Effects of poultry litter biochar supplementation on growth performance and gut microbiota modulation in the Yoruba ecotype chickens

Introduction

Poultry production represents a significant component of the agricultural sector in Nigeria, with indigenous chickens playing a crucial role in rural livelihoods and food security (Tenza et al., 2024; Cheruiyot et al., 2025) [49,10] . The Yoruba ecotype chicken, adapted to local conditions of tropical Nigerian climate, has prospects but often shows limited growth performance compared to the exotic chickens (Popoola et al., 2020; Kpomasse et al., 2023; Adjei-Mensah et al., 2024) [44,24,1] . Enhancing the productivity of these indigenous dual-purpose chickens (bred for meat and egg production) through sustainable interventions not only supports local economies and income of smallholder poultry farmers, but also contributes to food security by providing a reliable source of animal protein. Despite their advantages, Yoruba ecotype chickens are prone to various health issues, including metabolic disorders and infections (Ogie et al., 2013; Fatai, 2020; Bolarinwa, 2021; Oyeniran et al., 2022; Ohagenyi et al., 2023) [36,15,5,42,37] . Traditional antibiotic treatments have been widely used to mitigate these problems; however, concerns regarding antibiotic resistance and food safety have prompted a search for alternative solutions. Biochar, a dark and carbon enriched by-product from pyrolysis of biomass, has gained attention as a potential feed additive in poultry production (Willson et al., 2019; Kalus et al., 2020; Goiri et al., 2021) [54,22,16] . Poultry Litter Biochar (PLB), derived from thermochemical conversion of poultry litter waste, specifically possesses unique characteristics that are different from other animal-or plant-based biochars, including higher nutrient content, greater cation-exchange capacity, and the significant ability to adsorb metal ions (Sobik-Szołtysek et al., 2021; Masocha and Dikinya, 2022; Ngo et al., 2023) [48,29,33] . Recent research suggests biochar supplementation may influence gut health by modifying the intestinal environment, potentially affecting nutrient absorption and growth performance (Ayeneshet and Temesgen, 2025; Ohanaka et al., 2025) [4,38] . The addition of biochar to poultry diets has shown variable effects on production parameters. Some studies report that dietary biochar can increase feed conversion ratio and decrease body weight gain in broilers (Ichekor et al., 2024; Onwuegbuta et al., 2024) [19,40] . However, other research has demonstrated improved production parameters in chickens, including higher body weight gains (Elnaggar et al., 2022; El-Kelawy et al., 2024) [14,13] . These contrasting findings warrant further investigation, particularly in the Nigerian indigenous chicken breeds that have not been extensively studied in this context, such as the Yoruba ecotype chickens. Additionally, PLB supplementation may influence gut microbiota composition by providing habitat for beneficial bacteria and potentially adsorbing harmful compounds (Man et al., 2021; Al-Khalaifah and Al-Nasser, 2023) [27,2] . The carbon structure of biochar may create a favourable environment for microbial colonization, which could positively impact digestive efficiency. Dim et al. (2018) [11] attributed improved growth performance of broiler chickens fed 6% biochar per kg feed to a balanced intestinal microbiota resulting from biochar feeding. The specific effect of PLB on gut microbiota modulation remain unexplored in the Yoruba ecotype chickens. With the view to bridge this knowledge gap, the present study evaluated the effects of feed supplementation with 6% PLB on growth performance and gut microbiota composition in the Yoruba ecotype chickens. Evaluating growth performance and intestinal microbiota in poultry is crucial to advancing sustainable food security, especially as global demand for animal protein rises and food systems face mounting challenges from climate change, resource constraints, and population growth. Growth performance assessment-including metrics like body weight gain, feed conversion ratio, and survival rates-directly reflects the efficiency and productivity of poultry production systems (Chen et al., 2020; Marmelstein et al., 2024) [9,28] . Improved growth performance means more meat and eggs can be produced using fewer resources, which is vital for increasing food availability and affordability, particularly in resource-poor settings. Smallholder poultry production, in particular, plays a significant role in poverty reduction and nutrition by providing accessible animal protein and income for vulnerable populations (Wong et al., 2017; Birhanu et al., 2023) [55,6] . Intestinal microbiota evaluation is equally important because gut health underpins nutrient absorption, disease resistance, and overall productivity in poultry. A healthy and balanced gut microbiome enhances feed efficiency, reduces the need for antibiotics, and lowers the risk of infectious diseases, thereby supporting safer and more sustainable poultry production (Kogut, 2019; Wickramasuriya et al., 2022; Salem et al., 2023) [23, 53, 46] . This is especially critical as antimicrobial resistance and emerging diseases threaten both animal and human health within the food system. Together, these evaluations guide the development of resilient poultry breeds, optimized feeding strategies, and effective health management practices. Such innovations are essential for boosting production, reducing losses, and ensuring a stable supply of affordable, high-quality animal protein-key pillars of sustainable food security in both rural and urban communities worldwide. Moreover, by converting poultry litter waste into a value-added feed additive (such as PLB), this research contributes to meaningful knowledge about sustainable feeding practices for indigenous chicken production in Nigeria, addressing both agricultural productivity and challenges of environmental management in the poultry sector.

Materials and Methods

Ethical Statement

This study adhered to ethical principles for use of animals in research. All procedures were carried out in accordance with extant provisions by the Institutional Animal Care and Use Committee at the University of Nigeria.

Location of the Study

The study was conducted at the Poultry Unit of the Department of Animal Science, University of Nigeria, Nsukka. Nestled in the rainforest belt of Nigeria, Nsukka experiences a classic tropical savannah climate, marked by clear wet and dry seasons (Njokuocha and Akaegbobi, 2014; Ejikeme and Okonkwo, 2023) [34,12] . The rains typically begin in April and last through October, with a brief lull in August. From November to March, the weather shifts into a dry period, often accompanied by the dusty harmattan winds. During the wet season, rainfall is abundant, especially between July and September, when monthly averages can reach around 200 mm (Okoro et al., 2021; Nnaemeka-Okeke and Okeke, 2024) [39,35] . Humidity is also high, often ranging between 80% and 90%. As the dry season sets in, temperatures climb, humidity drops to about 50-80%, and rainfall becomes scarce, with December and January seeing as little as 8-20 mm of rain. Daytime temperatures fluctuate throughout the year, generally ranging from 22 °C during the coolest months to peaks of 35 °C in the hottest months, especially from February to April (Ukwu et al., 2024; Ugwu et al., 2025) [51,50] .

Duration and Timeline of Activities

The experiment lasted for 10 weeks, including a 7-day acclimatization period followed by 56 days of experimental feeding. The timeline of activities for week-1 included pen preparation and cleaning, sourcing of experimental birds, feed formulation and biochar preparation, and bird acclimatization. From weeks 2-9, the activities involved experimental feeding, daily data collection (feed intake), weekly data collection (body weights), and routine management practices. Week-10 was for collection of cloacal swabs, microbiological analysis, data compilation and analysis, as well as final report preparation.

Experimental Biochar

Production of the experimental poultry litter biochar (PLB) was carried out on-farm following the guidelines and specifications of the European Biochar Foundation (2012). Poultry litter waste from the grow-out cycle of a deep litter system was used as substrate for the pyrolytic production of experimental poultry litter biochar. The poultry litter waste was sun-dried on concrete floors for 14 days; then, emptied into a locally-made kiln for slow pyrolysis (i.e., thermochemical heating). The kiln was designed to have a funnel chamber, for loading the substrate, with an air-tight cover to limit oxygen conditions in the kiln during pyrolysis. At the base of the kiln is a heating chamber, with a manual rotatory-handle that stretched from top of the kiln into to the heating chamber. This was used for intermittent stirring of the pyrolysing substrate to ensure uniform charring during the thermochemical heating. The dried poultry litter waste was loaded into the kiln through the funnel chamber and ignited to burn slowly and consistently for 4 hours at approximately 600 °C. At intervals of 20 minutes, the pyrolysing substrate was stirred with the rotatory-handle until uniform pyrolysis was achieved. Afterwards, the resultant char was discharged from the kiln and spread on clean and dry floor to cool under room conditions. Later on, the cooled char was ground with a manual blender (Crown Nigeria Limited), and sieved to fine powder using a 0.2 mm sieve to obtain the experimental PLB. Proximate analysis of the PLB was carried out according to official methods of the Association of Official Analytical Chemists (2005) [3] .

Diet Formulation

The experimental diet was prepared using locally available ingredients according to National Research Council (1994) [32] recommendation for grower chickens reared in the tropical environment, ensuring that both the control diet and the 6% PLB-supplemented diet had equivalent energy and protein contents. This approach eliminated differences in nutrient density as a confounding factor, isolating the effects of PLB supplementation. The test inclusion level of PLB in the diet was based on strong evidence that 6% biochar supplementation per kg feed is effective in improving growth performance and health status of chickens reared under tropical conditions (Dim et al., 2018) [11] . Percentage compositions of the experimental diet is shown in Table 2. The dry ingredients were thoroughly mixed in a drum mixer to ensure even distribution of microingredients. The experimental diet was kept for use in a cool, dry place to prevent incidence of mold and nutrient deterioration. [25,43] . Sterile swab sticks were used to collect cloacal swabs from each chicken aseptically. The swabs were immediately placed in sterile tubes and keep in an ice chest until processing. Then, each swab was vortexed in 1 ml of sterile saline to release microbes from the swab. Serial ten-fold dilutions in the sterile saline were prepared for accurate enumeration of test bacteria population. The selective media and incubation conditions for the different bacterial groups are shown in Table 3. After incubation, colonies that showed typical morphology for each bacteria were counted and expressed as colony-forming units per swab (CFU/swab). For quality control, negative controls (sterile swabs) and positive controls (known strains) were included in each batch. Aseptic techniques were maintained to avoid contamination. The bacterial counts were logtransformed (log [10] CFU) for statistical analysis, and compared across the treatment groups (PLB vs. control).

Results and Discussion

Growth Performance

Table 4 presents results on the effect of PLB biochar supplementation on growth performance metrics of Yoruba ecotype chickens. The result showed that PLB-treated group achieved significantly (p<0.05) higher final body weight (2.34±0.18kg) compared to the control (2.05±0.15kg). This aligns with findings in broilers where biochar-enhanced diets improved weight gain through enhanced nutrient absorption (Dim et [11,47,13] . Daily feed intake (DFI) remained comparable between groups (112.00±5.00 g/day vs. 109.00±4.00 g/day for PLB and control, respectively), but the PLB group achieved accelerated growth rate (i.e., mean weekly body weight gains; p:<: 0.05), and a 19% lower FCR (3.10 vs. 3.80 in controls; p<0.01). This improvement likely stemmed from the effect of test PLB on digestive efficiency. The porous structure of test PLB possibly increased the effective surface area for enzymatic action in the gastrointestinal tract, as hypothesized in previous studies (Jandosova et al., 2017; Schmidt et al., 2019; Miao et al., 2020) [47, 30] . In addition, the reduced FCR translated to a 22% decrease in feed cost per kilogram gain (₦412 vs. ₦528), making PLB supplementation economically viable for smallholder farmers. At current market prices (₦1,200/kg for PLB vs. ₦850/kg for standard feed), the 6% supplementation increased feed costs by ₦22/kg. However, the improved FCR reduced total feed required per kilogram gain by 18%, yielding a net cost saving of ₦116/kg. For a typical smallholder flock of 50 birds, this equates to ₦34,800 savings over an 8-week cycle a significant economic incentive. These findings align with commercial poultry operations reporting 30% cost reductions through biochar integration (Orge and Abon, 2014; Huang et al., 2014; Rathnayake et al., 2023) [41,17,45] .

Gut Microbiota Modulation

According to Table 5, PLB supplementation at 6% per kg feed created substantial shifts in microbial populations. The PLB group showed a significant reduction in Escherichia counts (3.10 ×10 ³ CFU/swab vs. 1.40×10 ⁵ CFU/swab in controls; p<0.001), corroborating the capacity of biochar to adsorb pathogenic bacteria. Studies in broilers and local chickens found significant reductions in faecal E. coli and total coliforms following biochar supplementation (Ismail et al., 2016; Hien et al., 2018; Mohammadi-Aragh et al., 2023) [20,18,31] Conversely, Lactobacillus populations significantly (p<0.05) increased by 1.8-fold (8.70 ×10 ⁶ CFU/swab vs. 4.80×10 ⁶CFU / swab), while Bifidobacterium counts rose 2.1-fold (6.20× 10 [5] CFU/swab vs. 2.90×10 ⁵ CFU/swab; p<0.05). These changes suggest that test PLB acted as a prebiotic, providing a favourable habitat for probiotic colonization by supporting proliferation of beneficial gut bacteria like Lactobacillus and Bifidobacterium while suppressing harmful bacteria such as Escherichia and Clostridium. Test PLB might have achieved this microbial balance through pH modulation by the alkaline nature, inhibiting acid-sensitive pathogens like Escherichia. The alkaline nature of biochar can modulate gut pH, creating an unfavourable environment for pathogenic bacteria while supporting beneficial flora. The high surface area of test PLB likely bound enterotoxins and reduced their bioavailability, preventing enterocyte damage and maintained intestinal integrity. The porous structure and large surface area of biochar enable the capacity to adsorb toxins, pathogens, and excess ammonia in the gut, thereby reducing their negative impacts (Cai et al., 2022; Li et al., 2023) [7,26] . The carbon matrix of test PLB might have served as a scaffold for beneficial bacterial biofilm formation. Notably, Clostridium populations remained unchanged (p>0.05), indicating selective antimicrobial effects of test PLB. This lack of PLB effect on Clostridium populations warrants investigation as potential biofilm formation or spore resistance may necessitate higher PLB concentrations for full pathogen control. However, the microbiota shifts observed in the experimental chickens correlated with improved growth performance, as the improved gut health and microbial balance enhanced nutrient digestibility and retention, contributing to better growth performance and feed efficiency. Dim et al. (2018) [11] had reported that birds fed 6% biochar showed improved growth performance, including better feed conversion ratios (FCR), indicating enhanced feed efficiency. The authors attributed these benefits to ability of dietary biochar to modulate gut microbiota by reducing pathogenic bacteria and promoting beneficial microbes, as well as its adsorptive properties that detoxified harmful substances in the gut. Moreover, the genetic background of experimental chickens might have influenced how they responded to PLB supplementation. Local and indigenous breeds adapted to tropical environments, like the Yoruba ecotype chickens, often have robust gut microbiota and immune responses, which may have synergized with the prebiotic and adsorptive effects of test PLB. Genetic variations in gut morphology, immune function, and microbial colonization patterns can affect the impact of test feeding. For instance, indigenous breeds with naturally higher resilience to gut pathogens may experience greater improvements in growth performance when their diets are supplemented with biochar (Authors et al 2024; Authors et al 2025). There is emerging evidence that biochar supplementation may interact with host genes involved in gut barrier function and immune modulation, but more research is needed to clarify these genetic mechanisms. All the same, findings from this study suggest that PLB supplementation at 6% per kg feed can enhance growth performance and gut microbiota in Yoruba ecotype chickens reared in tropical environment. These benefits were mediated through toxin adsorption, prebiotic effects, and improved nutrient utilization, with the response influenced by the genetic adaptation of the experimental birds to local conditions. Nonetheless, careful attention to the inclusion levels in the feed, as well as the birds' genetics are very essential for optimizing outcomes.

Conclusion and Recommendation

This study demonstrated that supplementing Yoruba ecotype chicken diets with 6% poultry litter biochar significantly improved growth performance and beneficially modulated gut microbiota. The PLB-supplemented group showed higher final body weights and improved feed conversion ratios, indicating enhanced nutrient utilization and feed efficiency. The economic analysis further emphasized the practical value of PLB supplementation by showing reduced feed costs per kilogram of weight gain, making it a cost-effective strategy for smallholder poultry farmers. Microbiological analysis revealed a marked reduction in pathogenic Escherichia populations alongside increased counts of beneficial Lactobacillus and Bifidobacterium, highlighting the role of biochar in promoting a healthy intestinal environment in tropical chickens. These findings align with existing literature that attributes positive effects of dietary biochar to the adsorptive capacity, prebiotic properties, and ability to detoxify harmful substances in the gut.

References

1. Al-Khalaifah H, Al-Nasser A. Critical review on the use of biochar in poultry industry: Benefits, characteristics and applications. World's Poultry Science Journal. 2023;79(4):807833.https://DOI.org/10.1080/00439339.2023.2239776
2. Association of Official Analytical Chemists. Official Methods of Analysis of the Association of Official Analytical Chemists, 18th edition. Gaithersburg, MD: Association of Official Analysis Chemists Inc.; 2005.
3. Ayeneshet B, Temesgen T. Role of biochar as a feed additive on animal performance, digestibility, micro-biota dynamics, and reduction of enteric methane production. Advances in Agriculture. 2025;2025(1):9911760.https://DOI.org/10.1155/aia/99117 60
4. Bolarinwa MO. Performance of Three Nigerian Indigenous Chicken Strains Vaccinated against Newcastle Disease in Ovo and Post-Hatch. Ibadan, Nigeria: University of Ibadan; 2021.http://hdl.handle.net/123456789/1122
5. Birhanu MY, Osei-Amponsah R, Obese FY, Dessie T. Smallholder poultry production in the context of increasing global food prices: Roles in poverty reduction and food security. Animal Frontiers. 2023;13(1):1725.https://DOI.org/10.1093/af/vfac069
6. Cai Y, Zhu M, Meng X, Zhou JL, Zhang H, Shen X. The role of biochar on alleviating ammonia toxicity in anaerobic digestion of nitrogen-rich wastes: A review. Bioresource Technology. 2022;351:126924.
7. Chen Q, Saatkamp HW, Cortenbach J, Jin W. Comparison of Chinese broiler production systems in economic performance and animal welfare. Animals. 2020;10(3):491.https://DOI.org/10.3390/ani10030491
8. Cheruiyot JK, Kibett JK, Omunyin ME, Kere GM. Exploring the latent roles of indigenous chickens among rural households in Kericho County, Kenya: A grounded theory approach. East African Journal of Interdisciplinary Studies. 2025;8(1).https://DOI.org/10.37284/eajis.8.1.2653
9. Dim CE, Akuru EA, Egom MA, Nnajiofor NW, Ossai OK, Ukaigwe CG, Onyimonyi AE. Effect of dietary inclusion of biochar on growth performance, haematology and serum lipid profile of broiler birds. Agro-Science. 2018;17(2).https://DOI.org/10.4314/as.v17i2.2
10. Ejikeme JNU, Okonkwo UU. The effect of COVID-19 on tourism and the hospitality industry in Nsukka, Nigeria. American Research Journal of Humanities and Social Science. 2023;6(5):1-11.
11. El-Kelawy M, Elnaggar AS, Enass A. The influence of supplementing broiler chickens with humic acid or biochar as growth promoters on their productive performance, nutrient digestibility, and physiological performance. Egyptian Poultry Science Journal. 2024;44(1):123142.https://DOI.org/10.21608/epsj.2024.354787
12. Elnaggar AS, Elghalid O, Elsayed A, Harfoush A. Effect of graded levels of biochar supplementation as a growth promoter on productive and physiological performance of broiler chicks. Egyptian Poultry Science Journal. 2022;42(3):243263.https://DOI.org/10.21608/epsj.2022.263846
13. Goiri I, Ruiz R, Atxaerandio R, Lavin JL, de Otálora XD, García-Rodríguez A. Assessing the potential use of a feed additive based on biochar on broilers feeding upon productive performance, pH of digestive organs, cecum fermentation and bacterial community. Animal Feed Science and Technology. 2021;279:115039.https://DOI.org/10.1016/j.anifeedsci.20 21.115039
14. Huang Y, Anderson M, Lyons GA, McRoberts WC, Wang Y, McIlveen-Wright MR, Roskilly AP, Hewitt NJ. Technoeconomic analysis of biochar production and energy generation from poultry litter waste. Energy Procedia. 2014;61:714-717.
15. Hien NN, Dung NNX, Manh LH, Le-Minh BT. Effects of biochar inclusion in feed and chicken litter on growth performance, plasma lipids and fecal bacteria count of Noi lai chicken. Livestock Research for Rural Development. 2018;30(7):131.
16. Ichekor C, Okhale OE, Usieta OH, Obinyan F. Effect of infeed inclusion of biochar from mixture of wood and empty palm fruit bunch on growth performance of broiler chickens. Nigerian Journal of Animal Science and Technology. 2024;7(4):1-
17. Ismail A, Shoieb A, Prasher SO, Chénier MR, Patel PM. Evaluation of biochar soil amendments in reducing soil and water pollution from total and fecal Coliforms in poultry manure. Canadian Biosystems Engineering. 2016;58(1):1021-1031.
18. Jandosov JM, Mikhalovska LI, Howell CA, Chenchik DI, Kosher NK, Lyubchik SKB, Silvestre-Albero J, Ablaikhanova NT, Srailova GT, Tuleukhanov ST, Mikhalovsky SV. Synthesis, morphostructure, surface chemistry and preclinical studies of nanoporous rice huskderived biochars for gastrointestinal detoxification. Eurasian Chemico-Technological Journal. 2017;19(4).https://DOI.org/10.18321/ectj678
19. Kalus K, Konkol D, Korczyński M, Koziel JA, Opaliński S. Effect of biochar diet supplementation on chicken broilers performance, NH3 and odor emissions and meat consumer acceptance. Animals. 2020;10(9):1539.https://DOI.org/10.3390/ani10091539
20. Kogut MH. The effect of microbiome modulation on the intestinal health of poultry. Animal Feed Science and Technology. 2019;250:32-
21. https://DOI.org/10.1016/j.anifeedsci.2018.10.008 24. Kpomasse CC, Kouame YAE, N'nanle O, Houndonougbo FM, Tona K, Oke OE. The productivity and resilience of the indigenous chickens in the tropical environments: Improvement and future perspectives. Journal of Applied Animal Research. 2023;51(1):456-
22. Lee YJ, Yu WK, Heo TR. Identification and screening for antimicrobial activity against Clostridium difficile of Bifidobacterium and Lactobacillus species isolated from healthy infant faeces. International Journal of Antimicrobial Agents. 2003;21(4):340346.https://DOI.org/10.1016/S0924-8579(02)00389-8
23. Li Y, Cheng C, Wang H, Zhou L, Yang J, Zhang Y, Li H, Zhou D. Distribution, toxicity, and impacts of nano-biochar in mice following dietary exposure: Insights into environmental risks and mammalian effects. Environmental Pollution. 2023;338:122652.
24. Man KY, Chow KL, Man YB, Mo WY, Wong MH. Use of biochar as feed supplements for animal farming. Critical Reviews in Environmental Science and Technology. 2021;51(2):187217.https://DOI.org/10.1080/10643389.2020.1721980
25. Marmelstein S, Costa IPD, Terra AV, da Silva RF, Capela GPD, Moreira MAL, Junior CDR, Gomes CFS, dos Santos M. Advancing efficiency sustainability in poultry farms through data envelopment analysis in a Brazilian production system. Animals. 2024;14(5):726.https://DOI.org/10.3390/ani14050726
26. Masocha BL, Dikinya O. The role of poultry litter and its biochar on soil fertility and Jatropha curcas L. growth on sandy-loam soil. Applied Sciences. 2022;12(23):12294.https://DOI.org/10.3390/app12231229
27. Miao Z, Liu Y, Guo L, Zhao W, Zhang J. Effects of dietary chitosan on growth rate, small intestinal morphology, nutrients apparent utilization and digestive enzyme activities of growing Huoyan geese. Animal. 2020;14(12):26352641.https://DOI.org/10.1017/S1751731120001408
28. Mohammadi-Aragh MK, Linhoss JE, Marty CA, Evans JD, Purswell JL, Chaves-Cordoba B, Chesser Jr GD, Lowe JW. Evaluating the Effects of pine and miscanthus biochar on Escherichia coli, total aerobic bacteria, and bacterial communities in commercial broiler litter. Avian Diseases. 2023;67(1):10-19.
29. National Research Council. Nutrient Requirement of Poultry, 9th Revised Edition. Washington DC: National Academy Press; 1994.
30. Ngo DNG, Chuang XY, Huang CP, Hua LC, Huang C. Compositional characterization of nine agricultural waste biochars: The relations between alkaline metals and cation exchange capacity with ammonium adsorption capability. Journal of Environmental Chemical Engineering. 2023;11(3):110003.https://DOI.org/10.1016/j.jece.2023.11 0003
31. Njokuocha RC, Akaegbobi IM. A contribution to the Holocene vegetation history of Nigeria: Pollen from Ohe pond Nsukka, southeastern Nigeria. Quaternary International. 2014;338:2834.https://DOI.org/10.1016/j.quaint.2013.07.036
32. Nnaemeka-Okeke RC, Okeke FO. Assessing the influence of seasonal precipitation patterns on groundwater quality in the coal rich environment of Enugu, Nigeria. Discover Applied Sciences. 2024;6:208.https://DOI.org/10.1007/s42452-024-05837-x
33. Ogie AJ, Salako AE, Emikpe BO, Amosun EA, Adeyemo SA, Akinoluwa PO. The comparative susceptibility of commercial and Nigerian indigenous chicken ecotypes to Salmonella gallinarum infection. Sokoto Journal of Veterinary Sciences. 2013;11(2).https://DOI.org/10.4314/sokjvs.v11i2.8
34. Ohagenyi JI, Iregbu FC, Udeh VC. Estimation of genetic parameters for body weight and some colour traits in the seventh-generation index selected Nigeria heavy local chicken ecotype. Agricultural Science Digest. 2023;43(4):551-555.
35. Ohanaka AUC, Ohanaka JN, Nwogu CM, Ogbuewu IP, Etuk IF, Okoli IC. Growth promoting and health enhancing effects of aged palm sap-enriched activated biochar in broiler nutrition. Journal of Agriculture and Rural Development in the Tropics and Subtropics. 2025;126(1):13-24.https://DOI.org/10.17170/kobra2025011410815
36. Okoro EC, Ugwu EBI, Onah IG, Omeje LC. Rainfall and solar irradiance monitoring in Nsukka zone, Nigeria. European Journal of Statistics and Probability. 2021;9(2):110.https://ssrn.com/abstract=3909374
37. Onwuegbuta J, Ugwuowo L, Umeakuana D. Effect of biochar and clay on growth performance and organ weight of broiler. Egyptian Journal of Nutrition. 2024;39(2):133141.https://DOI.org/10.21608/enj.2024.356650
38. Orge RF, Abon JEO. Cogeneration of biochar and heat from rice hull: Its application in the poultry industry. OIDA International Journal of Sustainable Development. 2014;7(08):105-114.
39. Oyeniran VJ, Iyasere OS, Durosaro SO, Fasasi FB, Odetayo PO, Ogunfuyi SA, Odetunde PO, Akintayo TC, Daramola JO. An exploratory study on differences in maternal care between two ecotypes of Nigerian indigenous chicken hens. Frontiers in Veterinary Science. 2022;9.https://DOI.org/10.3389/fvets.2022.980609
40. Penders J, Vink C, Driessen C, London N, Thijs C, Stobberingh EE. Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR. FEMS Microbiology Letters. 2005;243(1):141147.https://DOI.org/10.1016/j.femsle.2004.11.052
41. Popoola MA, Lamidi LO, Raji AM, Adebisi GL, Onuoha CJ, Olaoye RO. Households' choice of genotypes and traits of preference in Yoruba ecotype chicken in Ibadan, Nigeria. Agricultural Science. 2020;2(1).https://DOI.org/10.30560/as.v2n1p23
42. Rathnayake D, Schmidt HP, Leifeld J, Mayer J, Epper CA, Bucheli TD, Hagemann N. Biochar from animal manure: A critical assessment on technical feasibility, economic viability, and ecological impact. GCB Bioenergy. 2023;15(9):1078-1104.
43. Salem HM, Saad AM, Soliman SM, Selim S, Mosa WFA, Ahmed EA, Al-Jaouni SK, Almuhayawi MS, Abd El-Hack ME, El-Tarabily KA, El-Saadony MT. Ameliorative avian gut environment and bird productivity through the application of safe antibiotics alternatives: A comprehensive review. Poultry Science. 2023;102(9):102840.https://DOI.org/10.1016/j.psj.2023.1 02840
44. Schmidt H, Hagemann N, Draper K, Kammann C. The use of biochar in animal feeding. PeerJ. 2019;7:e7373.https://DOI.org/10.7717/peerj.7373
45. Sobik-Szołtysek J, Wystalska K, Malińska K, Meers E. Influence of pyrolysis temperature on the heavy metal sorption capacity of biochar from poultry manure. Materials. 2021;14(21):6566.https://DOI.org/10.3390/ma14216566
46. Tenza T, Mhlongo LC, Ncobela CN, Rani Z. Village chickens for achieving sustainable development goals 1 and 2 in resource-poor communities: A literature review. Agriculture. 2024;14(8):1264.https://DOI.org/10.3390/agriculture1408 1264
47. Ugwu EC, Nweze NO, Nwadinigwe AO. Phenological responses of forest flora to the changing climate in derived savannas. Trends in Environmental Sciences. 2025;1(1):70-85.http://DOI.org/10.17311/tes.2025.70.85
48. Ukwu, U. N., Oburu, B. N., Udochukwu, D. P., Adewuyi, S. O., Muojiama, S. O., Osadebe, V. O., Ezeh, I. N., Ishieze, P. U., & Dauda, N. (2024). Performance of mungbean (Vigna radiata L.
49. Wilczek) accessions under intermittent water deficit stress in a tropical environment. Journal of Applied Biology & Biotechnology. 2024;X(XX):1-
50. Wickramasuriya SS, Park I, Lee K, Lee Y, Kim WH, Nam H, Lillehoj HS. Role of physiology, immunity, microbiota, and infectious diseases in the gut health of poultry. Vaccines. 2022;10(2):172.https://DOI.org/10.3390/vaccines1002017 2
51. Willson NL, Van TH, Bhattarai SP, Courtice JM, McIntyre JR, Prasai TP, Moore RJ, Walsh K, Stanley D. Feed supplementation with biochar may reduce poultry pathogens, including Campylobacter hepaticus, the causative agent of spotty liver disease. PLOS ONE. 2019;14(4):e0214471.https://DOI.org/10.1371/journal.pon e.0214471
52. Wong JT, de Bruyn J, Bagnol B, Grieve H, Li M, Pym R, Alders RG. Smallscale poultry and food security in resource-poor settings: A review. Global Food Security. 2017;15:43-52.https://DOI.org/10.1016/j.gfs.2017.04.003