Research Article

Fecal Microbiota Transplantation Improves Milk Yield and Feed Efficiency in Late-Lactation Cows

Nathiara Aidini Milati Fadillah, Bambang Waluyo Hadi Eko Prasetiyono*, Nuruliarizki Shinta Pandupuspitasari, Annisa Dhea Lathifa, Ali Azhar, Bilal Ahmed, Adi Rahman Satrio

Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Central Java, Indonesia (50275); 2Department of Transfusion Medicine and Clinical Microbiology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, 10330, Thailand; 3Food Research for Safety, Security, and Sustainability, Semarang, Indonesia; 4Tropic Research on Productivity, Genetic Enhancement, and Conservation of Local Livestock (TROPICAL), Diponegoro University, Semarang, Central Java, Indonesia.

Received | April 21, 2026; Accepted | May 11, 2026; Published | June 25, 2026

*Correspondence | Bambang Waluyo Hadi Eko Prasetiyono, Department of Animal Science, Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Central Java, Indonesia (50275); Email: [email protected]

Citation | Fadillah NAM, Prasetiyono BWHE, Pandupuspitasari NS, Lathifa AD, Azhar A, Ahmed B, Satrio AR (2026). Fecal microbiota transplantation improves milk yield and feed efficiency in late-lactation cows. Adv. Anim. Vet. Sci., 14(6):1372-1381.

DOI | https://dx.doi.org/10.17582/journal.aavs/2026/14.6.1372.1381

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Demand for milk as a highly nutritious food source continues to increase globally in line with population growth and changes in consumption patterns (FAO, 2023; OECD/FAO, 2022). Dairy cattle play a strategic role in supplying animal protein and serving as a source of income for farmers, particularly in mixed farming systems in tropical and subtropical regions (Thornton, 2010). Sustainable production systems have therefore become a major focus to improve the efficiency and stability of milk production (FAO, 2023).

Productivity of dairy cattle in tropical lowland areas remains relatively low and is often accompanied by reduced milk quality, including fat, protein, and lactose contents (Habimana et al., 2023). Howeve, heat stress is a major limiting factor that reduces feed intake, disrupts energy balance, and alters host metabolism (Bernabucci et al., 2010). The reduction in dry matter and organic matter intake under heat stress directly decreases the availability of fermentable substrates in the digestive tract, thereby reducing the efficiency of feed conversion into milk components (Sanz-Fernandez et al., 2024). These physiological changes also affect the composition and activity of the gut microbiota, which in turn lowers post-ruminal fermentation capacity and overall nutrient utilization efficiency (Holman and Gzyl, 2019).

The rumen is the primary site of fermentation in dairy cows, contributing approximately 70–80% of total SCFA production. However, the hindgut represents an important secondary fermentation site, processing rumen-escape substrates, including an estimated 20–30% of dietary NDF and 15–25% of starch (NRC, 2001). The hindgut microbiota play an important role in the further fermentation of feed substrates that escape ruminal fermentation, producing short-chain fatty acids (SCFA) that support energy metabolism, milk component synthesis, and intestinal mucosal integrity (Liu et al., 2023). Feed utilization efficiency in ruminants is determined not only by the amount of feed consumed, but also by the fermentative capacity of the hindgut microbial community, which independently contributes to the conversion of substrates into functional metabolites (Monteiro et al., 2022). Microbial dysbiosis, which commonly occurs under tropical stress conditions, may disrupt fermentation profiles and reduce metabolic efficiency (Zhao et al., 2019). While probiotics and prebiotics strategies have administered utility and restore health gut microbiome. FMT introduces intact polymicrobial communities and may offer complementary mechanisms for microbiota modulation (Weingarden and Vaughn, 2017). The relationship among microbiota, SCFA production, and milk synthesis is conceptually presented in Figure 1.

Fecal microbiota transplantation (FMT) offers a more comprehensive approach through the transfer of an intact microbial community from a high-performing donor to a recipient, thereby potentially improving microbiota stability and fermentative capacity (Weingarden and Vaughn, 2017). FMT has been associated with improvements in hindgut fermentation and may contribute to improved milk production through gluconeogenesis and lipogenesis pathways (Wang et al., 2026). Modulation of the hindgut microbiota through FMT may also improve feed conversion efficiency independently of feed intake level, since the composition of the hindgut microbial community has been shown to be more closely associated with nutrient utilization efficiency than with the quantity of feed consumed (Monteiro et al., 2022). The success of this intervention is strongly influenced by environmental factors and by compatibility between donor and recipient (Xu et al., 2021).

Studies on the role of FMT in improving feed intake, feed efficiency, milk production, and milk quality through modulation of the hindgut microbiota in dairy cows raised in tropical lowlands are still limited (Tapio et al., 2016). Therefore, this study aimed to evaluate the effects of FMT derived from high-performing Friesian Holstein cows on dry matter intake, organic matter intake, feed conversion, feed efficiency, and milk production and quality, as a basis for the development of more effective and sustainable microbiota-based strategies.

MATERIALS AND METHODS

Study site and ethical compliance

Experiments were conducted at the Research Farm of the Faculty of Animal and Agricultural Sciences, Universitas Diponegoro, Semarang, Indonesia. All animal handling and experimental procedures were approved by the Animal Ethics Committee of the Faculty of Animal and Agricultural Sciences, Universitas Diponegoro. Approval Number: 071/UPH-FPIK/EC/X/2023, dated October 12, 2023. Based on meteorological data from the Badan Meteorologi Klimatologi dan Geofisika (BMKG) Semarang station during the study period, mean daily temperature was approximately 28–32°C and mean relative humidity was 75–85%, yielding estimated Temperature-Humidity Index (THI) values of 78–84. A THI above 72 is generally considered to indicate heat stress in dairy cattle (NRC, 2001), confirming that the cows were exposed to mild to moderate heat stress conditions during the study.

Animal selection

The animals used in this study were Friesian Holstein (FH) cows in the late lactation phase as recipients, approximately 4 years of age, with body weights ranging from 370 to 430

 

kg and at the eighth month of lactation. In addition, one Friesian Holstein cow from a highland area was used as the donor. The donor cow was selected based on good production performance, good health status, and the absence of any history of gastrointestinal disorders. Donor suitability was confirmed through a comprehensive fecal health examination to minimize the risk of transferring undesirable microorganisms. The donor cow was an FH cow from PT Sumber Citarasa Alam (SCA), Bogor, West Java, with ear tag code F.920. The donor was in the third lactation phase and had a daily milk yield of 28 liters.

Experimental design

The study used a repeated-treatment Latin square design across experimental periods. Each animal received all treatments in rotation during different periods, allowing better control of individual animal variation and period effects. This approach was considered appropriate for exploratory studies in large animals with limited animal availability. The treatments consisted of T0 = basal ration + 0 g FMT, T1 = basal ration + 5 g FMT, and T2 = basal ration + 10 g FMT. A 14-day washout period was included between each treatment period. For a 3×3 Latin square, the error degrees of freedom = (n−1) (n−2) = 2; all results should therefore be interpreted as exploratory and preliminary. Cows were offered feed at 3% of body weight (as-fed basis) as a daily maximum allowance; actual dry matter intake (DMI) was calculated as feed offered minus orts, and was recorded daily for each animal (Table 1).

FMT administration

The FMT material was prepared from feces of healthy donor cows and mixed with glycerol (1:1, v/v) and physiological NaCl solution as chemical protectants, followed by thorough homogenization. The detailed preparation procedure is illustrated in Figure 2. The suspension was subsequently encapsulated in double-layered gelatin capsules to protect the microorganisms from gastrointestinal degradation during transit. Each capsule contained approximately 0.9 mL of suspension, equivalent to 1 g of FMT material. Oral administration was performed during the first 7 days of each 28-day experimental period at daily doses of 5 g (T1; 5 capsules) and 10 g (T2; 10 capsules) to ensure effective microbial delivery and colonization in the lower gastrointestinal tract. Based on published benchmarks for bovine fecal suspensions, estimated bacterial loads were in the range of 10⁸–10⁹ CFU per capsule (Tian et al., 2024). Direct microbial viability testing of the encapsulated FMT was not performed, which is acknowledged as a limitation.

 

Table 1: Composition and nutrient content of the concentrate and elephant grass.

Feed Ingredient	Proportion (% DM basis)
A: Concentrate composition and nutrient content
Cassava Pulp	30
Wheat Pollard	21
Coconut Meal	38
Extruded Soybean (Soyxyl)	7
Mineral Mix (St. Vit)	1
Calcium Carbonate	1
Salt	1
GoPro (RUP supplement)	1
B: Concentrate nutrient content (%)
Dry Matter	85.56
Ash	5.28
Crude Protein	17.08
Total Digestible Nutrient (TDN)	76.83
Crude Fat	8.38
Crude Fiber	15.04
Nitrogen-Free Extract	54.21
Basis	Based on analysis
C: Pennisetum purpureum nutrient content (%)
Dry Matter	19.900
Ash	12.76
Crude Protein	10.2
Total Digestible Nutrient (TDN)	45.99
Crude Fat	1.6
Crude Fiber	42.3
Nitrogen-Free Extract	33.14
Basis	Based on analysis

 

 

Milk sampling

Milk production data were recorded daily during each experimental cycle. Daily milk yield was calculated as the total volume obtained from the morning and afternoon milkings (daily milk yield = morning milk yield + afternoon milk yield). Milk quality analysis was conducted at the end of each cycle (day 28). A subsample equivalent to 10% of each individual milking session’s yield was collected from morning and afternoon milkings, pooled, homogenized, and a 50 mL aliquot was analyzed using a Lactoscan MCC (Milkotester, Bulgaria) calibrated for bovine milk (ISO/IEC 17025-accredited; reported accuracy: ±0.05% fat, ±0.04% protein, ±0.03% lactose) to determine lactose, fat, and protein contents.

Fecal sample collection

Fecal samples were collected from donor cows (Fe SCA cow), control cows (Fe cowT0), and treatment cows (Fe cowT1 and Fe cowT2). Samples were stored in RNA/DNA Shield tubes and sent to the laboratory. Genomic DNA was extracted from 1 g of feces using the ZymoBIOMICS Stool DNA Kit (catalog no. D4304) for metagenomic analysis, and DNA quality was evaluated using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Rumen sample collection

Rumen samples were collected from the control and FMT-treated cows during the experimental period for microbiota evaluation. Sampling was performed using an ororuminal stomach tube inserted into the rumen through the oral cavity, and the rumen fluid was aspirated into a sterile collection tube. The samples were subsequently transferred into tubes containing RNA/DNA Shield and transported to the laboratory for further analysis. Genomic DNA extraction and quality assessment were performed using the same general workflow as that used for fecal samples prior to shotgun metagenomic sequencing.

Shotgun metagenomic sequencing

Genomic deoxyribonucleic acid (DNA) was extracted and its integrity assessed using 1% agarose gel electrophoresis to ensure DNA quality for subsequent analysis. DNA fragmentation up to 350 bp was achieved using the Covaris M200 instrument; subsequently, paired-end libraries were constructed using Polymerase Chain Reaction (PCR) amplification to enrich the library template. Library quantity was determined by quantitative real-time PCR. High-quality libraries with a minimum sequencing depth of 10 Gb per sample were sequenced using the Nova HiSeq X-ten platform. Quality filtering was performed using fastp v0.23.2 (parameters: cut_tail, cut_window_size 4, cut_mean_quality 20, length_required 50). Host DNA decontamination was performed using Bowtie2 v2.4.5 against the Bos taurus genome (ARS-UCD1.2). Taxonomic classification was performed using Kraken2 v2.1.2 with the NCBI RefSeq database (release 2024), and abundance estimation was performed using Bracken v2.7 at the genus level. Microbiota similarity was assessed using Bray-Curtis dissimilarity at the genus level, with Principal Coordinates Analysis (PCoA) used for visualization.

Statistical analysis

Production data were analyzed by analysis of variance (ANOVA) using the F-test at the 5% significance level (α = 0.05). When a significant treatment effect was detected, Tukey’s Honest Significant Difference (HSD) test was applied at the 5% significance level to compare mean values among treatments, as it controls the family-wise error rate more conservatively than DMRT, which is appropriate given the small sample size. Prior to ANOVA, data were assessed for normality using the Shapiro-Wilk test and for homogeneity of variance using Levene’s test. Results confirmed that all main production variables did not significantly deviate from normality (W > 0.85, p > 0.05) and variance homogeneity was maintained (p > 0.05). It is noted that for a 3×3 Latin square, the error degrees of freedom = (n−1)(n−2) = 2, which limits statistical power; therefore, all results should be interpreted as exploratory and preliminary.

RESULTS AND DISCUSSION

Effect of FMT on hindgut microbiota, fermentation, and SCFA production

The results showed that fecal microbiota transplantation (FMT) significantly modulated the composition of the hindgut microbiota in dairy cows during the late lactation phase, particularly in the T1 treatment (5 g dose), which showed increased abundance of major fermentative genera such as Bacteroides, Prevotella, and Oscillospiraceae. Microbiota similarity was assessed using Bray-Curtis dissimilarity at the genus level (PCoA visualization). T1 showed the lowest mean Bray-Curtis dissimilarity to the donor profile (mean BC = 0.31), compared to T2 (mean BC = 0.47) and T0 (mean BC = 0.61), indicating the highest structural similarity to the donor in T1. In contrast, the control group (T0) showed a distinct profile, reflecting the dysbiosis condition commonly found in dairy cows under tropical environments (Lin et al., 2023). These compositional changes are illustrated in Figure 3.

These changes in microbiota composition were associated with increased fermentative activity. Direct SCFA measurements were not conducted in this study; the following discussion of SCFA roles is therefore based on the known metabolic functions of the identified bacterial taxa as reported in the literature, and represents an inferred mechanistic explanation rather than an observed finding. Short-chain fatty acids (SCFAs) serve as the primary link between microbiota activity and host energy metabolism (Liu et al., 2023). The genera Bacteroides and Prevotella are known to contribute to the degradation of complex polysaccharides and the production of SCFAs, particularly acetate and propionate (Zhao et al., 2022; Kou et al., 2024). Propionate serves as the primary precursor for gluconeogenesis in lactose synthesis (Wang et al., 2023), acetate plays a role in de novo lipogenesis for milk fat formation (Qi et al., 2023), while butyrate supports intestinal mucosal integrity and nutrient absorption efficiency (He and Dong, 2023).

Treatment T2 (10-gram dose) showed lower microbiota similarity compared to T1 (5-gram dose), indicating colonization limitations at higher doses. This reduced engraftment efficiency at higher doses may reflect multiple mechanisms including: (1) intermicrobial competition and niche saturation; (2) physical bulk effects of a higher inoculum volume potentially altering gastrointestinal transit time; and (3) possible immune tolerance limits at higher microbial loads. This aligns with the concept that microbial colonization success is influenced by intermicrobial competition and ecological niche capacity (Xu et al., 2021).

 

Effect of fecal microbiota transplantation on milk production, composition, and ecological dynamics

FMT administration had a significant effect (P < 0.05) on increasing milk production and quality in Friesian Holstein dairy cows during the late lactation phase (Table 2). The highest milk production was observed in T1 (6.96 L/cow/day), followed by T2 (6.55 L) and T0 (5.98 L), indicating that a moderate dose produced the optimal response.

The increased relative abundance of Prevotella and Bacteroides, which are known propionate-producing genera (Zhao et al., 2022; Kou et al., 2024), is consistent with the hypothesis that propionate-mediated gluconeogenesis may have contributed to the observed increase in lactose and milk volume in T1. However, direct propionate measurements were not conducted, and this mechanistic link remains inferential. Propionate is known to enhances the supply of glucose for lactose synthesis, which plays a role in regulating milk volume through osmotic pressure (Wang et al., 2023). This is reflected in the simultaneous increase in lactose concentration and milk volume at T1.

Table 2: Milk production and quality of Friesian Holstein cows in the late-lactation phase.

Parameters	T0	T1	T2	P value
Milk production (liters/head/day)	5.98b ± 1.82	6.96a ± 2.15	6.55ab ± 2.06	0.040
Milk fat (%)	4.74b ± 0.06	5.55a ± 0.29	4.98b ± 0.03	0.045
Milk protein (%)	2.99b ± 0,19	3.33a ± 0.08	3.01b ± 0.02	0.048
Lactose (%)	4.43b ± 0.03	5.08a ± 0.07	4.88a ± 0.17	0.019

The role of propionate as the primary precursor of hepatic gluconeogenesis is a key factor in increased milk production. Increased propionate production in the T1 group enhances the systemic glucose supply used in lactose synthesis in the mammary glands (Wang et al., 2023). Lactose functions as the primary regulator of milk volume through osmotic pressure mechanisms; thus, an increase in its concentration directly contributes to increased milk production. This finding suggests that the increase in production reflects metabolic efficiency rather than merely increased nutrient intake. The simultaneous increase in lactose (T1: 5.08% vs. T0: 4.43%) and milk volume (T1: 6.96 L vs. T0: 5.98 L) is consistent with this hypothesis.

Milk quality also showed a significant improvement, particularly in fat, protein, and lactose content. The highest milk fat content at T1 (5.55%) indicates increased de novo lipogenesis activity in mammary tissue triggered by the increased availability of acetate resulting from microbial fermentation. Acetate serves as the primary precursor in fatty acid synthesis and plays a role in activating the lipogenic signaling pathway via the GPR41 and GPR43 receptors, which increase the expression of genes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) (Qi et al., 2023).

The increase in milk protein content in T1 (3.33%) indicates improved nitrogen utilization efficiency and increased availability of amino acids for milk protein synthesis. Butyrate, produced by hindgut fermentation, is well documented to enhance intestinal mucosal integrity and nutrient absorption efficiency (He and Dong, 2023). While butyrate concentrations were not directly measured in this study, the observed increase in fermentative taxa associated with butyrate production suggests this mechanism may have been active in T1, warranting investigation in future studies with direct metabolite quantification.

The lower response in the T2 group compared to T1 suggests that increasing the FMT dose does not always improve production performance. This inefficiency is related to the ecological dynamics of the microbiota, influenced by intermicrobial competition and ecological niche limitations within the hindgut ecosystem. An increase in inoculum volume without accompanying community stability leads to reduced colonization efficiency and metabolic function (Xu et al., 2021).

These ecological mechanisms indicate that the success of FMT is determined by the balance between microbial colonization capacity, substrate availability, and community stability. Donor microbes with high metabolic capacity are able to occupy suboptimal niches under conditions of dysbiosis, thereby enhancing fermentation efficiency and the production of energy metabolites. Limited niche capacity acts as a limiting factor at high doses, so the effectiveness of FMT exhibits a non-linear response pattern with optimal results at moderate doses.

Overall, the increase in milk production and quality reflects the integration of microbiota modulation, enhanced fermentation activity, and the optimization of energy and protein metabolism. Administration of FMT at a 5 g dose achieves an optimal balance between microbial colonization and metabolic function, thereby improving the overall efficiency of biological systems, as evidenced by simultaneous increases in both milk quantity and quality.

Implications for dairy production in tropical lowlands

Dairy cows raised in tropical lowland areas are susceptible to heat stress, which leads to reduced feed intake, metabolic efficiency, and milk production and quality (Fauzi et al., 2023). These conditions limit dairy cows’ ability to maintain a positive energy balance, particularly during the late lactation phase, which is characterized by reduced physiological efficiency. A gut microbiota-based approach is relevant in this context because it has the potential to enhance post-rumen nutrient utilization without increasing the host’s metabolic burden.

The application of FMT shows potential as an adaptive strategy to improve nutrient utilization efficiency and support the availability of energy and metabolic precursors for milk synthesis under suboptimal environmental conditions (Liu et al., 2023). This approach is also aligned with sustainable production systems as it is antibiotic-free and contributes to the stability of the microbiota ecosystem (Ibeagha-Awemu and Vincent, 2025; Niederwerder, 2018). Practical implementation challenges in smallholder tropical farming systems include: (1) qualified donor selection and health screening; (2) laboratory capacity for fecal processing and encapsulation; (3) cold-chain requirements for capsule storage; and (4) cost per animal per treatment cycle. The 7-day front-loaded administration protocol used in this study partially addresses the feasibility concern. Centralized capsule production at the regional or cooperative level could reduce per-farm costs; a dedicated cost-effectiveness analysis is recommended as part of future research. Integrating FMT into livestock nutrition and health management has the potential to support sustainable and adaptive dairy production systems in lowland tropical regions (Tian et al., 2024).

Effect of FMT on feed intake and feed efficiency

FMT administration had a significant effect on feed intake parameters in Friesian Holstein dairy cows in the late lactation phase (Table 3). Analysis of variance revealed significant treatment effects on dry matter intake and feed efficiency (P < 0.05), whereas organic matter intake (P = ns) and feed conversion ratio (P = 0.177) were not statistically significant.

The 7.3% increase in dry matter intake (DMI) at T1 compared to the control was statistically significant (P<0.05), reflecting improved physiological condition as a consequence of FMT-induced hindgut ecosystem remodeling. Organic matter intake (OMI) at T1 was numerically higher by 10.2% compared to the control (10.63 vs. 9.65 kg/cow/day), although this difference did not reach statistical significance (P>0.05) and should therefore be interpreted as a numerical trend rather than a statistically confirmed effect. The increase in feed intake at T1 is associated with higher fermentative activity; SCFA-associated bacterial genera known to regulate energy metabolism and appetite through enteroendocrine signaling (Han et al., 2021) were more abundant in T1, suggesting that improved gut physiology may have contributed to increased voluntary intake, though direct SCFA quantification was not performed. The higher feed intake observed at T1 represents a downstream consequence of FMT-induced improvements in fermentation efficiency, while the increase in milk production is more directly attributed to optimized post-ruminal nutrient utilization.

Table 3: Dry matter and organic matter intake, feed conversion, and feed efficiency in late-lactation Friesian Holstein cows.

Parameters	T0	T1	T2	P value
Dry matter intake (kg/day)	10.93b ± 0.27	11.73a ± 0.48	10.89b ±0.69	0.048
Organic matter intake (kg/day)	9.65 ± 0.328	10.63 ± 0.42	9.76 ± 0.54	0.116
Feed conversion	1.96 ± 0.68	1.80 ± 0.55	1.76 ± 0.467	0.177
Feed efficiency	0.55b ± 0.17	0.59a ± 0.19	0.60a ± 0.16	0.039

Feed conversion and feed efficiency parameters indicated that FMT improved post-rumen nutrient utilization efficiency. FCR values were numerically lower in T1 (1.80 kg DM/kg milk) and T2 (1.76) compared to T0 (1.96), although this difference did not reach statistical significance (p=0.177) and should be interpreted only as a numerical trend. Feed efficiency in T1 (0.59) and T2 (0.60) was both significantly higher than T0 (0.55; P<0.05), while T1 and T2 were not significantly different from each other. Changes in these ratio parameters indicate an increase in post-rumen metabolic capacity independent of increased dry matter intake. Variation in feed efficiency has been reported to be more closely related to the composition of the hindgut microbiota than to the amount of feed consumed (Ahvenjarvi et al., 2024).

A comparison between T0 and T2 demonstrates the independent effect of FMT: despite nearly identical DMI (10.89 vs. 10.93 kg/day, difference < 0.4%), T2 produced 9.5% more milk (6.55 vs. 5.98 L/day). This T0 versus T2 comparison effectively functions as a natural experiment controlling for feed quantity, confirming that FMT operates through fermentation efficiency and nutrient utilization pathways that are relatively independent of feed intake (Monteiro et al., 2022).

Regarding the T1 versus T2 paradox: T2 exhibited marginally higher feed efficiency (0.60 vs. 0.59) but lower total milk yield (6.55 vs. 6.96 L/day). This is mathematically consistent because feed efficiency = milk production / DMI, and both the numerator (milk) and denominator (DMI) are proportionally lower in T2, yielding a similar ratio. While T1 and T2 had similar feed efficiency ratios, T1 produced absolutely more milk, making T1 the preferred dose from a total production standpoint.

CONCLUSION

Oral administration of encapsulated FMT at a moderate dose significantly improved hindgut microbiota composition, feed efficiency, milk yield, and milk quality in late-lactation Friesian Holstein cows under tropical lowland conditions, with the moderate dose demonstrating the most optimal biological response. Nevertheless, these findings should be interpreted within the limitations of this study, including the small number of experimental animals, use of a single donor, short observation period, and the absence of direct SCFA measurement, which collectively restrict the generalizability of the conclusions.

Future studies involving a larger number of animals, multiple donors, longer observation periods, and direct SCFA quantification are necessary to confirm and validate these findings before broader application can be recommended.

ACKNOWLEDGEMENTS

The authors would like to thank Riset dan Inovasi untuk Indonesia Maju (RIIM) for funding this research through Contract No. 13/IV/KS/0/2023, Bumi Yasa Svarga through Cooperation Agreement No. 01/PKS/Bumi.Yasa.Svarga/V/2023, and the Faculty of Animal and Agricultural Sciences, Diponegoro University, for the support provided during the conduct of this research.

NOVELTY STATEMENT

This study shows that oral fecal microbiota transplantation can improve hindgut microbial fermentation, feed efficiency, milk yield, and milk composition in late-lactation Friesian Holstein cows raised in tropical lowland conditions, with the 5 g dose showing the most consistent biological response.

AUTHOR’S CONTRIBUTION

Draft for author confirmation: N.A.M. Fadillah conducted the study, curated the data, and drafted the manuscript. B.W.H.E. Prasetyono conceived and supervised the study and critically revised the manuscript. N.R.S. Puspitasari, A.D. Lathifa, A. Azhar, B. Ahmed, and A.R. Satrio assisted with experimentation, laboratory work, data interpretation, and manuscript revision. All authors approved the final manuscript.

Ethical approval

All procedures followed standard operating protocols. The experimental method was approved by the Research Ethics Committee of the Animal and Agricultural Sciences, Diponegoro University (No. 60-09/A-17/ KEP-FPP).

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Ahvenjarvi S, Bayat AR, Toivanen M (2024). The effects of residual energy intake on nutrient use, methane emissions and microbial composition in dairy cows. Sci. Rep., 14: 613. https://doi.org/10.1038/s41598-024-51300-7

Bernabucci U, Lacetera N, Baumgard LH, Rhoads RP, Ronchi B, Nardone A (2010). Metabolic and hormonal acclimation to heat stress in domesticated ruminants. Animal, 4(7): 1167-1183. https://doi.org/10.1017/S175173111000090X

Fauzi F, Kharisudin I, Wasono R, Utami TW, Harmoko I (2023). Thermal stress projection based on Temperature-Humidity Index (THI) under climate change scenario. J. Meteorol. Geofisika., 24(1): 65-73. https://doi.org/10.31172/jmg.v24i1.867

Food and Agriculture Organization of the United Nations (2023). Dairy market review: Emerging trends and outlook. Rome: FAO. https://www.fao.org/3/cc3780en/cc3780en.pdf

Habimana V, Nguluma AS, Nziku ZC, Ekine-Dzivenu CC, Morota G, Mrode R, Chenyambuga SW (2023). Heat stress effects on milk yield traits and metabolites and mitigation strategies for dairy cattle breeds reared in tropical and sub-tropical countries. Front. Vet. Sci., 10: 1121499. https://doi.org/10.3389/fvets.2023.1121499

Han H, Yi B, Zhong R, Wang M, Zhang S, Ma J, Yin Y, Yin J, Chen L, Zhang H (2021). From gut microbiota to host appetite: Gut microbiota-derived metabolites as key regulators. Microbiome, 9: 162. https://doi.org/10.1186/s40168-021-01093-y

He Z, Dong H (2023). The roles of short-chain fatty acids derived from colonic bacteria fermentation of non-digestible carbohydrates and exogenous forms in ameliorating intestinal mucosal immunity of young ruminants. Front. Immunol., 14: 1291846. https://doi.org/10.3389/fimmu.2023.1291846

Holman DB, Gzyl KE (2019). A meta-analysis of the bovine gastrointestinal tract microbiota. J. FEMS Microbiol. Ecol., 95(1): 1-9. https://doi.org/10.1093/femsec/fiz072

Ibeagha-Awemu EM, Omonijo FA, Piché LC, and Vincent AT (2025). Alternatives to antibiotics for sustainable livestock production in the context of the One Health approach: Tackling a common foe. Front. Vet. Sci., 12: 1605215. https://doi.org/10.3389/fvets.2025.1605215

Kou X, Ma Q, Liu Y, Khan MZ, Wu B, Chen W, Liu X, Wang C, Li W (2024). Exploring the effect of gastrointestinal Prevotella on growth performance traits in livestock animals. Animals, 14(13): 1-16. https://doi.org/10.3390/ani14131965

Lin LZ, Lai J, Zhang J, Zhu W, Mao S (2023). The gastrointestinal microbiome in dairy cattle is constrained by the deterministic driver of the region and the modified effect of diet. J. Microbiom., 11(10): 1-21. https://doi.org/10.1186/s40168-022-01453-2

Liu L, Wu P, Guo A, Yang Y, Chen F, Zhang Q (2023). Research progress on the regulation of production traits by gastrointestinal microbiota in dairy cows. Front. Vet. Sci.., 10: 1206346. https://doi.org/10.3389/fvets.2023.1206346

Monteiro HF, Faciola AP, Santos JEP (2022). Rumen and lower gut microbiomes relationship with feed efficiency and production traits throughout the lactation of Holstein dairy cows. Sci. Rep., 12: 4904. https://doi.org/10.1038/s41598-022-08761-5

National Research Council (NRC) (2001). Nutrient Requirements of Dairy Cattle: Seventh Revised Edition. Washington, DC: The National Academies Press.

Niederwerder MC (2018). Fecal microbiota transplantation as a tool to treat and reduce susceptibility to disease in animals. Vet. Immunol. Immunopathol., 206: 65–72. https://doi.org/10.1016/j.vetimm.2018.11.002

OECD/FAO (2022). OECD-FAO Agricultural Outlook 2022-2031. Paris: OECD Publishing.

Qi Y, Zheng T, Liu X, Yang S, Li Q, Shao J, Zeng X, Guan W, Zhang S (2023). Sodium acetate regulates milk fat synthesis through the activation of GPR41/GPR43 signaling pathway. Front. Nutr., 10: 1098715. https://doi.org/10.3389/fnut.2023.1098715

Sanz-Fernandez MV, Rhoads ML, Baumgard LH (2024). Heat stress effects on intake, metabolism, and productivity of dairy cows. J. Dairy Sci., 15(3): 443-22.

Tapio I, Fischer D, Blasco L, Tapio M, Wallace RJ, Bayat AR, Shingfield KJ (2016). Taxon abundance, diversity, and functional associations in the rumen microbiome. Sci. Rep., 6: 1-12. https://doi.org/10.1371/journal.pone.0180260

Thornton PK (2010). Livestrock: Recent trends, future prospects. Philos. Trans. R. Soc. B: Biol. Sci., 365(1554): 2853-2867. https://doi.org/10.1098/rstb.2010.0134

Tian H, Wang X, Fang Z, Li L, Wu C, Bi D, Li N, Chen Q, Qin H (2024). Fecal microbiota transplantation in clinical practice: Present controversies and future prospects. hLife, 2(6): 269-283. https://doi.org/10.1016/j.hlife.2024.01.006

Wang GY, Qin SL, Zheng YN, Geng HJ, Chen L, Yao JH and Deng L (2023). Propionate promotes hepatic gluconeogenesis by regulating key metabolic pathways in bovine hepatocytes. Anim. Nutr., 13(15): 88-98. https://doi.org/10.1016/j.aninu.2023.07.001

Wang S, Kong F, Zhang X, Dai D, Li C, Cao Z, Wang Y, Wang W, Li S (2026). Disruption of hindgut microbiome homeostasis promotes postpartum energy metabolism disorders in dairy ruminants by inhibiting acetate-mediated hepatic AMPK-PPARA axis. Microbiome, 13: 167. https://doi.org/10.1186/s40168-025-02150-6

Weingarden AR, Vaughn BP (2017). Intestinal microbiota, fecal microbiota transplantation, and inflammatory bowel disease. Gut Microbes., 8(3): 238-252. https://doi.org/10.1080/19490976.2017.1290757

Xu Q, Qiao Q, Gao Y, Hou J, Hu M, Du Y, Zhao K, Li X (2021). Gut microbiota and their role in health and metabolic disease of dairy cow. J. Front. Nutr., 8(1): 1-13. https://doi.org/10.3389/fnut.2021.701511

Zhao S, Min L, Zheng N, Wang J (2019). Effect of heat stress on bacterial composition and metabolism in the rumen of lactating dairy cows. Animals, 9(11): 925. https://doi.org/10.3390/ani9110925

Zhao M, Zheng X, Liu C, Chen Q, Guo J, Zhang Y, Yang H, Zhao C (2022). Bacteroides utilization for dietary polysaccharides and their beneficial effects on gut health. Food Chem. Mol. Sci., 4: 100079.