shipping, influencing poultry production. Embryonic manipulation may develop resistance against several stressors. This study investigates the effects of thermoneutral temperature (T0; 37.8°C) with no injection (N0) (T0N0), T0 with 0.6 ml of 10% in ovo gamma-aminobutyric acid (GABA) supplementation (N1) at 17.5th embryonic day (ED) (T0N1), thermal manipulation (T1) at 39.6°C from the 10th to 18th ED (6 h/day) with N0 (T1N0), and T1 with N1 (T1N1) on hatchability parameters and hepatic expression of stress-related genes in day-old Arbor Acres chicks. The parameters determined were hatchability, body weight (BW), organ weight, hepatic malondialdehyde (MDA), and antioxidant-related gene expression. Percent hatchability was calculated on a fertile egg basis. Growth performance was analyzed using each chick as an experimental unit. Eight birds per group were used for organ weight. Two-way ANOVA was used taking temperature and GABA as the main effect for growth performance and gene expression studies. Analysis was performed using an IBM SPSS statistics software package 25.0 (IBM software, Chicago, IL, USA). Hatchability was similar in all the groups and was slightly lower in the T1N1. Higher BW was recorded in both T1 and N1. Intestinal weight and MDA were higher in T0N1 against T0N0 and T1N1, respectively. The expression of HSP70, HSP90, NOX1, and NOX4 genes was higher and SOD and CAT genes were lower in the T1 group. The present results show that T1 and N1 independently improve the BW of broiler chicks at hatch, but T1 strongly regulates stress-related gene expression and suggests that both T1 and N1 during incubation can improve performance and alleviate stress after hatch.