Prehatch thermal manipulation of embryos and posthatch baicalein supplementation increased liver metabolism, and muscle proliferation in broiler chickens.

The exposure of broiler chickens to high ambient temperatures causes heat stress (HS), negatively affecting their health and production performance. To mitigate heat stress in broilers, various strategies, including dietary, managerial, and genetic interventions, have been extensively tested with varying degrees of efficacy. For sustainable broiler production, it is imperative to develop an innovative approach that effectively mitigates the adverse effects of HS. Our previous studies have provided valuable insights into the effects of prehatch embryonic thermal manipulation (TM) and posthatch baicalein supplementation on embryonic thermotolerance, metabolism, and posthatch growth performance. This follow-up study investigated the effect of these interventions on gluconeogenesis and lipid metabolism in the liver, as well as muscle proliferation and regeneration capacity in heat-stressed broiler chickens. A total of six-hundred fertile Cobb 500 eggs were incubated for 21 d. After candling, 238 eggs were subjected to TM at 38.5°C with 55% relative humidity (RH) from embryonic day (ED) 12 to 18. These eggs were transferred to the hatcher and kept at a standard temperature (37.5°C) from ED 19 to 21, while 236 eggs were incubated at a controlled temperature (37.5°C) till hatch. After hatching, 180 day-old chicks from both groups were raised in 36 pens treatment (n = 10 birds/pen, 6 replicates per treatment). The treatments were: 1) Control, 2) TM, 3) Control heat stress (CHS), 4) Thermal manipulation heat stress (TMHS), 5) Control heat stress supplement (CHSS), and 6) Thermal manipulation heat stress supplement (TMHSS). Baicalein was added to the treatment group diets starting from d 1. All birds were raised under the standard environment for 21 d, followed by chronic heat stress from d 22 to 35 (32-33 °C for 8 h) in the CHS, TMHS, CHSS, and TMHSS groups. A thermoneutral (22-24°C) environment was maintained in the Control and TM groups. RH was constant (50 ± 5%) throughout the trial. In the liver, TM significantly increased (P < 0.05) IGF2 expression. Baicalein supplementation significantly increased (P < 0.05) HSF3, HSP70, SOD1, SOD2, TXN, PRARα, and GHR expression. Moreover, the combination of TM and baicalein supplementation significantly increased (P < 0.05) the expression of HSPH1, HSPB1, HSP90, LPL, and GHR. In the muscle, TM significantly increased (P < 0.05) HSF3 and Myf5 gene expression. TM and baicalein supplementation significantly increased (P < 0.05) the expression of MyoG and significantly (P < 0.05) decreased mTOR and PAX7. In conclusion, the prehatch TM of embryos and posthatch baicalein supplementation mitigated the deleterious effects of HS on broiler chickens by upregulating genes related to liver gluconeogenesis, lipid metabolism, and muscle proliferation.

Embryonic thermal manipulation: a potential strategy to mitigate heat stress in broiler chickens for sustainable poultry production.

Due to high environmental temperatures and climate change, heat stress is a severe concern for poultry health and production, increasing the propensity for food insecurity. With climate change causing higher temperatures and erratic weather patterns in recent years, poultry are increasingly vulnerable to this environmental stressor. To mitigate heat stress, nutritional, genetic, and managerial strategies have been implemented with some success. However, these strategies did not adequately and sustainably reduce the heat stress. Therefore, it is crucial to take proactive measures to mitigate the effects of heat stress on poultry, ensuring optimal production and promoting poultry well-being. Embryonic thermal manipulation (TM) involves manipulating the embryonic environment's temperature to enhance broilers' thermotolerance and growth performance. One of the most significant benefits of this approach is its cost-effectiveness and saving time associated with traditional management practices. Given its numerous advantages, embryonic TM  is a promising strategy for enhancing broiler production and profitability in the poultry industry. TM increases the standard incubation temperature in the mid or late embryonic stage to induce epigenetic thermal adaption and embryonic metabolism. Therefore, this review aims to summarize the available literature and scientific evidence of the beneficial effect of pre-hatch thermal manipulation on broiler health and performance.

Effect of in ovo feeding of xylobiose and xylotriose on plasma immunoglobulin, cecal metabolites production, microbial ecology, and metabolic pathways in broiler chickens.

BACKGROUND: Dietary supplementation of xylooligosaccharides (XOS) has been found to influence gut health by manipulating cecal microbiota and producing microbe-origin metabolites. But no study investigated and compared the effect of in ovo feeding of xylobiose (XOS2) and xylotriose (XOS3) in chickens. This study investigated the effect of in ovo feeding of these XOS compounds on post-hatch gut health parameters in chickens. A total of 144 fertilized chicken eggs were divided into three groups: a) non-injected control (CON), b) XOS2, and c) XOS3. On the 17th embryonic day, the eggs of the XOS2 and XOS3 groups were injected with 3 mg of XOS2 and XOS3 diluted in 0.5 mL of 0.85% normal saline through the amniotic sac. After hatching, the chicks were raised for 21 d. Blood was collected on d 14 to measure plasma immunoglobulin. Cecal digesta were collected for measuring short-chain fatty acids (SCFA) on d 14 and 21, and for microbial ecology and microbial metabolic pathway analyses on d 7 and 21. RESULTS: The results were considered significantly different at P < 0.05. ELISA quantified plasma IgA and IgG on d 14 chickens, revealing no differences among the treatments. Gas chromatography results showed no significant differences in the concentrations of cecal SCFAs on d 14 but significant differences on d 21. However, the SCFA concentrations were lower in the XOS3 than in the CON group on d 21. The cecal metagenomics data showed that the abundance of the family Clostridiaceae significantly decreased on d 7, and the abundance of the family Oscillospiraceae increased on d 21 in the XOS2 compared to the CON. There was a reduction in the relative abundance of genus Clostridium sensu stricto 1 in the XOS2 compared to the CON on d 7 and the genus Ruminococcus torques in both XOS2 and XOS3 groups compared to the CON on d 21. The XOS2 and XOS3 groups reduced the genes for chondroitin sulfate degradation I and L-histidine degradation I pathways, which contribute to improved gut health, respectively, in the microbiome on d 7. In contrast, on d 21, the XOS2 and XOS3 groups enriched the thiamin salvage II, L-isoleucine biosynthesis IV, and O-antigen building blocks biosynthesis (E. coli) pathways, which are indicative of improved gut health. Unlike the XOS3 and CON, the microbiome enriched the pathways associated with energy enhancement, including flavin biosynthesis I, sucrose degradation III, and Calvin-Benson-Bassham cycle pathways, in the XOS2 group on d 21. CONCLUSION: In ovo XOS2 and XOS3 feeding promoted beneficial bacterial growth and reduced harmful bacteria at the family and genus levels. The metagenomic-based microbial metabolic pathway profiling predicted a favorable change in the availability of cecal metabolites in the XOS2 and XOS3 groups. The modulation of microbiota and metabolic pathways suggests that in ovo XOS2 and XOS3 feeding improved gut health during the post-hatch period of broilers.

Embryonic thermal manipulation reduces hatch time, increases hatchability, thermotolerance, and liver metabolism in broiler embryos.

The broilers' health and growth performance are affected by egg quality, incubation conditions, and posthatch management. Broilers are more susceptible to heat stress because they have poor thermoregulatory capacity. So, it is crucial to develop a strategy to make chicks thermotolerant and cope with heat stress in post-hatch life. This study investigated the effects of embryonic thermal manipulation (TM) on different hatching parameters (hatch time, hatchability, and hatch weight), brain thermotolerance, and liver metabolism. Six hundred fertile Cobb 500 eggs were incubated for 21 d. After candling on embryonic day (ED) 10, 238 eggs were thermally manipulated at 38.5°C with 55% relative humidity (RH) from ED 12 to 18, then transferred to the hatcher (ED 19-21, standard temperature, 37.5°C) and 236 eggs were incubated at a standard temperature (37.5°C) till hatch. The samples were collected from the Control and TM groups on ED 15 and 18 of the embryonic periods. Hatchability was significantly higher (P < 0.05) in the TM group (94.50%) than in the control group (91.0%). Hatch weight did not differ significantly between the TM group (50.54 g) and the Control group (50.39 g). Most importantly, hatch time was significantly lower (P < 0.05) in the TM group than in the Control. In the D15 embryo brain, the mRNA expression of TRPV1,TRPV2, TRPV3, and the epigenetic marker H3K27 were significantly lower (P < 0.05) in the TM group compared to the Control group. However, in the D18 brain, the expression of TRPV1, TRPV2, and CRHR1 was significantly higher (P < 0.05) in the TM group than in the Control group. In the liver, the mRNA expression of SLC6A14 was significantly lower (P < 0.05) in the D15 TM group than in the D15 Control group. Conversely, the DIO3 mRNA expression was significantly higher (P < 0.05) in the D15 TM group than in the D15 Control group. The expression of GPX3, FOXO1, IGF2, and GHR in the liver was significantly higher in the D18 TM group compared to the D18 Control group (P < 0.05). In conclusion, increased expression of the aforementioned markers during the later embryonic period has been linked to reduced hatch time by increasing liver metabolism and thermotolerance capacity in the brain.

Scanning electron microscope-based evaluation of eggshell quality.

The eggshell is the outermost covering of an egg that provides physical and chemical protection. It is a major source of calcium and minerals for the growing embryo during incubation. The egg industry suffers from a considerable economic loss due to poor eggshell quality. Therefore, developing an accurate and precise method of determining eggshell quality is crucial in improving eggshells in subsequent generations of breeding stock. Hence, this study aimed to develop a method to accurately and precisely determine 1) eggshell thickness using a scanning electron microscope (SEM) and 2) eggshell mineral components using an SEM-Energy Dispersive Spectrometry (EDS) system. Four types of table eggs (N = 48; 12 eggs/group): Cage-Free Organic from the US Mainland (CFO-M) and Hawaii (CFO-H), Caged Non-Organic from the US Mainland (CNO-M), and Hawaii (CNO-H) were sourced from the grocery store. Approximately 0.5 mm2 pieces of eggshells from the equator region of the egg were taken and processed for visualization under the SEM. Three distinct layers of eggshell were identified under SEM: the outermost cuticle, the middle palisade, and the innermost mammillary region. The results showed that CFO-H eggs have a greater eggshell thickness (380.43 ± 2.69 µm) and effective thickness (306.28 ± 4.15 µm). Similarly, the mammillary knob count was denser in CNO-H eggs (186 ± 23.02 knobs/0.5 mm2). Calcium (97.36 ± 0.17%) was the highest among minerals in lower palisades (LP). The magnesium concentration was lowest in the LP region, whereas the phosphorus concentration was highest in the upper palisades. Our study established a scientific method to assess the eggshell quality and biochemical characteristics of eggs through SEM and EDS. This method can be used as a marker for selecting superior parent stock to improve eggshell quality in subsequent generations of breeding stock.

Epigen enhances the developmental potential of in vitro fertilized embryos by improving cytoplasmic maturation.

Numerous growth factors contribute to oocyte maturation and embryonic development in vivo; however, only a few are understood. One such factor is epigen, a new member of the epidermal growth factor (EGF) family that is secreted by the granulosa cells of immature oocytes. We hypothesized that epigen may play a role in oocyte maturation, specifically in the nuclear and cytoplasmic aspects. This study aimed to investigate the effects of epigen on porcine oocyte maturation and embryo development in vitro. In this study, three different concentrations of epigen (3, 6, and 30 ng/mL) were added to tissue culture medium-199 (TCM-199) during in vitro maturation of porcine oocytes. A control group that did not receive epigen supplementation was also included. Mature porcine oocytes were fertilized, and the resulting zygotes were cultured until day 7. The levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured in the in vitro matured oocytes. At the same time, the expression patterns of genes related to apoptosis were detected in day 7 blastocysts (BLs) using real-time quantitative PCR Apoptosis was detected by annexin-V assays in mature oocytes. Data were analyzed using ANOVA and Duncan's test on SPSS, and results are presented as mean ± SEM. The group that received 6 ng/mL epigen had a significantly lower rate of germinal vesicle breakdown (GVBD) than the control group without affecting the nuclear maturation among the experimental groups. Among the treatment groups, the 6 ng/mL epigen group showed significantly higher levels of intracellular GSH and lower ROS production. Supplementation with 6 ng/mL epigen significantly improved blastocyst (BL) formation rates compared to those in the control and 3 ng/mL groups. Additionally, the blastocyst expansion rate was significantly higher with epigen supplementation (6 ng/mL). In the fertilization experiment, the group supplemented with 6 ng/mL epigen exhibited significantly higher levels of monospermy and fertilization efficiency and lower levels of polyspermy than the control group. This study indicated that adding epigen at a concentration of 6 ng/mL can significantly enhance the developmental potential of porcine oocytes fertilized in vitro. Specifically, the study found that epigen improves cytoplasmic maturation, which helps prevent polyspermy and emulates monospermic penetration.

Pre-hatch thermal manipulation of embryos and post-hatch baicalein supplementation mitigated heat stress in broiler chickens.

BACKGROUND: High environmental temperatures induce heat stress in broiler chickens, affecting their health and production performance. Several dietary, managerial, and genetics strategies have been tested with some success in mitigating heat stress (HS) in broilers. Developing novel HS mitigation strategies for sustaining broiler production is critically needed. This study investigated the effects of pre-hatch thermal manipulation (TM) and post-hatch baicalein supplementation on growth performance and health parameters in heat-stressed broilers. RESULTS: Six hundred fertile Cobb 500 eggs were incubated for 21 d. After candling on embryonic day (ED) 10, 238 eggs were thermally manipulated at 38.5 °C with 55% relative humidity (RH) from ED 12 to 18, then transferred to the hatcher (ED 19 to 21, standard temperature) and 236 eggs were incubated at a controlled temperature (37.5 °C) till hatch. After hatch, 180-day-old chicks from both groups were raised in 36 pens (n = 10 birds/pen, 6 replicates per treatment). The treatments were: 1) Control, 2) TM, 3) control heat stress (CHS), 4) thermal manipulation heat stress (TMHS), 5) control heat stress supplement (CHSS), and 6) thermal manipulation heat stress supplement (TMHSS). All birds were raised under the standard environment for 21 d, followed by chronic heat stress from d 22 to 35 (32-33 °C for 8 h) in the CHS, TMHS, CHSS, and TMHSS groups. A thermoneutral (22-24 °C) environment was maintained in the Control and TM groups. RH was constant (50% ± 5%) throughout the trial. All the data were analyzed using one-way ANOVA in R and GraphPad software at P < 0.05 and are presented as mean ± SEM. Heat stress significantly decreased (P < 0.05) the final body weight and ADG in CHS and TMHS groups compared to the other groups. Embryonic TM significantly increased (P < 0.05) the expression of heat shock protein-related genes (HSP70, HSP90, and HSPH1) and antioxidant-related genes (GPX1 and TXN). TMHS birds showed a significant increment (P < 0.05) in total cecal volatile fatty acid (VFA) concentration compared to the CHS birds. The cecal microbial analysis showed significant enrichment (P < 0.05) in alpha and beta diversity and Coprococcus in the TMHSS group. CONCLUSIONS: Pre-hatch TM and post-hatch baicalein supplementation in heat-stressed birds mitigate the detrimental effects of heat stress on chickens' growth performance, upregulate favorable gene expression, increase VFA production, and promote gut health by increasing beneficial microbial communities.

Effects of microalgae, with or without xylanase supplementation, on serum immunoglobulins, cecal short-chain fatty acids, microbial diversity, and metabolic pathways of broiler chickens.

Modern broilers are highly susceptible to environmental and pathogenic threats, leading to gut disorders and poor nutrient utilization if not managed properly. Nutritional programming using several feedstuffs and coproducts to manage gut health has been studied. This study used microalgae as a functional compound and xylanase enzyme in broilers' diets as a strategy to manage gut health. A total of 162 one-day-old unsexed Cobb 500 broiler chicks were randomly assigned to 1 of the 3 dietary treatments: a) corn-soybean meal-based control diet (CON), b) 3% microalgae (MAG), and c) MAG with xylanase enzyme (MAG+XYN). The chicks were reared for 35 days (d) on a floor pen system maintaining standard environment conditions to evaluate the effects of microalgae, with or without xylanase supplementation, on serum immunoglobulins, cecal short-chain fatty acids (SCFA) production, cecal microbial diversity, and metabolic pathways. No significant differences were found for serum immunoglobulin and cecal SCFA among the treatment groups (P > 0.05). Relative microbial abundance at the genus level showed that MAG and MAG+XYN groups had a diverse microbial community on d 3 and d 35. However, no bacterial genus had a significant difference (P > 0.05) in their relative abundance on d 3, but 16 genera showed significant differences (P < 0.05) in their relative abundance among the dietary treatments on d 35. Most of these bacteria were SCFA-producing bacteria. Moreover, MAG and MAG+XYN-fed broilers had better responses than CON groups for metabolic pathways (D-mannose degradation, pectin degradation I and II, ß-1-4-mannan degradation, tetrahydrofolate biosynthesis, glutathione biosynthesis, glutathione-peroxide redox reactions, lactate fermentation to propionate, acetate, and hydrogen, etc.) both on d 3 and d 35. The results suggest that using microalgae, with or without xylanase, had no statistical impact on serum immunoglobulins and cecal SCFA production in broilers. However, an improvement in the cecal microbial diversity and metabolic pathways, which are essential indicators of gut health and nutrient utilization, was observed. Most of the improved metabolic pathways were related to fiber utilization and oxidative stress reduction.

Effects of microalgae, with or without xylanase supplementation, on growth performance, organs development, and gut health parameters of broiler chickens.

Microalgae are becoming potential sustainable feed ingredients, whereas terrestrial feedstuffs are becoming scarce and costly. They are rich in nutritional and functional values but have lower digestibility. This study evaluated the effects of microalgae with or without xylanase supplementation on growth performance and gut health of broiler chickens. A total of 162-day-old Cobb 500 chicks were raised for 35 d. Birds were fed with either 1 of the 3 dietary treatments: 1) corn-soybean meal-based diet (CON), 2) CON + 3% microalgae (MAG), and 3) MAG + xylanase (MAG+XYN) in 2 phases (starter: d 0-21 and finisher: d 22-35) in mash form. Each dietary treatment had 6 replicates, with 9 birds in each replicate. The level of significance was considered at the P value <0.05. The BW, ADG, and ADFI were significantly higher in MAG by 50%, 52.5%, and 42.4%, respectively, and MAG+XYN by 44.1%, 49.7%, and 38.6%, respectively, compared to the CON group. No significant difference was observed for FCR; however, FCR was reduced by 6.3% in both MAG and MAG+XYN groups compared to the CON group. The carcass and organ weight relative to the total body weight were not significantly different among the treatments. The expressions of Zonula occludens 1 (ZO1), Cluster of differentiation 56 (CD56), and Solute carrier family 7 member 7 (SLC7A7) were significantly modulated, for example, by 3.7, 3.9, and 3.3 folds, respectively, in the MAG group compared to CON and 0.8, 0.6, and 1.1 folds, respectively, in the MAG group compared to MAG+XYN groups on d 35. Villi surface area (VSA) of ileum tended to increase on d 3 (P = 0.0725) and d 35 (P = 0.0785) in the MAG and MAG+XYN groups, compared to the CON group. The results suggest that adding microalgae with or without xylanase to broiler's diet could promote growth performance and show a tendency to improve gut health parameters. The nutrient profile and its functional properties make microalgae a valuable resource to the poultry industry as a part substitution of corn and soybean meal and a functional feed supplement to modulate the gut health of broilers.

Dietary supplementation of microalgae mitigates the negative effects of heat stress in broilers.

Heat stress in poultry is a serious concern, affecting their health and productivity. To effectively address the issue of heat stress, it is essential to include antioxidant-rich compounds in the poultry diet to ensure the proper functioning of the redox system. Microalgae (Spirulina platensis) are rich in antioxidants and have several health benefits in humans and animals. However, its role in health and production and the underlying mechanism in heat-stressed broilers are poorly understood. This study aimed to determine the effect of microalgae supplementation on the health and production of heat-stressed broilers. Cobb500 day-old chicks (N = 144) were raised in litter floor pens (6 pens/treatment and 8 birds/pen). The treatment groups were: 1) no heat stress (NHS), 2) heat stress (HS), and 3) heat stress + 3% microalgae (HS+MAG). The broilers in the HS+MAG group were fed a diet supplemented with 3% microalgae, whereas NHS and HS groups were fed a standard broiler diet. Broilers in the NHS were raised under standard temperature (20°C-24°C), while HS and HS+MAG broilers were subjected to cyclic heat stress from d 22 to 35 (32°C-33°C for 8 h). Heat stress significantly decreased the final body weight, whereas the supplementation of microalgae increased the final body weight of broilers (P < 0.05). The expressions of ileal antioxidant (GPX3), immune-related (IL4), and tight-junction (CLDN2) genes were increased in microalgae-supplemented broilers compared to heat-stressed broilers (P < 0.05). The ileal villus height to crypt depth ratio was improved in microalgae-supplemented broilers (P < 0.05). In addition, microbial alpha, and beta diversities were higher in the HS+MAG group compared to the HS group (P < 0.05). There was an increase in volatile fatty acid-producing bacteria at the genus level, such as Ruminococcus, Ocillospira, Lactobacillus, Oscillobacter, Flavonifractor, and Colidextribacter in the group that received microalgae supplementation. In conclusion, dietary supplementation of microalgae improved the growth performances of heat-stressed broilers by improving their physiogenomics. Thus, the dietary inclusion of microalgae can potentially mitigate heat stress in broilers.

Application of botanical products as nutraceutical feed additives for improving poultry health and production.

Poultry is one of the most consumed sources of animal protein around the world. To meet the global demands for poultry meat and eggs, it is necessary to improve their nutrition to sustain the poultry industry. However, the poultry industry faces several challenges, including feedstuff availability, the banning of antibiotics as growth promoters, and several environmental stressors. Therefore, there is a critical need to include available nutraceuticals in the diet to sustain the poultry industry. Nutraceuticals are natural chemical substances that positively influence animal physiological and productive traits. Botanical products (such as fenugreek seeds, ginger roots, and olive leaves) are among the most commonly used nutraceuticals and are gradually gaining popularity in the poultry industry due to their immense benefits in nutrition and therapeutic properties. They can be added to the diet separately or in combination (as a natural antioxidant and immunostimulant) to improve poultry health and production. Botanical products are rich in essential oils and essential fatty acids, which have multiple benefits on the animal's digestive system, such as activating the digestive enzymes and restoring microbiota balance, enhancing poultry health, and production. These nutraceuticals have been shown to stimulate the expression of several genes related to growth, metabolism, and immunity. In addition, the essential oil supplementation in poultry diets up-regulated the expression of some crucial genes associated with nutrient transportation (such asglucose transporter-2 and sodium-glucose cotransporter-1). Previous studies have suggested that supplementation of botanical compounds increased broiler body weight and hen egg production by approximately 7% and 15%, respectively. Furthermore, the supplementation of botanical compounds enhanced the reproductive efficiency of hens and the semen quality of roosters by 13%. This review article discusses the significant effects of some botanical products in the poultry industry and how they can benefit poultry, especially in light of the ban on antibiotics as growth promoters.

Genomic and probiotic attributes of Lactobacillus strains from rice-based fermented foods of North Eastern India.

The probiotic attributes and genomic profiles of amylase-producing Lactobacillus strains from rice-based fermented foods of Meghalaya in the North-Eastern India were evaluated in the study. A preliminary screening of 17 lactic acid bacteria strains was performed based on their starch hydrolysis and glucoamylase activities. Out of 17 strains, 5 strains (L. fermentum KGL4, L. rhamnosus RNS4, L. fermentum WTS4, L. fermentum KGL2, and L. rhamnosus KGL3A) were selected for further characterization of different probiotic attributes. Whole-genome sequencing of two of the best strains was carried out using a shotgun sequencing platform based on their rich probiotic attributes. The EPS production was in the range of 2.89-3.92 mg/mL. KGL2 (41.5%) and KGL3A (41%) showed the highest antioxidant activity. The highest antibiotic susceptibility was exhibited by all the five Lactobacillus strains against ampicillin, ranging from 24.66 to 27.33 mm. The lactobacilli isolates used in the study could survive the simulated gastric/intestinal juices. Genomic characterization of KGL4 and KGL3A illustrated their possible adherence to the intestinal wall, specialized metabolic patterns, and possible role in boosting host immunity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05633-8.

Anti-Inflammatory, ACE Inhibitory, Antioxidative Activities and Release of Novel Antihypertensive and Antioxidative Peptides from Whey Protein Hydrolysate with Molecular Interactions.

OBJECTIVE: The aim of the study was to evaluate the whey protein hydrolysate with bio-functional attributes viz. antioxidative, anti-inflammatory and ACE inhibition efficacy and release of bioactive peptides with antioxidative and ACE-inhibitory activity by employing Pepsin. METHOD: The antioxidant, Anti-inflammatory, ACE inhibitory and proteolytic activities of the whey protein hydrolysates were studied followed by SDS-PAGE analysis and IEF. Anti-inflammatory activity of whey protein hydrolysate was also studied on RAW 264.7 cell line. The separation of the bioactive peptides from whey protein hydrolysate was achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS. RESULTS: WPC (Whey protein concentrate) hydrolysate with pepsin showed proteolytic activity ranging between 14.46 and 18.87 mg/ml. Using the ABTS assay, the highest antioxidative activity was observed in 10 kDa retentate (84.50%) and 3 kDa retentate (85.96%), followed by the highest proteolytic activity (13.83 mg/ml) and ACE inhibitory activity (58.37%) in a 5% WPC solution at 65 °C after 8 h of pepsin hydrolysis. When the protein hydrolysate concentration was low, the production of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages (RAW 264.7) was reduced. SDS-PAGE results exhibited very little protein bands when comparing with WPC hydrolysates to insoluble WPC. There were no protein spots on 2 D gel electrophoresis and "in-solution trypsin digestion" technique have been utilized to digest protein samples directly from WPC hydrolysates. Novel antioxidative peptides and ACE inhibitory peptides were also observed by comparing two databases, i.e., BIOPEP and AHTPDB respectively. The peptide sequences used in this study were found to have excellent potential to be used as inhibitors of hACE as all of them were able to show substantial interactions against the enzyme's active site. CONCLUSIONS: The antihypertensive and antioxidative peptides from whey protein hydrolysates may be beneficial for the future development of physiologically active functional foods. Further, in vivo investigations are required to establish the health claim for each individual bioactive peptide from whey protein hydrolysate.Supplemental data for this article is available online at.

Advanced Practical Strategies to Enhance Table Egg Production.

The global demand for table eggs has increased exponentially due to the growing human population. To meet this demand, major advances in hen genetics, nutrition, and husbandry procedures are required. Developing cost-effective and practically applicable strategies to improve egg production and quality is necessary for the development of egg industry worldwide. Consumers have shown a strong desire regarding the improvement of hens' welfare and egg quality. They also become interested in functional and designer foods. Modifications in the nutritional composition of laying hen diets significantly impact egg nutritional composition and quality preservation. According to previous scientific research, enriched egg products can benefit human health. However, producers are facing a serious challenge in optimizing breeding, housing, and dietary strategies to ensure hen health and high product quality. This review discussed several practical strategies to increase egg production, quality, and hens' welfare. These practical strategies can potentially be used in layer farms for sustainable egg production. One of these strategies is the transition from conventional to enriched or cage-free production systems, thereby improving bird behavior and welfare. In addition, widely use of plant/herbal substances as dietary supplements in layers' diets positively impacts hens' physiological, productive, reproductive, and immunological performances.

Maternal immunization against myostatin suppresses post-hatch chicken growth.

Myostatin (MSTN) is a negative regulator of skeletal muscle growth, thus it was hypothesized that immunization of hens against MSTN would enhance post-hatch growth and muscle mass via suppression of MSTN activity by anti-MSTN IgY in fertilized eggs. This study investigated the effects of immunization of hens against chicken MSTN (chMSTN) or a MSTN fragment (Myo2) on the growth and muscle mass of offspring. In Experiment 1, hens mixed with roosters were divided into two groups and hens in the Control and chMSTN groups were immunized with 0 and 0.5 mg of chMSTN, respectively. In Experiment 2, hens in the chMSTN group were divided into chMSTN and Myo2 groups while the Control group remained the same. The Control and chMSTN groups were immunized in the same way as Experiment 1. The Myo2 group was immunized against MSTN peptide fragment (Myo2) conjugated to KLH. Eggs collected from each group were incubated, and chicks were reared to examine growth and carcass parameters. ELISA showed the production of IgYs against chMSTN and Myo2 and the presence of these antibodies in egg yolk. IgY from the chMSTN and Myo2 groups showed binding affinity to chMSTN, Myo2, and commercial MSTN in Western blot analysis but did not show MSTN-inhibitory capacity in a reporter gene assay. In Experiment 1, no difference was observed in the body weight and carcass parameters of offspring between the Control and chMSTN groups. In Experiment 2, the body weight of chicks from the Myo2 group was significantly lower than that of the Control or chMSTN groups. The dressing percentage and breast muscle mass of the chMSTN and Myo2 groups were significantly lower than those of the Control group, and the breast muscle mass of Myo2 was significantly lower than that of the chMSTN. In summary, in contrast to our hypothesis, maternal immunization of hens did not increase but decreased the body weight and muscle mass of offspring.

RNA-Sequencing based analysis of bovine endometrium during the maternal recognition of pregnancy.

BACKGROUND: Maternal recognition is the crucial step for establishing pregnancy in cattle. This study aims to identify endometrial genes and biological pathways involved in the maternal recognition of pregnancy. Caruncular endometrial tissues were collected from Day 15-17 of gestation (pregnant), non-pregnant (absence of conceptus), and cyclic (non-bred) heifers. RESULTS: Total RNAs were isolated from the caruncular endometrial tissues of pregnant, non-pregnant, and cyclic heifers, and were subjected to high-throughput RNA-sequencing. The genes with at least two-fold change and Benjamini and Hochberg p-value ≤ 0.05 were considered differentially expressed genes and further confirmed with quantitative real-time PCR. A total of 107 genes (pregnant vs cyclic) and 98 genes (pregnant vs non-pregnant) were differentially expressed in the pregnant endometrium. The most highly up-regulated genes in the pregnant endometrium were MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, EIF3M, PRSS22, MS4A8, and TINAGL1. Interferon signaling, immune response, nutrient transporter, synthesis, and secretion of proteins are crucial pathways during the maternal recognition of pregnancy. CONCLUSIONS: The study demonstrated that the presence of conceptus at Day 15-17 of gestation affects the endometrial gene expression related to endometrial remodeling, immune response, nutrients and ion transporters, and relevant signaling pathways in the caruncular region of bovine endometrium during the maternal recognition of pregnancy.

Starchy and fibrous feedstuffs differ in their in vitro digestibility and fermentation characteristics and differently modulate gut microbiota of swine.

BACKGROUND: Alternative feedstuffs may contribute to reducing feed costs of pig production. But these feedstuffs are typically rich in fiber and resistant starch (RS). Dietary fibers and RS are fermented in the gastrointestinal tract (GIT) and modulate the microbial community. Certain microbes in the GIT can promote host health, depending on the type of fermentation substrates available. In this study, six alternative feedstuffs (three starchy: Okinawan sweet potato, OSP; yam, and taro, and three fibrous: wheat millrun, WMR; barley brewers grain, BBG; and macadamia nut cake, MNC) were evaluated for their in vitro digestibility and fermentation characteristics and their effects on pig's hindgut microbial profile. After 2 steps of enzymatic digestion assay, residues were fermented using fresh pig feces as microbial inoculum, and gas production was recorded periodically for 72 h and modeled for fermentation kinetics. After fermentation, the residual liquid phase was analyzed for short-chain fatty acid (SCFA), and the solid phase was used to determine the nutrient's digestibility and microbial community. RESULTS: In vitro ileal digestibility of dry matter and gross energy was higher in starchy than fibrous feedstuffs. Total gas and SCFA production were significantly higher (P < 0.001) in starchy feedstuffs than fibrous feedstuffs. Both acetate and propionate production was significantly higher (P < 0.001) in all starchy feedstuffs than BBG and MNC; WMR was in between. Overall alpha diversity was not significantly different within and between starchy and fibrous feedstuffs. Beta diversity (measured using bray Curtis dissimilarity distance) of starchy feedstuffs was significantly different (P < 0.005) than fibrous feedstuffs. CONCLUSION: Starchy feedstuffs acted as a substrate to similar types of microbes, whereas fibrous feedstuffs resulted in a more diverse microbial population. Such alternative feedstuffs may exert comparable beneficial effects, thus may be included in swine diets to improve gut health.

Mathematical progression of avian egg shape with associated area and volume determinations.

Development of nondestructive techniques for estimating egg parameters requires a comprehensive approach based on mathematical theory. Basic properties used to solve theoretical and applied problems in this respect are volume (V) and surface area (S). There are respective formulae for calculating V and S of spherical, ellipsoidal, and ovoid eggs in classical egg geometry; however, the mathematical description and calculation of these parameters for pyriform eggs have remained elusive. In the present study, we derived the appropriate formulae and established that this would be not only applicable and valid for the category of pyriform eggs, but also universal and explicit for all other naturally occurring avian egg shapes. Thus, we have demonstrated "mathematical progression" of this natural object, considering the egg as a sequence of geometric figures that transform from one to another in the following sequence of shapes: sphere → ellipsoid → ovoid (whose profile corresponds to Hügelschäffer's model) → pyriform ovoid.

Effects of in ovo delivered xylo- and mannan- oligosaccharides on growth performance, intestinal immunity, cecal short-chain fatty acids, and cecal microbiota of broilers.

BACKGROUND: This study investigated a novel in ovo feeding strategy to determine the prebiotic effects of xylo- and mannan- oligosaccharides (XOS and MOS) differing in the degree of polymerization. A total of 192 fertilized eggs were divided into 6 treatment groups: i) normal saline control (NSC), ii) xylotriose (XOS3), iii) xylotetraose (XOS4), iv) mannotriose (MOS3), v) mannotetraose (MOS4), and vi) no injection control (NIC), each containing 4 replicate trays with 8 eggs per replicate. On d 17 of incubation, 3 mg of oligosaccharides (except for controls) dissolved in 0.5 mL of 0.85% normal saline were injected into the amnion of Cobb 500 broilers eggs. After hatch, the chicks were raised for 28 d under standard husbandry practices and were fed a commercial broilers diet ad libitum, and samples were collected periodically. RESULTS: The hatchability, growth performance, and relative weights of breast, drumstick, liver, and proventriculus were not different among the treatments (P > 0.05). The XOS3 injection increased the total short-chain fatty acid production at d 28 compared with both control groups (P < 0.05). The villus height to crypt depth ratio was significantly higher in the XOS4 group than both controls on the hatch day (P < 0.01) but were not different among any treatments on d 7 and 28 (P > 0.05). On the hatch day, the expression level of the CD3 gene (a T cell marker) was increased by XOS3, while the IL-10 gene (a marker of anti-inflammatory cytokine) was reduced by MOS4 (P < 0.05) compared with both controls. Compared with both controls, XOS3 exhibited a trend of reduction for IL-10 (P = 0.074). No cytokines or lymphocyte markers were affected by the treatments on d 7 (P > 0.05), except XOS4 increased IL-4 compared with NSC (P < 0.05). The broilers in the MOS4 group had higher operational taxonomic units (OTUs) and had more differentially abundant taxa, including order Lactobacillales and family Leuconostocaceae (P < 0.05) than both controls on d 28. The predictive functional profiling indicated that the linoleic acid metabolism pathway was enriched in the cecal microbiota of the XOS3 group compared with both controls (P < 0.05). CONCLUSION: The effects of these XOS and MOS on ileal mucosa and immunity are transient, but the effects on fermentation and cecal microbiota are prolonged, and further research is warranted to determine their use as a gut health promoter in poultry.

Novel insights into the regulation of LATS2 kinase in prehierarchical follicle development via the Hippo pathway in hen ovary.

The large tumor suppressor homolog 2 (LATS2), one of the central regulators of the Hippo/MST signaling pathway, plays an inhibitory role in ovarian function and different organ development and growth in mammals. However, the exact roles and molecular regulatory mechanisms of LATS2 in chicken granulosa cell (GC) proliferation, differentiation, and steroidogenesis required for ovarian follicle growth, development, and follicular selection remain poorly understood. This study demonstrated that the LATS2 protein was predominantly localized in the oocytes and undifferentiated GCs of various-sized prehierarchical follicles of the hen ovary. Expression levels of LATS2 mRNA were significantly higher in the smaller follicles (from 1 mm to 5.9 mm in diameter) and the GCs than in the larger follicles (6-6.9 mm in diameter up to F1). Moreover, we found that high levels of LATS2 suppressed the GC proliferation and the mRNA and protein expression of the genes serving as the biomarkers of follicle selection, GC differentiation, and steroidogenesis in the GCs, including FSHR, STAR, CYP11A1, ESR1, and ESR2. Interestingly, the LATS2 significantly downregulated SAV1 and YAP1 transcripts but upregulated the expression of STK3, STK4, TEAD1, and TEAD3 mRNA. Our study provided evidences that STK3/4-LATS2-YAP1 not only acts as a suppressor of cell proliferation and follicle selection but also LATS2 may serve as an enhancer in cell proliferation and follicle selection through the YAP1-LATS2 and the LATS2-STK3/4 feedback loops by promoting the expression of TEAD1/3 but inhibiting the expression of SAV1 transcripts in the prehierarchical follicle development of hen ovary. Taken together, the present study initially revealed the pivotal role and molecular mechanism of LATS2 in the regulation of hen prehierarchical follicle development by controlling GC proliferation, differentiation, steroidogenesis, and follicle selection via the Hippo/MST signaling pathway.