AAVS_13_12_2716-2727

INTRODUCTION

Ruminant nutrition research increasingly focuses on strategies to improve feed efficiency while mitigating greenhouse gas (GHG) emissions (Janssen, 2010; Jayanegara et al., 2021). Dairy cattle are of particular concern due to their high energy requirements during lactation and their significant contribution to methane (CH₄) emissions (Battini et al., 2016; Despal et al., 2017). Methane production represents an energetic loss of 2–12% of gross energy intake (Blaxter and Clapperton, 1965; Wang et al., 2025), thereby lowering feed efficiency (Waghorn and Hegarty, 2011). Moreover, methane has a global warming potential ~28 times higher than CO₂ (Battini et al., 2016). Therefore, nutritional interventions that redirect hydrogen (H₂) away from methanogenesis toward more efficient fermentation pathways are crucial for sustainable dairy production (Janssen, 2010).

One promising approach involves dietary fat supplementation (Despal et al., 2022; Riestanti et al., 2024; Zahera et al., 2024). Lipids not only increase dietary energy density (Jayanegara et al., 2021; Riestanti et al., 2021; Zahera et al., 2024) but also alter microbial populations and serve as alternative H₂ sinks through biohydrogenation (Kjeldsen et al., 2024). Palm fatty acid distillate (PFAD), a low-cost by-product of the palm oil industry, has been identified as a potential feed supplement for ruminants (Moate et al., 2004; Riestanti et al., 2021). However, free unsaturated fatty acids can impair fiber digestion and microbial activity (Despal et al., 2022; Riestanti et al., 2024). To minimize this risk, protection technologies such as calcium soap formation are used to bypass ruminal degradation (Riestanti et al., 2023; Zahera et al., 2024). Protected fats not only improve energy supply in lactating cows but may also affect volatile fatty acid (VFA) patterns particularly acetate (C2) and propionate (C3) thereby influencing glucogenic efficiency and methane formation (Rabiee et al., 2012).

Trace minerals such as selenium (Se), copper (Cu), and zinc (Zn) play important roles in rumen metabolism and microbial antioxidant defense. Selenium supports glutathione peroxidase activity to maintain cellular oxidative balance (Calamari et al., 2011), while Cu and Zn are key components of superoxide dismutase and other antioxidant metalloenzymes that protect rumen microbes from oxidative stress (Uchida et al., 2001). Adequate supplementation of these minerals has been shown to enhance microbial protein synthesis and fermentation stability, particularly when dietary lipids are included (Nocek et al., 2006). In the present experiment, Se, Cu, and Zn were supplied at three graded inclusion levels following the recommendations of Nasem (2021): 0.15–0.60 mg/kg DM for Se, 6–24 mg/kg DM for Cu, and 25–100 mg/kg DM for Zn. These inclusion levels remain well below established toxicity thresholds for ruminants (Suttle, 2010) and are not expected to induce mineral antagonism, as interactions among Se, Cu, and Zn are minimal when provided within recommended physiological ranges. Therefore, the combined supplementation of these minerals is expected to strengthen antioxidant capacity and support more stable rumen fermentation.

The integration of these microminerals with PFAD supplied in calcium soap form (protected PFAD) is expected to improve rumen fermentation efficiency, stabilize microbial populations, and mitigate methane emissions by redirecting reducing equivalents (H₂) toward propionate formation (Li et al., 2019; Nocek et al., 2006; Razzaghi et al., 2022), although systematic evaluations of their combined effects under in vitro conditions remain limited.

Although both protected fat and microminerals have been individually studied in ruminant nutrition, their potential synergistic effects on rumen fermentation remain poorly described. The complementary roles of protected PFAD and microminerals are proposed to support rumen fermentation through related biochemical pathways in the rumen. PFAD, when converted to calcium soap, acts as a hydrogen sink during the biohydrogenation of unsaturated fatty acids, thereby reducing the available hydrogen for methanogenesis and shifting volatile fatty acid production toward propionate formation (Jenkins, 1993; Sun et al., 2022). Meanwhile, Se, Cu, and Zn function as cofactors for redox and antioxidant enzymes (e.g., glutathione peroxidase, superoxide dismutase, cytochrome oxidase) (Spears, 2003; Vigh et al., 2023), maintaining microbial oxidative balance and stabilizing the rumen’s redox potential (Suttle, 2010). Consequently, the combined supplementation of PFAD and trace minerals is expected to enhance microbial fermentation stability, reduce oxidative stress, and sustain nutrient digestibility and energy recovery within the rumen ecosystem.

Therefore, this study was conducted to evaluate the combined effects of micromineral mixtures and protected PFAD supplementation on rumen fermentation (pH, protozoa, VFA, NH₃), methane mitigation, in vitro digestibility, gas production, and energy partitioning.

MATERIALS AND METHODS

Diet and supplement preparations

The basal substrate was formulated according to NRC (2001) requirements for lactating dairy cows. The diet consisted of 60% elephant grass (Pennisetum purpureum) and 40% concentrate (Anzhany et al., 2022). Palm fatty acid distillate (PFAD) was processed into calcium soap form to provide a protected fat source similar to the method used by Zahera et al. (2024). Four levels of PFAD were tested: 0, 1, 2, and 3% of diet DM. Nutrient content of the diet after protected PFAD supplementation is shown in Table 1.

The micromineral mixture was specifically formulated to deliver three graded supplementation levels of selenium (Se), copper (Cu), and zinc (Zn). In this formulation, selenium was provided as organic selenium yeast (Sel-Plex), copper was sourced from copper sulfate pentahydrate (CuSO₄·5H₂O), and zinc was supplied in the form of zinc oxide (ZnO). The detailed mineral supply per kilogram of diet DM for each dose (D0–D3) is presented in Table 2.

Table 1: Nutrient content of the treatment ration.

PFAD (%)	Nutrient and energy content
PFAD (%)	Dry matter (%)	Ash (% DM)	Crude protein (% DM)	Ether extract (% DM)	Crude fiber (% DM)	ADF (%)	NDF (%)	Hemi-cellulose (%)	Gross energy (MJ/kg DM)
0	90.56	9.56	13.20	3.50	20.66	27.16	49.84	22.68	17.18
1	90.65	9.46	13.07	4.46	20.45	26.87	49.31	22.44	17.41
2	90.75	9.37	12.94	5.43	20.25	26.61	48.83	22.22	17.63
3	90.84	9.27	12.80	6.40	20.04	26.33	48.32	22.00	17.85

Note: DM= dry matter, MJ= Mega joule, ADF= acid detergent fiber, NDF= neutral detergent fiber, PFAD= Palm fatty acid distillate.

Table 2: Composition of micromineral substrates at different doses.

No.	Component	Micromineral
No.	Component	Dose 0	Dose 1	Dose 2	Dose 3
1	Selenium (mg/kg)	0	0.15	0.30	0.60
2	Copper (mg/kg)	0	6	12	24
3	Zinc (mg/kg)	0	25	50	100

In vitro preparation and incubation

Fresh rumen inoculum was collected from three fistulated Friesian Holstein bulls housed in the Dairy Nutrition Division Closed-House Barn, Faculty of Animal Science, IPB University. Samples were taken before morning feeding, strained through four layers of cheesecloth, and immediately mixed with a mineral–carbonate buffer (1:2, v/v) under continuous CO₂ flushing.

In vitro incubations followed the two-stage method of Tilley and Terry (1963) with modifications. Substrates (0.5 g) were incubated with buffered rumen fluid at 39 °C for 48 h (Stage 1), followed by 48 h digestion with pepsin–HCl (Stage 2). Dry matter (IVDMD) and organic matter digestibility (IVOMD) were determined from dried and ashed residues. Each treatment was run in duplicate with blanks, and the experiment was repeated on separate donor days.

Fermentation characteristics measured after 4 h of Stage 1 incubation included pH (digital pH meter), ammonia-N (Conway diffusion), total VFA (steam distillation), and partial VFA (acetate, propionate, butyrate, isoacids) determined by gas chromatography (Yulistiani et al., 2017). Methane (CH₄) was estimated from VFA profiles using the stoichiometric equation CH₄ = 0.45 × acetate – 0.275 × propionate + 0.40 × butyrate (Moss et al., 2000). Protozoa were enumerated microscopically (log CFU/mL) before terminating fermentation with saturated HgCl₂.

A parallel set of 100-ml calibrated glass syringes was used for the Hohenheim gas test, with gas volume recorded at 24 h. Digestible energy (DE), metabolizable energy (ME), net energy for lactation (NEl), and methane energy were estimated from gas production and diet composition using (Menke et al., 1979): DE = ME/0.82; ME = 0.145 × GP + 4.12 × CP + 6.5 × CP² + 20.6 × EE + 1.54; NEl = 0.096 × GP + 0.0038 × CP + 0.000173 × EE² + 0.54; and methane energy = CH₄ × 0.089. Here, DE, ME, and NEl are expressed in MJ/kg DM; CP and EE are crude protein and ether extract (g/g DM); GP is 24-h gas production (ml/200 mg DM); and CH₄ is methane concentration (mmol/50 ml) estimated from stoichiometry.

Design experiment and data analysis

The study used a 4 × 4 factorial experiments arranged under a randomized complete block design (RCBD). Two experimental factors were tested: micromineral dose (four levels: 0, 1, 2, and 3) as Factor A, and protected palm fatty acid distillate (PFAD; 0, 1, 2, and 3% of dry matter) as Factor B, experiment structure shown in Figure 1. The experiment consisted of four replications, with each replication corresponding to one incubation run using rumen fluid collected weekly. At each sampling, rumen fluid was collected from two donor cows and composited in equal proportions to form a homogeneous inoculum. The sampling week was treated as the blocking factor to account for potential variation in rumen microbial activity across collection periods.

All variables were analyzed using two-way ANOVA to determine the main effects of PFAD, micromineral dose, and their interaction (PFAD × mineral). When significant differences were detected (p < 0.05), mean comparisons were performed using orthogonal contrasts to separate linear and quadratic effects across PFAD levels (0–3%). Polynomial regression analysis was used to identify quadratic trends and to estimate the optimum inclusion level by solving the vertex of the fitted model (x = –b/2a). The goodness of fit of each model was evaluated based on the coefficient of determination (R²). The model was descriptive; inferences relied on two-way ANOVA.

All analyses were conducted in SPSS (version 25, IBM Corp., Armonk, NY, USA), and mean comparisons followed Steel and Torrie (1980) for a valid interpretation of treatment effects.

RESULTS AND DISCUSSION

In vitro conditions and fermentation product

The effects of combined micromineral mixtures and protected PFAD supplementation on in vitro fermentation of dairy cattle diets are summarized in Table 3. Rumen pH values remained stable (6.83–7.07) across treatments, indicating that supplementation neither induced acidification nor disrupted the microbial ecosystem. A pH above 6.2 is considered optimal for fibrolytic bacterial activity, and the buffering system in vitro likely contributed to maintaining this stability (McDonald et al., 2021). These observations are consistent with previous in vitro studies (Anzhany et al., 2022; Despal et al., 2022; Riestanti et al., 2024), which also highlighted the importance of buffering systems in sustaining ruminal pH balance.

Protozoa count showed no significant differences across treatments (6.30–6.35 log CFU/mL), indicating that neither protected PFAD nor mineral supplementation measurably suppressed their growth. Although unsaturated fatty acids are toxic to protozoa (Anzhany et al., 2022; Jenkins, 1993; Riestanti et al., 2024), the protected PFAD likely mitigated this effect. At rumen pH (6.0–7.0), Ca-soaps are relatively insoluble, reducing the release of free fatty acids into the aqueous phase and thereby limiting direct membrane toxicity. Their lower availability to microbial lipases and hydrogenating bacteria further decreases the risk of protozoal inhibition. Once in the abomasum (pH < 6), soaps dissociate and release fatty acids for digestion. This protection enabled protozoa

Table 3: In vitro conditions and fermentation products of dairy cow rations supplemented with a mixture of minerals and protected PFAD.

Parameter	Micro-mineral dose	0% PFAD	1% PFAD	2% PFAD	3% PFAD	Mean (Dose)
pH	Dose-0	6.86 ± 0.18	7.07 ± 0.06	6.89 ± 0.10	6.88 ± 0.12	6.93 ± 0.12
	Dose-1	6.85 ± 0.14	6.88 ± 0.39	6.89 ± 0.13	6.87 ± 0.14	6.92 ± 0.22
	Dose-2	6.89 ± 0.12	6.88 ± 0.17	6.92 ± 0.18	6.83 ± 0.28	6.87 ± 0.18
	Dose-3	6.90 ± 0.15	6.88 ± 0.16	6.87 ± 0.22	6.88 ± 0.20	6.90 ± 0.17
Mean (PFAD)		6.88 ± 0.14	6.93 ± 0.22	6.89 ± 0.15	6.87 ± 0.18
Protozoa (log CFU/mL)	Dose-0	6.36 ± 0.28	6.38 ± 0.16	6.37 ± 0.23	6.33 ± 0.11	6.36 ± 0.19
	Dose-1	6.22 ± 0.13	6.29 ± 0.21	6.31 ± 0.17	6.34 ± 0.28	6.29 ± 0.19
	Dose-2	6.41 ± 0.14	6.22 ± 0.08	6.37 ± 0.22	6.23 ± 0.22	6.31 ± 0.18
	Dose-3	6.21 ± 0.19	6.41 ± 0.20	6.18 ± 0.43	6.50 ± 0.19	6.32 ± 0.28
Mean (PFAD)		6.30 ± 0.20	6.33 ± 0.17	6.31 ± 0.26	6.35 ± 0.21
Total VFA (mM)	Dose-0	103.90 ± 16.34	123.44 ± 16.01	107.44 ± 18.52	124.01 ± 43.64	114.70 ± 25.36
	Dose-1	141.64 ± 44.89	125.35 ± 18.50	113.08 ± 35.47	134.78 ± 22.32	128.71 ± 30.74
	Dose-2	141.80 ± 36.34	136.19 ± 25.63	101.88 ± 9.69	118.62 ± 24.05	124.62 ± 28.14
	Dose-3	106.53 ± 32.96	102.74 ± 28.32	134.19 ± 26.21	124.70 ± 32.79	117.04 ± 30.13
Mean (PFAD)		123.47 ± 35.97	121.93 ± 23.82	114.15 ± 25.22	125.53 ± 29.11
Ammonia-N (NH₃–N)	Dose-0	9.76 ± 2.79	9.72 ± 4.32	7.67 ± 2.53	8.05 ± 1.54	8.80 ± 2.83
	Dose-1	8.23 ± 2.95	8.07 ± 1.09	6.92 ± 0.86	7.03 ± 1.01	7.56 ± 1.65
	Dose-2	7.47 ± 2.20	7.41 ± 1.68	7.21 ± 0.76	7.37 ± 1.70	7.36 ± 1.50
	Dose-3	7.52 ± 5.31	6.46 ± 1.12	5.90 ± 2.80	7.83 ± 3.33	6.93 ± 3.21
Mean (PFAD)		8.25 ± 3.29	7.92 ± 2.51	6.92 ± 1.89	7.57 ± 1.91

Note: Values represent mean ± standard deviation (SD) of four replicates. PFAD = protected palm fatty acid distillate; micromineral = mixture of Se, Cu, and Zn.

to maintain their role in fiber degradation while still contributing hydrogen for methanogenesis (Despal et al., 2022). Previous studies also reported that inclusion of Ca-soap or protected fats up to 2–3% of dietary dry matter is safe and does not impair microbial growth, fermentation, or digestibility (Behan et al., 2024; Riestanti et al., 2023; Zahera et al., 2024). Collectively, these findings confirm that protected PFAD provides effective rumen protection, ensuring energy delivery without compromising protozoal populations or fermentation stability.

Total volatile fatty acid (VFA) concentrations ranged from 102.7 to 141.8 mM, with mean values of 114.2–125.5 mM. Compared with the control (114.7 mM), supplementation tended to increase total VFA at Dose-1 (128.7 mM) and Dose-2 (124.6 mM), though variability among replicates limited consistency. These findings suggest that protected lipid and mineral supplementation did not compromise fermentability and may enhance microbial activity at certain dose combinations (Riestanti et al., 2021; Zahera et al., 2024). The micromineral mix enhances microbial activity because trace minerals serve as vital enzyme cofactors (Calamari et al., 2011), protect microbes from stress (Uchida et al., 2001), and improve energy metabolism (Overton and Yasui, 2014). Together with protected fat, they create a more favorable fermentation environment, supporting higher total VFA production. This indicates a complementary synergy between protected PFAD and microminerals, where PFAD regulates hydrogen flow through biohydrogenation while trace minerals maintain microbial redox balance under the altered fermentation environment.

Ammonia-N (NH₃-N) concentrations ranged from 5.90 to 9.76 mM, with means of 6.92–8.25 mM. Supplementation consistently lowered NH₃-N compared with the control (8.25 mM), yielding values within the optimal range (5–8 mM) for microbial growth and indicating more efficient nitrogen utilization and microbial protein synthesis (Despal et al., 2023). This effect likely reflects improved capture of NH₃-N by microbes when sufficient energy is available, supported by protected fat that sustains microbial activity and enhances nitrogen assimilation. In addition, microminerals such as Zn, Cu, Mn, Co, and Fe act as cofactors for nitrogen-metabolizing enzymes (e.g., glutamate dehydrogenase, glutamine synthetase), facilitating conversion of NH₃ into organic compounds. Although the effects of microminerals were modest, slightly lower NH₃-N at higher doses was consistent with their enzymatic role in nitrogen metabolism (Bach et al., 2005).

Overall, supplementation-maintained rumen pH and protozoa within normal ranges, while improving VFA production and optimizing ammonia utilization. These results highlight the potential of this strategy to enhance rumen fermentation efficiency without disturbing microbial stability.

Volatile fatty acid profile and estimate methane production

The effects of combined micromineral mixtures and protected PFAD supplementation on the partial volatile fatty acid (VFA) profile and estimated methane production are presented in Table 4. Acetate concentrations showed a significant difference (p<0.05) across treatments, with the lowest value at 1% PFAD (51.5%) and higher values at 0% (63.1%) and 3% PFAD (62.4%). Propionate showed the opposite trend, with a significant increase at 1% PFAD (29.0%), compared to only 21.6% and 21.9% at 0% and 3% PFAD, respectively. These results indicate that moderate PFAD supplementation promotes a shift toward propionate formation. For dairy cattle, however, maintaining a sufficient acetate supply is crucial since acetate is the main precursor for milk fatty acid synthesis (Yu et al., 2024). Thus, while propionate improvement enhances glucogenic energy, too low acetate levels may compromise milk fat yield if not properly balanced (Seymour et al., 2005; Suttle, 2010; Sutton et al., 2003).

The acetate-to-propionate (C₂:C₃) ratio varied significantly among treatments, reaching its lowest value at 1% PFAD (1.95) and the highest at 0% PFAD (3.22). The observed range of ratios aligns closely with previous in vitro findings reported by Anzhany et al. (2022) and Rosmalia et al. (2022), confirming the consistency of the fermentation pattern across similar experimental conditions. A lower ratio reflects increased propionate production and reduced hydrogen availability for methanogenesis, which can improve energy efficiency (Beauchemin et al., 2008), but may risk insufficient acetate for milk fat synthesis if the ratio falls below 2.0 (Bauman and Griinari, 2001). In contrast, the control diet exceeded the recommended range of 2.5–3.0 for optimal energy utilization (Firkins and Mitchell, 2023), indicating an excessive acetate-to-propionate ratio. The 2% PFAD treatment (2.52) was well aligned with this optimal range, suggesting a balanced supply of acetate and propionate for both milk fat and lactose synthesis. Similarly, 3% PFAD (3.06) remained within the upper end of the range, maintaining sufficient acetate for milk fat synthesis while still enhancing energy efficiency (Allen, 2000). These findings indicate that protected PFAD supplementation at 2–3% inclusion provides the most favorable rumen fermentation balance for sustaining both milk yield and milk fat content.

Estimated methane production varied significantly among treatments, with the lowest value observed at 1% PFAD (20.5 mM) compared with 0% (26.7 mM) and 3% PFAD (26.4 mM). This pattern corresponded with a lower acetate-to-propionate ratio, indicating that increased propionate formation acts as an alternative

Table 4: Partial VFA profile as affected by the treatments.

Parameter	Micromineral dose	0% PFAD	1% PFAD	2% PFAD	3% PFAD	Mean (Dose)
Acetate (C₂)	Dose-0	60.17 ± 12.03	50.09 ± 9.19	56.74 ± 14.06	59.38 ± 11.10	56.59 ± 11.26
	Dose-1	67.86 ± 13.08	52.72 ± 11.63	60.57 ± 14.81	64.75 ± 8.33	61.47 ± 12.39
	Dose-2	59.55 ± 13.65	57.20 ± 9.85	52.66 ± 17.33	65.68 ± 20.08	58.77 ± 14.86
	Dose-3	64.71 ± 14.69	46.02 ± 4.51	60.43 ± 12.56	59.70 ± 14.70	57.71 ± 13.21
Mean (PFAD)		63.07 ± 12.49ᵃ	51.51 ± 9.22ᵇ	57.60 ± 13.64ᵇ	62.38 ± 13.08ᵃ
Propionate (C₃)	Dose-0	22.41 ± 4.47	29.40 ± 6.90	26.35 ± 5.27	24.14 ± 3.17	25.57 ± 5.33
	Dose-1	19.61 ± 5.22	28.40 ± 9.38	23.07 ± 5.08	22.02 ± 2.39	23.27 ± 6.35
	Dose-2	23.41 ± 4.57	26.33 ± 7.68	26.53 ± 8.18	18.93 ± 6.01	23.80 ± 6.83
	Dose-3	20.81 ± 7.63	31.85 ± 6.27	23.44 ± 4.13	22.50 ± 2.67	24.65 ± 6.61
Mean (PFAD)		21.56 ± 5.25ᵇ	28.99 ± 7.14ᵃ	24.85 ± 5.50ᵃ	21.90 ± 3.95ᵇ
C₂:C₃ Ratio	Dose-0	2.81 ± 0.90	1.81 ± 0.67	2.29 ± 0.97	2.53 ± 0.74	2.36 ± 0.83
	Dose-1	3.75 ± 1.48	2.13 ± 1.20	2.81 ± 1.11	3.00 ± 0.73	2.92 ± 1.20
	Dose-2	2.70 ± 1.12	2.36 ± 0.94	2.29 ± 1.57	4.01 ± 2.30	2.84 ± 1.58
	Dose-3	3.61 ± 1.94	1.50 ± 0.41	2.69 ± 0.95	2.69 ± 0.79	2.62 ± 1.30
Mean (PFAD)		3.22 ± 1.36ᵃ	1.95 ± 0.83ᵇ	2.52 ± 1.08ᵇ	3.06 ± 1.33ᵃ
Isobutyrate (iC₄)	Dose-0	2.78 ± 2.35	2.88 ± 3.06	2.16 ± 1.16	2.22 ± 1.56	2.51 ± 1.96
	Dose-1	1.73 ± 1.21	2.26 ± 0.97	2.05 ± 1.07	1.68 ± 0.95	1.93 ± 0.98
	Dose-2	1.93 ± 1.23	2.57 ± 1.22	2.02 ± 1.39	1.57 ± 1.44	2.02 ± 1.24
	Dose-3	1.58 ± 0.80	2.51 ± 1.30	2.05 ± 1.19	2.21 ± 1.85	2.09 ± 1.25
Mean (PFAD)		2.00 ± 1.43	2.55 ± 1.66	2.07 ± 1.08	1.92 ± 1.36
Butyrate (nC₄)	Dose-0	11.18 ± 7.08	13.63 ± 4.46	11.81 ± 7.33	11.32 ± 6.39	11.98 ± 5.83
	Dose-1	8.65 ± 6.30	13.17 ± 3.26	11.52 ± 7.65	9.43 ± 4.43	10.69 ± 5.39
	Dose-2	12.28 ± 7.34	10.91 ± 4.42	15.17 ± 9.75	11.01 ± 10.20	12.34 ± 7.59
	Dose-3	10.28 ± 6.05	15.48 ± 5.14	11.44 ± 7.19	12.13 ± 9.38	12.33 ± 6.67
Mean (PFAD)		10.60 ± 6.16	13.30 ± 4.26	12.48 ± 7.37	10.97 ± 7.18
Isovalerate (iC₅)	Dose-0	2.06 ± 1.35	2.28 ± 1.13	1.59 ± 0.99	1.67 ± 1.02	1.90 ± 1.05
	Dose-1	1.26 ± 0.97	2.05 ± 1.06	1.60 ± 1.14	1.16 ± 0.58	1.52 ± 0.93
	Dose-2	1.52 ± 1.10	1.62 ± 0.39	1.93 ± 1.61	1.51 ± 1.48	1.65 ± 1.12
	Dose-3	1.45 ± 0.97	2.16 ± 0.90	1.41 ± 0.86	2.01 ± 2.17	1.76 ± 1.25
Mean (PFAD)		1.57 ± 1.04	2.03 ± 0.86	1.63 ± 1.08	1.59 ± 1.32
Valerate (nC₅)	Dose-0	1.41 ± 0.75	1.73 ± 0.80	1.36 ± 0.78	1.28 ± 0.71	1.45 ± 0.70
	Dose-1	0.89 ± 0.67	1.40 ± 0.54	1.19 ± 0.86	0.97 ± 0.49	1.11 ± 0.62
	Dose-2	1.30 ± 0.82	1.36 ± 0.29	1.69 ± 1.35	1.29 ± 1.21	1.41 ± 0.91
	Dose-3	1.17 ± 0.78	1.98 ± 0.95	1.23 ± 0.83	1.45 ± 1.21	1.46 ± 0.92
Mean (PFAD)		1.19 ± 0.71	1.62 ± 0.67	1.37 ± 0.90	1.25 ± 0.88
Estimated CH₄ (mM)	Dose-0	25.38 ± 3.57	19.91 ± 5.84	23.01 ± 5.03	24.61 ± 3.20	23.23 ± 4.60
Estimated CH₄ (mM)	Dose-1	28.60 ± 4.84	21.18 ± 7.06	25.52 ± 5.06	26.85 ± 2.67	25.54 ± 5.41
	Dose-2	25.27 ± 4.52	22.86 ± 5.23	22.47 ± 6.52	28.75 ± 6.67	24.84 ± 5.80
	Dose-3	27.51 ± 6.70	18.14 ± 4.04	25.32 ± 4.06	25.53 ± 3.46	24.13 ± 5.61
Mean (PFAD)		26.69 ± 4.74ᵃ	20.53 ± 5.36ᶜ	24.08 ± 4.89ᵇ	26.44 ± 4.17ᵃ

Note: Values are mean ± standard deviation (SD) of four replicates. PFAD = protected palm fatty acid distillate; micromineral = mixture of selenium, copper, and zinc. Superscripts (a–c) within a row indicate significant differences (p < 0.05) among PFAD levels.

hydrogen (H₂) sink, thereby reducing substrate availability for methanogenesis (Pereira et al., 2022; Wang et al., 2018). The stoichiometric model of Moss et al. (2000) assumes that H₂ generated during acetate and butyrate formation is used for methane synthesis, while in lipid-supplemented diets part of the H₂ may be diverted to reductive acetogenesis, biohydrogenation, or microbial synthesis (Jenkins, 1993; Patra, 2013). Consequently, methane estimates may be slightly lower than actual values; however, the reduced CH₄ observed at moderate PFAD inclusion indicates an effective redirection of hydrogen flow toward propionate formation and lipid hydrogenation, thereby lowering methane losses and improving rumen energy efficiency.

The significant differences observed in acetate, propionate, the C2:C3 ratio, and methane production highlight the strong effect of PFAD supplementation on rumen fermentation. A 1% inclusion level shifted fermentation toward a more glucogenic profile and reduced methane production, but it also lowered acetate, which is essential for milk fat synthesis. For dairy cattle, the practical implication is that while lipid supplementation can improve energy efficiency and environmental sustainability, feeding strategies must ensure that acetate supply remains sufficient to sustain optimal milk fat production.

Polynomial orthogonal analysis revealed that acetate (C2), propionate (C3), the C2:C3 ratio, and estimated methane (CH₄) all responded quadratically to protected PFAD supplementation (Figure 2). The fitted curves clearly identified extreme points (vertex values) calculated from the first derivative of each quadratic equation.

The refined quadratic models generated in this study further reinforce the central pattern that protected PFAD supplementation exerts a consistent curvilinear influence on rumen fermentation. Acetate followed the model Y = 4.0863x² − 11.858x + 62.123 (R² = 0.79), reaching its predicted minimum of approximately 53.5 mol% at around 1.45% PFAD. In contrast, propionate exhibited the opposite trend, with the model Y = −2.5958x² + 7.4739x + 22.199 (R² = 0.77) identifying a maximum of approximately 27.6 mol% at a nearly identical inclusion level of 1.44% PFAD. Consistent with these patterns, the acetate-to-propionate ratio (C₂:C₃) declined sharply along the model Y = 0.4512x² − 1.3461x + 3.1273 (R² = 0.82), achieving its lowest predicted value of ~2.1 at 1.49% PFAD. Methane production also displayed a curvilinear reduction described by Y = 2.1312x² − 6.1146x + 26.147 (R² = 0.76), with the minimum predicted value of approximately 21.8 mM occurring at ~1.44% PFAD. Collectively, these regression outputs clearly converge on a narrow optimal range of roughly 1.4-1.5% PFAD, which represents the point of maximal glucogenic shift in rumen fermentation characterized by the lowest acetate levels, the lowest C₂:C₃ ratio, the most pronounced suppression of methane, and the greatest enhancement of propionate formation.

This metabolic shift aligns with improved hydrogen utilization efficiency, where reducing equivalents (H₂) are redirected from methanogenesis toward propionate synthesis. However, such an extreme shift toward glucogenic end-products may constrain acetate availability, which is metabolically important for de novo milk fat synthesis. Consequently, although 1.4–1.5% PFAD marks the mechanistic optimum for altering fermentation stoichiometry at the in vitro level, slightly higher inclusion levels (approximately 2–3%) are likely more suitable in practical dairy production because they maintain sufficient acetate output while still providing meaningful methane mitigation. This interpretation is consistent with previous observations by Riestanti et al. (2021, 2023, 2024), who reported that moderate PFAD supplementation supports stable rumen fermentation, maintains milk fat yield, and enhances overall feeding efficiency in dairy cows.

In vitro digestibility

The effects of micromineral mixtures and protected PFAD supplementation on in vitro dry matter digestibility (DMD), organic matter digestibility (OMD), and gas production are presented in Table 5. Dry matter digestibility ranged from 59.9–64.2%, with treatment averages between 61.9–62.9%. There were no significant differences (p > 0.05) among PFAD or micromineral levels, indicating that supplementation did not adversely affect the overall digestibility of dietary dry matter. The relatively consistent digestibility values indicate that protected PFAD did not hinder microbial adherence to fibrous substrates, which is

Table 5: Effect of treatments on in vitro digestibility and gas production.

Parameter	Micro-mineral dose	0% PFAD	1% PFAD	2% PFAD	3% PFAD	Mean (Dose)
Dry matter digestibility (DMD, %)	Dose-0	59.96 ± 5.53	61.88 ± 1.04	60.41 ± 2.04	59.93 ± 3.96	60.55 ± 3.31
	Dose-1	63.77 ± 1.99	62.91 ± 3.54	62.01 ± 2.82	61.39 ± 3.16	62.52 ± 2.78
	Dose-2	63.86 ± 1.93	63.32 ± 2.49	62.65 ± 3.22	62.02 ± 4.60	62.96 ± 2.97
	Dose-3	62.23 ± 1.21	62.68 ± 2.35	62.45 ± 5.32	64.22 ± 2.26	62.90 ± 2.95
Mean (PFAD)		62.45 ± 3.26	62.70 ± 2.31	61.88 ± 3.31	61.89 ± 3.60
Organic matter digestibility (OMD, %)	Dose-0	65.24 ± 4.89	66.44 ± 1.15	64.49 ± 1.98	64.19 ± 4.16	65.09 ± 3.18
	Dose-1	68.40 ± 1.84	67.26 ± 3.05	66.51 ± 2.60	65.63 ± 3.00	66.95 ± 2.61
	Dose-2	68.40 ± 2.11	67.78 ± 2.66	66.90 ± 3.10	66.47 ± 4.52	67.39 ± 2.99
	Dose-3	66.96 ± 1.18	67.40 ± 2.54	66.85 ± 5.11	68.66 ± 2.63	67.47 ± 2.95
Mean (PFAD)		67.25 ± 2.90	67.22 ± 2.25	66.19 ± 3.21	66.24 ± 3.68
Gas production (mL / 200 mg DM)	Dose-0	41.31 ± 3.41	40.82 ± 3.34	38.07 ± 6.67	38.28 ± 0.65	39.62 ± 3.48
	Dose-1	41.36 ± 3.28	42.27 ± 5.50	42.94 ± 0.18	43.93 ± 1.99	42.63 ± 2.73
	Dose-2	41.48 ± 10.38	36.15 ± 10.32	33.49 ± 6.87	28.50 ± 0.13	34.91 ± 7.90
	Dose-3	46.28 ± 3.45	36.04 ± 10.15	35.89 ± 10.44	33.11 ± 6.71	37.83 ± 8.20
Mean (PFAD)		42.61 ± 5.04	38.82 ± 6.68	37.60 ± 6.52	35.96 ± 6.71

Note: Values are means ± standard deviation (SD) of four replicates. PFAD = protected palm fatty acid distillate; micromineral = mixture of Se, Cu, and Zn.

often a concern with lipid supplementation. This finding agrees with previous evidence showing that rumen-protected fats have negligible adverse effects on fiber degradation compared with unprotected oils (Jenkins, 1993). The DMD values obtained in the present work were also in close agreement with earlier reports by Riestanti et al. (2024) and Zahera et al. (2024).

Organic matter digestibility (OMD) ranged from 64.2–68.7% with averages of 66.2–67.3%, showing no significant effects (p > 0.05) of PFAD or mineral supplementation. OMD values were slightly higher than DMD, reflecting the greater solubility and fermentability of organic fractions and the exclusion of indigestible ash. Thus, OMD better represents the digestible portion of the feed, while DMD underestimates true fermentability. Higher OMD over DMD was also reported in a previous study (Al-Arif et al., 2017; Salas et al., 2019).

Gas production varied between 28.5 and 46.3 ml/200 mg DM, decreasing with increasing PFAD from 42.6 ml at 0% to 36.0 ml at 3%. As in vitro gas yield is widely used to estimate OMD and metabolizable energy (ME) (Menke and Steingass, 1988), this reduction suggests a lower fermentation-derived energy yield. Interestingly, OMD remained relatively stable at ~66–67%, indicating that PFAD did not diminish the proportion of digested organic matter but rather altered fermentation stoichiometry. This outcome is consistent with greater diversion of hydrogen toward non-gaseous sinks particularly biohydrogenation of unsaturated fatty acids thereby reducing gas formation (Jenkins, 1993; Pereira et al., 2022).

These findings align with the VFA data, where moderate PFAD inclusion increased propionate and reduced methane estimation. Thus, although gas production decreased, overall fermentation efficiency may have improved, with more energy conserved as VFAs rather than lost as gas. From a dairy cow nutrition perspective, this outcome is favorable as it maintains digestibility while reducing gaseous energy losses (Alvarez-Hess et al., 2019; Yang et al., 2022).

Energy partition

The effects of micromineral mixtures and protected PFAD supplementation on gross energy, digestible energy, methane energy loss, metabolizable energy, and net energy for lactation are presented in Table 6. Gross energy (GE) values were consistent across treatments (17.2–17.9 MJ/kg DM), reflecting the fixed caloric density of the diets. Digestible energy (DE) ranged from 9.3 to 12.0 MJ/kg DM, with numerically higher values observed at 1% PFAD and lower values at 2–3% PFAD. However, these differences were not statistically significant (p > 0.05), indicating that neither protected PFAD nor micromineral supplementation markedly altered the digestible energy fraction of the diet. The lack of an increase in DE despite PFAD supplementation can be attributed to the low ruminal fermentability of Ca-soap fat, which contributes little to VFA formation in vitro. PFAD may also replace a portion of fermentable carbohydrates, thereby reducing the substrate available for VFA production. This mechanism is

Table 6: Energy partition of dairy cattle ration supplemented with micro mineral mix and protected PFAD and different level application.

Parameter	Micro-mineral dose	0% PFAD	1% PFAD	2% PFAD	3% PFAD	Mean (Dose)
Digestible energy (DE, MJ/kg DM)	Dose-0	10.86 ± 0.60	11.01 ± 0.59	10.76 ± 1.18	11.03 ± 0.11	10.91 ± 0.56
	Dose-1	10.87 ± 0.58	11.27 ± 0.97	11.62 ± 0.03	12.03 ± 0.35	11.45 ± 0.64
	Dose-2	10.89 ± 1.84	10.18 ± 1.82	9.95 ± 1.21	9.30 ± 0.02	10.08 ± 1.24
	Dose-3	11.74 ± 0.61	10.16 ± 1.80	10.37 ± 1.85	10.11 ± 1.19	10.60 ± 1.31
Mean (PFAD)		11.09 ± 0.89	10.66 ± 1.18	10.67 ± 1.15	10.62 ± 1.19
Methane energy (MJ/kg DM)	Dose-0	2.43 ± 0.05	1.66 ± 0.78	1.97 ± 0.50	2.24 ± 0.16	2.08 ± 0.47
	Dose-1	2.85 ± 0.08	2.09 ± 0.79	2.58 ± 0.09	2.33 ± 0.12	2.46 ± 0.43
	Dose-2	2.16 ± 0.35	2.35 ± 0.20	2.38 ± 0.62	2.64 ± 0.18	2.38 ± 0.34
	Dose-3	2.37 ± 0.92	1.45 ± 0.22	2.22 ± 0.58	2.31 ± 0.35	2.09 ± 0.59
Mean (PFAD)		2.46 ± 0.46	1.89 ± 0.57	2.29 ± 0.44	2.38 ± 0.24
Metabolizable energy (ME, MJ/kg DM)	Dose-0	8.91 ± 0.49	9.03 ± 0.48	8.82 ± 0.97	9.04 ± 0.09	8.95 ± 0.46
	Dose-1	8.92 ± 0.47	9.24 ± 0.80	9.53 ± 0.03	9.86 ± 0.29	9.39 ± 0.52
	Dose-2	8.93 ± 1.50	8.35 ± 1.50	8.16 ± 1.00	7.62 ± 0.02	8.27 ± 1.02
	Dose-3	9.63 ± 0.50	8.34 ± 1.47	8.50 ± 1.51	8.29 ± 0.97	8.69 ± 1.07
Mean (PFAD)		9.10 ± 0.73	8.74 ± 0.97	8.75 ± 0.95	8.71 ± 0.97
Net energy for lactation (NEl, MJ/kg DM)	Dose-0	4.51 ± 0.33	4.46 ± 0.32	4.20 ± 0.64	4.22 ± 0.06	4.34 ± 0.33
	Dose-1	4.51 ± 0.31	4.60 ± 0.53	4.66 ± 0.02	4.76 ± 0.19	4.63 ± 0.26
	Dose-2	4.52 ± 1.00	4.01 ± 0.99	3.76 ± 0.66	3.28 ± 0.01	3.89 ± 0.76
	Dose-3	4.98 ± 0.33	4.00 ± 0.97	3.99 ± 1.00	3.72 ± 0.64	4.17 ± 0.79
Mean (PFAD)		4.63 ± 0.48	4.27 ± 0.64	4.15 ± 0.63	3.99 ± 0.64

Notes: Values are means ± standard deviation (SD) of four replicates. PFAD = protected palm fatty acid distillate; micromineral = mixture of Se, Cu, and Zn. No significant (p > 0.05) effects of PFAD, mineral dose, or their interaction were observed on DE, ME, or Nel.

consistent with Ueda et al. (2003), who reported that dietary oil supplementation reduced ruminal fiber digestion and acetate production due to lower availability of fermentable carbohydrate substrate. As a result, DE measured in vitro primarily reflects fermentation-derived energy and does not capture the post-ruminal utilization of fat that occurs under in vivo conditions.

Methane energy losses (CH₄) ranged from 1.45 to 2.85 MJ/kg DM (equivalent to ~13–20% of DE), with numerically lower values observed at 1% PFAD compared with other treatments. Although this pattern was consistent with the earlier trend in methane estimates from the VFA data, the differences were not statistically significant, indicating that PFAD inclusion did not consistently affect methane energy loss. The methane losses recorded in this experiment were higher than those reported by Morgavi et al. (2023), most likely reflecting differences in the basal diet composition. In the present work, diets were formulated using tropical forages, which are typically rich in structural carbohydrates and classified as third-grade or lower relative forage value (RFV). In contrast, Morgavi et al. (2023) utilized temperate forages with prime or first-grade RFV, known to promote more efficient ruminal fermentation. The higher fiber content of tropical forages favors greater acetate and butyrate formation, both of which generate more hydrogen and consequently enhance methane production (Firkins and Mitchell, 2023; Wang et al., 2018; Yulistiani et al., 2017).

Metabolizable energy (ME) values ranged from 7.6 to 9.9 MJ/kg DM, while net energy for lactation (NEl) ranged from 3.3 to 4.8 MJ/kg DM. Numerically, 1% PFAD yielded slightly higher ME and NEl compared with 2–3% PFAD, though the differences were not statistically significant (p > 0.05). This suggests that the efficiency of converting feed energy into metabolizable and milk-usable energy was maintained across treatments. The ME values observed in this study are comparable to those reported previously (Jayasinghe et al., 2022; Olfaz et al., 2018).

Although numerical variation was observed in DE, CH₄, ME, and NEl, statistical analysis showed no significant treatment effects. These findings suggest that supplementation with micromineral mixtures and protected PFAD up to 3% of the diet dry matter did not compromise energy partitioning in vitro. The maintenance of energy recovery across treatments complements the earlier results on digestibility and fermentation, confirming that the supplementation strategy was well tolerated by the rumen microbial ecosystem.

CONCLUSION

Supplementation of dairy cattle diets with micromineral mixtures and protected PFAD resulted in no significant differences in rumen pH and protozoal populations, indicating a well-balanced microbial ecosystem. Volatile fatty acid profiles exhibited clear quadratic responses, where moderate PFAD inclusion (~1.5–2%) enhanced propionate production and reduced methane estimates, while higher levels (2–3%) sustained adequate acetate supply for milk fat synthesis. Dry matter and organic matter digestibility were unaffected, although gas production declined slightly, indicating a shift in fermentation pathways rather than reduced nutrient utilization. Energy parameters (DE, ME, NEl, CH₄) showed no significant differences, confirming that supplementation up to 3% PFAD did not compromise overall energy recovery. Collectively, the results suggest that moderate supplementation with protected PFAD (2–3% of diet DM), in combination with micromineral mixtures, can be integrated into dairy diets to enhance fermentation efficiency, mitigate methane emissions, and support sustainable milk production without disrupting rumen stability.

ACKNOWLEDGEMENT

The authors gratefully acknowledge financial support from the Indonesian Ministry of Education, Culture, Research, and Technology under the Dissertation Study Program scheme (Grant No. 23109/IT3.D10/PT.01.03/P/B/2025).

Novelty Statement

This work presents the first integrated evaluation of micromineral mixtures (Se–Cu–Zn) combined with protected PFAD as a synchronized rumen-modulating strategy. The study demonstrates that this dual-supplement system can shift fermentation toward a more energy-efficient propionate pathway, suppress methane formation, and maintain digestibility and microbial stability, revealing a new and promising approach for advancing sustainable dairy nutrition and mitigation of ruminal energy loss

AUTHORS CONTRIBUTION

RAG conducted experimental work, performed laboratory analyses, and drafted the initial manuscript. D conceptualized and supervised the study, provided guidance on methodology and data interpretation, and critically revised the manuscript. DP contributed to study design, data validation, and interpretation of results. DMF served as technical supervisor, providing oversight of experimental procedures and ensuring methodological accuracy. SM assisted in sample preparation, laboratory analyses, and data collection. All authors reviewed, edited, and approved the final version of the manuscript.

Ethical clearance

This study was reviewed and approved by the IPB University Animal Ethics Committee guidelines (No. 113/KEH/SKE/IX/2023) and followed the animal use procedures in accordance with the Guide for the Care and Use of Laboratory Animals.

Generative AI and AI-assisted technology statement

Generative AI tools were used only to assist in grammar refinement, formatting, and improvement of language clarity during manuscript review. No AI tools were used for data generation, statistical analysis, experimental interpretation, or formulation of scientific results. All research findings, data processing, and conclusions are fully the work of the authors.

Conflict of interest

We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.

REFERENCES

Al-Arif MA, Suwanti LT, Estoepangestie AS, Lamid M (2017). The nutrients contents, dry matter digestibility, organic matter digestibility, total digestible nutrient, and NH3 rumen production of three kinds of cattle feeding models. KnE. Life Sci., 3: 338. https://doi.org/10.18502/kls.v3i6.1142

Allen MS (2000). Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J. Dairy Sci., 83: 1598–1624. https://doi.org/10.3168/jds.S0022-0302(00)75030-2.

Alvarez-Hess PS, Williams SRO, Jacobs JL, Hannah MC, Beauchemin KA, Eckard RJ, Wales WJ, Morris GL, Moate PJ (2019). Effect of dietary fat supplementation on methane emissions from dairy cows fed wheat or corn. J. Dairy Sci., 102: 2714–2723. https://doi.org/10.3168/jds.2018-14721

Anzhany D, Toharmat T, Despal (2022). Ration to produce milk high in conjugated linoleic acid at smallholder dairy farm: An in vitro reconstruction. Am. J. Anim. Vet. Sci., 17: 130–138. https://doi.org/10.3844/ajavsp.2022.130.138

Bach A, Calsamiglia S, Stern MD (2005). Nitrogen metabolism in the rumen. J. Dairy Sci., 88: E9–E21. https://doi.org/10.3168/jds.S0022-0302(05)73133-7

Battini F, Agostini A, Tabaglio V, Amaducci S (2016). Environmental impacts of different dairy farming systems in the Po Valley. J. Clean Prod., 112: 91–102. https://doi.org/10.1016/j.jclepro.2015.09.062

Bauman DE, Griinari JM (2001). Regulation and nutritional manipulation of milk fat: Low-fat milk syndrome. Livest Prod. Sci., 70: 15–29. https://doi.org/10.1016/S0301-6226(01)00195-6

Beauchemin KA, Kreuzer M, O’Mara F, McAllister TA (2008). Nutritional management for enteric methane abatement: A review. Aust. J. Exp. Agric., 48: 21–27. https://doi.org/10.1071/EA07199

Behan AA, Chwen LT, Kaka U, Muhammad AI, Samsudin AA (2024). Effect of rumen-protected fat on in vitro rumen fermentation and apparent biohydrogenation of fatty acids. J. Indones. Trop. Anim. Agric., 49: 252–263. https://doi.org/10.14710/jitaa.49.3.252-263

Blaxter KL, Clapperton JL (1965). Prediction of the amount of methane produced by ruminants. Br. J. Nutr., 19: 511–522. https://doi.org/10.1079/BJN19650046

Calamari L, Petrera F, Abeni F, Bertin G (2011). Metabolic and hematological profiles in heat-stressed lactating dairy cows fed diets supplemented with different selenium sources and doses. Livest. Sci., 142: 128–137. https://doi.org/10.1016/j.livsci.2011.07.005

Despal D, Irmadani D, Permana IG, Zahera R, Nuraina N (2022). Effect of different unsaturated fatty acids sources on in vitro fermentability and digestibility of ration in dairy cattle. Online J. Anim. Feed Res., 12: 154–159. https://doi.org/10.51227/ojafr.2022.20

Despal D, Permana IG, Toharmat T, Amirroennas DE (2017). Pemberian pakan sapi perah. 1st ed. IPB Press, Bogor.

Despal D, Yulianti YI, Zahera R, Agustiyani I, Rosmalia A, Afnan IM, Zain M, Tanuwiria UH (2023). Comparison of chemical composition, in vitro digestibility, and NIRS in estimating in situ rumen degradable protein of tropical foliage. Trop. Anim. Sci. J., 46: 211–220. https://doi.org/10.5398/tasj.2023.46.2.211

Firkins JL, Mitchell KE (2023). Invited review: Rumen modifiers in today’s dairy rations. J. Dairy Sci., 106: 3053–3071. https://doi.org/10.3168/jds.2022-22644

Janssen PH (2010). Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim. Feed Sci. Technol., 160: 1–22. https://doi.org/10.1016/j.anifeedsci.2010.07.002

Jayanegara A, Anzhany D, Despal D (2021). Maggot oil as a feed supplement for reducing methanogenesis of rumen microbial culture in vitro. IOP Conf. Ser. Mater Sci. Eng., 1098: 042100. https://doi.org/10.1088/1757-899X/1098/4/042100

Jayasinghe P, Ramilan T, Donaghy DJ, Pembleton KG, Barber DG (2022). Comparison of nutritive values of tropical pasture species grown in different environments and implications for methane production: A meta-analysis. Animals, 12: 1806. https://doi.org/10.3390/ani12141806

Jenkins TC (1993). Lipid metabolism in the Rumen. J. Dairy Sci., 76:3851–3863. https://doi.org/10.3168/jds.S0022-0302(93)77727-9

Kjeldsen MH, Weisbjerg MR, Larsen M, Højberg O, Ohlsson C, Walker N, Hellwing ALF, Lund P (2024). Gas exchange, rumen hydrogen sinks, and nutrient digestibility and metabolism in lactating dairy cows fed 3-nitrooxypropanol and cracked rapeseed. J. Dairy Sci., 107: 2047–2065. https://doi.org/10.3168/jds.2023-23743

Li XP, Tan ZL, Jiao JZ, Long DL, Zhou CS, Yi KL, Liu CH, Kang JH, Wang M, Duan FH, Tang SX, He ZX, Han XF (2019). Supplementation with fat-coated rumen-protected glucose during the transition period enhances milk production and influences blood biochemical parameters of liver function and inflammation in dairy cows. Anim. Feed Sci. Technol., 252: 92–102. https://doi.org/10.1016/j.anifeedsci.2019.04.010

McDonald P, Greenhalgh JFD, Morgan C, Edwards R, Sinclair L, Wilkinson R (2021). Animal Nutrition. 8th ed. Pearson Education, Harlow, UK.

Menke KH, Raab L, Salewski A, Steingass H, Fritz D, Schneider W (1979). The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric Sci., 93: 217–222. https://doi.org/10.1017/S0021859600086305

Menke KH, Steingass H (1988). Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev., 28: 7–55.

Moate PJ, Chalupa W, Jenkins TC, Boston RC (2004). A model to describe ruminal metabolism and intestinal absorption of long chain fatty acids. Anim. Feed Sci. Technol., 112: 79–105. https://doi.org/10.1016/j.anifeedsci.2003.10.007

Morgavi DP, Cantalapiedra-Hijar G, Eugène M, Martin C, Noziere P, Popova M, Ortigues-Marty I, Muñoz-Tamayo R, Ungerfeld EM (2023). Review: Reducing enteric methane emissions improves energy metabolism in livestock: is the tenet right? Animal., 17: 100830. https://doi.org/10.1016/j.animal.2023.100830

Moss AR, Jouany JP, Newbold J (2000). Methane production by ruminants: Its contribution to global warming. Anim. Res., 49: 231–253. https://doi.org/10.1051/animres:2000119

Nasem (2021). Nutrient requirements of dairy cattle. 8th ed. National Academies Press.

Nocek JE, Socha MT, Tomlinson DJ (2006). The effect of trace mineral fortification level and source on performance of dairy cattle. J. Dairy Sci., 89: 2679–2693. https://doi.org/10.3168/jds.S0022-0302(06)72344-X

NRC (2001). Nutrient requirements of dairy cattle. 7th ed. National Academies Press. Washington, D.C.

Olfaz M, Kilic U, Boga M, Abdi AM (2018). Determination of the in vitro gas production and potential feed value of olive, mulberry and sour orange tree leaves. Open Life Sci., 13: 269–278. https://doi.org/10.1515/biol-2018-0033

Overton TR, Yasui T (2014). Practical applications of trace minerals for dairy cattle. J. Anim. Sci., 92: 416–426. https://doi.org/10.2527/jas.2013-7145

Patra AK (2013). The effect of dietary fats on methane emissions, and its other effects on digestibility, rumen fermentation and lactation performance in cattle: A meta-analysis. Livest Sci., 155: 244–254. https://doi.org/10.1016/j.livsci.2013.05.023

Pereira AM, de Lurdes Nunes Enes Dapkevicius M, Borba AES (2022). Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission? Anim. Microbiome, 4: 1–12. https://doi.org/10.1186/s42523-021-00153-w

Rabiee AR, Breinhild K, Scott W, Golder HM, Block E, Lean IJ (2012). Effect of fat additions to diets of dairy cattle on milk production and components: A meta-analysis and meta-regression. J. Dairy Sci., 95: 3225–3247. https://doi.org/10.3168/jds.2011-4895

Razzaghi A, Leskinen H, Ahvenjärvi S, Aro H, Bayat AR (2022). Energy utilization and milk fat responses to rapeseed oil when fed to lactating dairy cows receiving different dietary forage to concentrate ratio. Anim. Feed. Sci. Technol., 293: 115454. https://doi.org/10.1016/j.anifeedsci.2022.115454

Riestanti LU, Despal, Oktavianti BP, Toharmat T, Retnani Y (2023). Effects of Ca-soap protected vegetable oil in dairy ration on rumen fermentability and in vitro digestibility. IOP Conf. Ser. Earth. Environ. Sci., 1168: 012023. https://doi.org/10.1088/1755-1315/1168/1/012023

Riestanti LU, Despal, Retnani Y (2021). Supplementation of prill fat derived from palm oil on nutrient digestibility and dairy cow performance. Am. J. Anim. Vet. Sci., 16: 172–184. https://doi.org/10.3844/ajavsp.2021.172.184

Riestanti LU, Despal, Retnani Y, Andarwulan N (2024). Unsaturated fat supplemented in the form of Ca-soap and prill fat in dairy cattle ration: In vitro study. BIO Web. Conf., 123: 01016. https://doi.org/10.1051/bioconf/202412301016

Rosmalia A, Permana IG, Despal D (2022). Synchronization of rumen degradable protein with non-fiber carbohydrate on microbial protein synthesis and dairy ration digestibility. Vet. World, 15: 252–261. https://doi.org/10.14202/vetworld.2022.252-261

Salas H, Castillejos L, López-Suárez M, Ferret A (2019). In vitro digestibility, in situ degradability, rumen fermentation and nitrogen metabolism of camelina co-products for beef cattle using a dual-flow continuous culture system. Animals, 9: 1079. https://doi.org/10.3390/ani9121079

Seymour WM, Campbell DR, Johnson ZB (2005). Relationships between rumen volatile fatty acid concentrations and milk production in dairy cows: A literature review. Anim. Feed Sci. Technol., 119: 155–169. https://doi.org/10.1016/j.anifeedsci.2004.10.001

Spears JW (2003). Comparative trace element nutrition: Trace mineral bioavailability in ruminants. J. Nutr., 133: 1506S–1509S. https://doi.org/10.1093/jn/133.5.1506S

Steel RGD, Torrie JH (1980). Principles and procedures of statistics: A biometrical approach, 2nd ed. McGraw-Hill, New York.

Sun X, Wang Y, Ma X, Li S (2022). Producing natural functional and low-carbon milk by regulating the diet of the cattle. The fatty acid associated rumen fermentation, biohydrogenation, and microorganism response. Front. Nutr., 9: 955846. https://doi.org/10.3389/fnut.2022.955846

Suttle NF (2010). Mineral nutrition of livestock. 4th ed. CABI, Wallingford, UK. https://doi.org/10.1079/9781845934729.0000

Sutton JD, Dhanoa MS, Morant SV, France J, Napper DJ, Schuller E (2003). Rates of production of acetate, propionate, and butyrate in the rumen of lactating dairy cows given normal and low-roughage diets. J. Dairy Sci., 86: 3620–3633. https://doi.org/10.3168/jds.S0022-0302(03)73968-X

Tilley JMA, Terry RA (1963). A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl Soc., 18: 104–111. https://doi.org/10.1111/j.1365-2494.1963.tb00335.x

Uchida K, Mandebvu P, Ballard CS, Sniffen CJ, Carter MP (2001). Effect of feeding a combination of zinc, manganese and copper amino acid complexes, and cobalt glucoheptonate on performance of early lactation high producing dairy cows. Anim. Feed Sci. Technol., 93: 193–203. https://doi.org/10.1016/S0377-8401(01)00279-6

Ueda K, Ferlay A, Chabrot J, Loor JJ, Chilliard Y, Doreau M. (2003). Effect of linseed oil supplementation on ruminal digestion in dairy cows fed diets with different forage:concentrate ratios. Journal of Dairy Science, 86 (12): 3999–4007. https://doi.org/10.3168/jds.S0022-0302(03)74011-9

Vigh A, Criste AD, Corcionivoschi N, Gerard C (2023). Rumen solubility of copper, manganese and zinc and the potential link between the source and rumen function: A systematic review. Agriculture, 13: 2198. https://doi.org/10.3390/agriculture13122198

Waghorn GC, Hegarty RS (2011). Lowering ruminant methane emissions through improved feed conversion efficiency. Anim. Feed Sci. Technol., 166–167: 291–301. https://doi.org/10.1016/j.anifeedsci.2011.04.019

Wang J, Liu T, Xu J, Liu H, Sun K, Fan H, Zhu S, Ran H, Zheng C (2025). Mannan oligosaccharides reduce the carbon footprint by decreasing methane emission and nitrogen excretion in Xiangdong black goats. Anim. Feed Sci. Technol., 328: 116464. https://doi.org/10.1016/j.anifeedsci.2025.116464

Wang K, Nan X, Chu K, Tong J, Yang L, Zheng S, Zhao G, Jiang L, Xiong B (2018). Shifts of hydrogen metabolism from methanogenesis to propionate production in response to replacement of forage fiber with non-forage fiber sources in diets in vitro. Front Microbiol., 9: 2764. https://doi.org/10.3389/fmicb.2018.02764

Yang Z, Liu S, Xie T, Wang Q, Wang Z, Yang H, Li S, Wang W (2022). Effect of unsaturated fatty acid ratio in vitro on rumen fermentation, methane concentration, and microbial profile. Fermentation, 8: 540. https://doi.org/10.3390/fermentation8100540

Yu Y, Fu R, Jin C, Gao H, Han L, Fu B, Qi M, Li Q, Suo Z, Leng J (2024). Regulation of milk fat synthesis: Key genes and microbial functions. Microorganisms, 12: 2302. https://doi.org/10.3390/microorganisms12112302

Yulistiani D, Puastuti W, Haryanto B, Purnomoadi A, Kurihara M, Thalib A (2017). Complete rumen modifier supplementation in corn cob silage basal diet of lamb reduces methane emission. Indones. J. Agric. Sci., 18: 33–42. https://doi.org/10.21082/ijas.v18n1.2017.p33-42

Zahera R, Pratiwi MI, Fitri A, Koike S, Permana IG, Despal (2024). Coconut fatty acid distillate Ca-soap with different calcium sources: Effects of varied proportions of protected and unprotected fat supplementation in dairy rations. Dairy, 5: 542–554. https://doi.org/10.3390/dairy5030041