Integration of Bacteriophages and Mannanase as Sustainable Feed Additives for Non-Ruminant Animals Under Low-Carbon Diet Regimes

다국어 초록 (Multilingual Abstract)

EXPERIMENT 1: Integrating Bacteriophage with High-and Low-protein feeding Strategies Enhances Performance, Gut health, and Environmental Sustainability in Ross 308 Broilers A 35-day growth trial was conducted to evaluate the effects of dietary protein density and bacteriophage (BP) supplementation on growth performance, nutrient digestibility, gas emissions, organ development, meat quality, and cecal microbiota in ROSS 308 broilers. A total of 1,440 chicks (47.87 ± 1.50 g initial BW) were randomly allocated to four treatments in a 2 × 2 factorial design: high protein (HP) and low protein (LP) diets, with or without 10 mg/kg BP, each with 20 replicates of 18 birds. During the grower phase (days 9 to 20), birds fed diet supplemented with BP showed tendency to improved bodyweight gain (BWG) and feed conversion ratio (FCR). Also, during the finisher phase, birds receiving BP supplementation demonstrated a significantly greater BWG and a tendency toward increased feed intake, with no differences in FCR. Furthermore, over the entire experimental period, final BW and cumulative BWG were significantly increased in birds fed BP supplementation. At the end of the trial, BP supplementation has tended to improve dry matter and significant increase in nitrogen digestibility in broilers. Moreover, ammonia emission was significantly reduced in birds fed LP diets. However, relative organ weights and meat quality traits (pH, water-holding capacity, cooking loss, drip loss, and color) were unaffected by dietary treatments. The alpha diversity of Chao1, observed features, Shannon’s index, Simpson’s index, and Pielou’s evenness were highly abundance in birds fed LP diet with BP. Principal coordinate analyses (Bray–Curtis and Unweighted UniFrac) indicated treatment-dependent clustering, with LP+ showing the most distinct separation from HP groups. The cecal microbiota was dominated by Firmicutes (54.5%) and Bacteroidota (36.9%), followed by minor phyla (<3%). At the genus level, Bacteroides (17.5%) and Lactobacillus (9.1%) were predominant. In summary, BP supplementation improved growth performance, nitrogen digestibility, and enhanced cecal microbial diversity, while LP diet reduced ammonia emissions without compromising productivity. No negative effects were observed on organ development or meat quality, suggesting BP as a beneficial feed additive across protein densities in broiler production.

EXPERIMENT 2: Effect of different levels of protein with and without Bacteriophage supplementation on growth performance, nutrient digestibility, gas emission, and gut health in weaning pigs
A 6-week growth trial was conducted using four groups of 50 weaning pigs [200 head [Duroc × (Landrace × Yorkshire)] with initial BW 6.61 ± 0.62 kg] to evaluate the effects of bacteriophage (BP) supplementation under different crude protein (CP) levels on their growth performance, nutrient digestibility, noxious gas emissions, and gut health. The pens of pigs (2 gilts and 3 barrows /pen) were randomly allocated into four treatment groups in a randomized complete block design, with 10 replicates/treatment. The experiment followed a 2 × 2 factorial arrangement, consisting of two dietary protein levels: high-protein diet (HP) and low-protein diet (LP, reduced by 2% CP) and two additives with (10 mg/kg) and without (0) BP. At week 3, pigs fed HP diets with BP supplementation had significantly higher BW than those fed LP diets without BP (p < 0.05). By week 6, pigs receiving BP supplementation, regardless of dietary protein level, tended to show greater BW (p < 0.05). Average daily gain was consistently higher in pigs fed HP+ BP during weeks 3, 6, and the overall period, although the difference was not significant (p > 0.05). Feed intake and feed conversion ratio were unaffected by either protein level or BP supplementation. In contrast, nitrogen digestibility was significantly greater (p < 0.05) in pigs fed HP+ BP compared with LP− BP, suggesting improved nutrient utilization. Furthermore, ammonia emission was markedly reduced (p < 0.05) in the LP+ BP group, indicating a potential benefit of BP in mitigating environmental nitrogen loss. Microbial diversity analyses revealed that alpha diversity of Simpson, indices were greatest in HP+, indicating enhanced microbial richness. Bray Curtis and Unweighted UniFrac analyses showed distinct clustering, with HP+ group forming a clear cluster separation from other groups highlighting that HP+ diet markedly influenced gut microbial structure of weaning pigs. At the phylum level, HP+ group pigs exhibited a higher proportion of Firmicutes_A, while Bacteroidota was enriched in LP- BP group pigs. Prevotella and Lactobacillus were the dominant genera, followed by Clostridium, Collinsella, and Faecalibacterium. Also, Prevotella abundance peaked in HP+, whereas Lactobacillus and Faecalibacterium were higher in LP−. LEfSe analysis further revealed that HP+ promoted fiber-fermenting and lactic acid–producing bacteria such as Clostridium, Limosilactobacillus, and Ruminococcus. HP − was enriched in Prevotella and Campylobacterales, whereas LP+ showed enrichment in anaerobic taxa like Mycoplasma and Oxalobacter. LP− was dominated by Lactobacillus johnsonii and Streptococcus. Collectively, these findings suggest that dietary BP supplementation, particularly under HP conditions, enhances growth performance, nutrient utilization, and beneficial microbial communities, while LP diet with BP significantly reduce gas emission in weaning pigs.

EXPERIMENT 3: Multi-scale effects of β-mannanase in finishing pigs: enhanced of growth performance, nutrient digestibility, fecal gas mitigation, and regulated gut microbiome
The aim of this study was to investigate the effects of high and low protein diet with or without mannanase supplementation on growth performance, nutrient digestibility, fecal gas emission, meat quality, and gut microbiome in finishing pigs. A total of 200 finishing pigs [(Landrace × Yorkshire) × Duroc; 55.02 ± 3.35kg (Starting Weight)] were used in a 10 weeks trial. Finishing pigs were randomly allotted into 4 treatments according to their initial body weight (BW). There were 10 replications in each treatment, with 5 pigs/pen. Dietary treatment groups were as follows: 1) HP, Basal diet; 2) HPM, Basal diet + 0.1% Mannanase; 3) LP, Low crude protein diet (-2% CP); 4) LPM, Low crude protein diet (-2% CP) + 0.1% Mannanase. Pigs fed high and low protein diet supplemented with enzyme showed significantly (p<0.05) increased ADG at the end of week 5, 10, and overall experimental period. Similarly, pigs fed high and low protein diet supplemented with enzyme showed significantly (p<0.05) increased Nitrogen digestibility at the end of trial. In addition, at the end of week 6, pigs fed low protein diet showed decreased (P < 0.05) ammonia emission compared to those fed high protein diet, and the inclusion of β-mannanase to low protein diet showed a significant decrease (P < 0.05) in NH3. Moreover, the results of alpha diversity indicated a significant difference (P < 0.05) in the Chao1 index, Simpson's index, Shannon index, and Pielou's evenness index in groups HP and LP compared to HPM and LPM. In addition, beta diversity analysis of Bray–Curtis and Unifrac distances showed a significant difference (P < 0.05) in HP and LP compared to HPM and LPM. These findings suggest that supplementing mannanase in high and low protein diets can improve the gut microbiome and nutrient digestibility of finishing pigs while reducing environmental impact, providing a viable strategy for pig production.

Conclusion
Adding bacteriophages to the feed of broilers and weaned pigs feed improves growth and nutrient digestibility. While this effect is most pronounced with high-protein feed, studies have also observed that low-protein feed increases gut microbial diversity and reduces ammonia emissions. optimizing protein levels in feed combined with bacteriophage supplementation is a promising approach to enhance production efficiency and reduce environmental impact.
Mannanase decomposes mannan and regulates the intestinal microbial environment, thereby improving nitrogen utilization efficiency and reducing ammonia (NH₃) production. Adding mannanase to feed can improve growth performance and reduce nitrogen excretion in both growing and fattening pigs.
* Low-protein feeds supplemented with phage and mannanase have the advantages of reducing feed costs for both pigs and chickens, easing environmental impact by reducing nitrogen and greenhouse gas emissions, and support health and growth.

목차 (Table of Contents)

• CHAPTER 1
• LITERATURE REVIEW 1
• 1. INTRODUCTION . 2
• 2. OVERVIEW OF PHAGE: HISTORY, STRUCTURE, AND LIFE CYCLE 3
• 3. APPLICATION OF PHAGE IN MONOGASTRIC ANIMAL 5
• 4. BACTERIOPHAGES: DUAL PERSPECTIVES ON THEIR BENEFITS AND LIMITATIONS 13
• 5. REFERENCES 15
• CHAPTER 2
• INTEGRATING BACTERIOPHAGE WITH HIGH-AND LOW-PROTEIN FEEDING STRATEGIES ENHANCES PERFORMANCE, GUT HEALTH, AND ENVIRONMENTAL SUSTAINABILITY IN ROSS 308 BROILERS 24
• 1. INTRODUCTION 25
• 2. MATERIALS AND METHODS 27
• 3. RESULTS 31
• 4. DISCUSSION 34
• 5. REFERENCES 38
• CHAPTER 3
• EFFECT OF DIFFERENT LEVELS OF PROTEIN WITH AND WITHOUT BACTERIOPHAGE SUPPLEMENTATION ON GROWTH PERFORMANCE, NUTRIENT DIGESTIBILITY, GAS EMISSION, AND GUT HEALTH IN WEANING PIGS 55
• 1. INTRODUCTION 56
• 2. MATERIALS AND METHODS 58
• 3. RESULTS AND DISCUSSION 62
• 4. REFERENCES 65
• CHAPTER 4
• OPTIMIZING ENERGY-PROTEIN EFFICIENCY AND GUT MICROBIAL STABILITY THROUGH Β-MANNANASE SUPPLEMENTATION IN FINISHING PIGS FED LOW-CARBON DIETS 79
• 1. INTRODUCTION 80
• 2. MATERIALS AND METHODS 82
• 3. RESULTS 87
• 4. DISCUSSION 89
• 5. REFERENCES 93