JAHP_14_1_305-312

INTRODUCTION

Recent consumers are increasingly demanding healthier animal products, emphasizing improved nutritional profiles and reduced health risks. Consequently, meat producers and the food industry are under growing pressure to deliver functional, health-promoting products (Espinales et al., 2024). Ruminant-derived foods, such as meat and milk, are major dietary components globally; enhancing their fatty acid composition, especially by increasing beneficial lipids like polyunsaturated fatty acids (PUFAs), offers a clear opportunity to meet these health-driven market trends (Ponnampalam et al., 2024). Dietary manipulation remains one of the most effective tools to positively modify the fatty acid profile of ruminant products (Savoini et al., 2016). Vegetable oils derived from seeds such as soybean, flax (linseed), and sunflower are commonly used in ruminant diets, owing to their high levels of PUFAs and monounsaturated fatty acids (MUFAs) (Castro et al., 2019). Sunflower seed oil, for instance, is rich in PUFAs (51.41%) and MUFAs (41.69%), while containing relatively low concentrations of saturated fatty acids (SFAs; 6.90%). Supplementation of vegetable oils in ruminant diets not only modifies the fatty acid composition of products but has also been associated with reduced methane production, increased propionate formation, and maintenance of feed digestibility, thereby enhancing feed efficiency (Consolo et al., 2014; Petraru et al., 2021; Correa et al., 2022).

Despite these advantages, dietary lipids undergo extensive modification in the rumen. Free PUFAs are toxic to ruminal microorganisms and are rapidly hydrogenated by bacteria such as Butyrivibrio spp., resulting predominantly in stearic acid (C18:0) and, to a lesser extent, vaccenic acid (C18:1) (Jayanegara et al., 2011; Nesic et al., 2024). Consequently, unprotected oils yield little PUFA available for absorption in the small intestine. Although rumen-protected fats can bypass microbial metabolism, chemical protection strategies such as formaldehyde and calcium salts are limited in practice due to toxicity concerns (Harvatine and Allen, 2006; Palmquist and Jenkins, 2017).

Plant secondary metabolites, particularly polyphenols, have emerged as natural alternatives to manipulate ruminal lipid metabolism. Polyphenolic compounds can alter microbial cell morphology and disrupt enzymatic processes, thereby reducing biohydrogenation and methanogenesis (Jafari et al., 2016; Toral et al., 2018; Vasta et al., 2019). In addition, polyphenols enhance oxidative stability of dietary lipids and increase the proportion of beneficial fatty acids in ruminant products (Toral et al., 2018). Their antimicrobial activity has also been linked to induction of reactive oxygen species in bacterial cells, leading to impaired microbial growth or cell death (Efenberger-Szmechtyk et al., 2021).

Marine red seaweeds such as Gelidium sp., which are abundant in Indonesian waters, represent a novel source of polyphenolic compounds. Gelidium contains carotenoids, polyphenols, phycobiliproteins, and mycosporine-like amino acids, and extracts of this genus have been shown to exhibit antioxidant, antimicrobial, and anti-enzymatic activities (Castejon et al., 2021; Meinita et al., 2023; McReynolds et al., 2023). For example, Gelidium sesquipedale ethanol extracts contain high concentrations of polyphenols (94.18 ± 0.02 mg GAE/g dry matter), while dichloromethane and hexane extracts yield lower amounts (Taouam et al., 2024). In addition, pigments such as phycoerythrin and catechins contribute antioxidant and antibacterial properties (Leksono et al., 2018).

Based on these characteristics, Gelidium sp. may represent a natural source of bioactive compounds capable of modulating ruminal lipid metabolism and overcoming the limitations of chemical fat protection strategies. However, limited information is available regarding the use of Gelidium extracts in ruminant diets, particularly in high–unsaturated fatty acid feeding systems. Therefore, the objective of this study was to evaluate the effects of Gelidium sp. extract supplementation on nutrient digestibility, gas production, and the ruminal fatty acid profile in vitro. We hypothesized that supplementation with Gelidium sp. extract would reduce ruminal biohydrogenation, increase PUFA preservation, and improve fermentation efficiency.

MATERIALS AND METHODS

The experiment was conducted in the Laboratory of Tropical Animal Research Center and Laboratory of Nutritional Biochemistry, Faculty of Animal Science, Universitas Gadjah Mada. The experimental materials included Gelidium sp. was collected from Sundak Beach, Gunungkidul, Indonesia, as a source of phenolic compounds. Rumen fluid was obtained from fistulated adult female Balinese cattle and served as the inoculum for in vitro fermentation. The experiment was approved by Ethical Clearance Committee in the Integrated Laboratory for Research and Testing Universitas Gadjah Mada (00007/III/UN1/LPPT/EC/2024).

Experimental design

A completely randomized design was used for in vitro incubation. The experiment included three independent runs of 48-h in vitro batch cultures to measure cumulative gas production. In vitro nutrient digestibility was determined using a two-stage culture system consisting of stage I (48 h) and stage II (96 h). Each culture included four dietary treatments with two replicates per treatment. The dietary treatments consisted of increasing Gelidium sp. extract supplementation levels (0, 1, 2 and 3% of diet DM or equal to 0, 90.4, 190.8, and 271.2 mg phenol/100 g diet DM, respectively), defined as Control, GE1, GE2, and GE3, respectively. The ingredient and nutrient composition of the experimental diets are presented in Tables 1 and 2.

Table 1: Diet composition.

Feedstuff composition	Treatment
Feedstuff composition	GE0	GE1	GE2	GE3
Elephant grass (%)	60	60	60	60
Wheat pollard (%)	35	35	35	35
Soybean meal (%)	3	3	3	3
Sunflower oil (%)	2	2	2	2
Gelidium sp. Extract (%)	0	1	2	3

GE0= Control/0% Gelidium sp. Extract; GE1= Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2= Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

Extraction of Gelidium sp.

Fresh Gelidium sp. were oven-dried at 55 °C for 3 d and ground to pass through a 1-mm screen. The powder was macerated in 98% methanol at a ratio of 1:5 (w/v) for 3 d at room temperature. The extract was filtered, frozen, and lyophilized using a freeze dryer (Lyovapor L-200, Büchi, Switzerland). Total phenol concentration of the extract was determined spectrophotometrically (UV-vis, 725 nm) according to Singleton and Rossi (1965).

Table 2: Nutrient composition of the diet.

Nutrient composition	Treatment
Nutrient composition	GE0	GE1	GE2	GE3
Dry matter (%)1	91.74	91.87	91.65	91.64
Organic matter (%)1	86.49	86.33	86.16	85.91
Crude protein (%)1	10.59	10.74	11.37	11.99
Ether extract (%)1	4.84	5.11	5.13	5.71
Crude fiber (%)	21.25	20.13	20.04	19.44
Ash (%)1	13.51	13.67	13.84	14.09
Nitrogen-free extract (%)2	49.80	50.34	49.62	48.46
TDN (%)2	66.20	67.42	67.57	68.44

Source: 1Proximate analysis at Laboratory of Nutritional Biochemistry. Faculty of Animal Science. Universitas Gadjah Mada (2024). 2NFE and TDN was measured using Hartadi (2005) formula. GE0 = Control/0% Gelidium sp. Extract; GE1 = Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2 = Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

In vitro incubation

In vitro gas production was conducted according to Menke and Steingass (1988). Rumen fluid was collected before the morning feeding, filtered through four layers of cheesecloth, and maintained at 39 °C in insulated flasks. The inoculum was prepared by mixing rumen fluid and buffer (1:2, v/v) while continuously flushing with CO₂ to maintain anaerobic conditions. Substrates (300 mg DM) were placed on 100-mL glass syringes, and 30 mL of inoculum-buffer mixture was added. Syringes were incubated at 39 °C for 48 h and manually agitated every 8 h. Gas volume was recorded at 0, 2, 4, 6, 12, 24, 36, and 48 h. At 48 h, gas was collected in a vacuum container for methane analysis by gas chromatography. Curve fitting was performed using the FitCurve program (Chen, 1996). At the end of incubation, syringe contents were filtered through pre-weighed crucibles with glass wool for determination of in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD). The filtrate was used to measure CMCase (Halliwell and Lovelady, 1981), and protease enzyme activity (Bergmeyer et al., 1983), protozoa population (Diaz et al., 1993), and fatty acid profile and methane by gas chromatography.

In vitro digestibility

Nutrient digestibility was determined using the two-stage in vitro method of Tilley and Terry (1963). The fermentation medium consisted of rumen fluid and McDougall’s buffer (1:4, v/v) under continuous CO₂ flushing. For stage I, 25 mL of inoculum-buffer mixture was incubated with 250 mg DM substrate (50-mL tubes) for IVDMD and IVOMD determination, while 50 mL inoculum-buffer mixture was incubated with 500 mg DM substrate (100-mL tubes) for in vitro crude protein digestibility (IVCPD) and crude fiber digestibility (IVCFD). Tubes were sealed with rubber stoppers, incubated at 39 °C for 48 and 96 h, and shaken every 8 h. For stage II, after 48 h incubation, samples were acidified with HCl and pepsin (3:1) to reduce pH to 3.5, simulating abomasal conditions, and further incubated for 96 h. Residues were filtered through pre-weighed crucibles with glass wool to determine IVDMD, IVOMD, and IVCFD, whereas IVCPD was measured on filter-paper residues according to AOAC (2005).

Fatty acid profile analysis

After 48 h incubation, 10 mL of rumen fluid was collected from each syringe and stored at –20 °C. Lipids were extracted using a chloroform–methanol method (2:1 v/v), and fatty acid methyl esters (FAMEs) were prepared via base- and acid-catalyzed transesterification. Fatty acid composition was determined using a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu Corp., Kyoto, Japan) equipped with a flame ionization detector. Separation was performed on a SP-2560 capillary column (100 m × 0.25 mm i.d., 0.20 μm film thickness; Supelco, Bellefonte, PA, USA) designed for cis/trans isomer resolution. The oven initial temperature program was: 140 °C (5 min hold), ramped at 4 °C/min to 240 °C, final hold 15 min. Helium was used as the carrier gas (1.0 mL/min). Injector and detector temperatures were maintained at 250 °C. Individual fatty acids were identified by retention time relative to external standards (Supelco 37 Component FAME Mix), and results were expressed as a percentage of total fatty acids (Pecka-Kiełb et al., 2023).

Statistical analysis

Data were analyzed by one-way ANOVA in a completely randomized design using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). When significant treatment effects were detected, means were separated using Duncan’s multiple range test (DMRT). Statistical significance was declared at P < 0.05.

RESULTS

Supplementation of Gelidium sp. extract significantly affected protease activity and protozoa population but had no effect on CMCase activity (Table 3). Protease activity decreased at the highest inclusion level (GE3; 7.07 U/g) compared with the control and lower inclusion levels (GE0–GE2; P = 0.05). In contrast, CMCase activity was not influenced by supplementation and remained similar across treatments (8.74–9.22 U/g; P= 0.12). Protozoa population was progressively reduced with increasing levels of Gelidium sp. extract, with the lowest value observed at GE3 (3.68 × 10⁴/mL) compared with GE0 (4.81 × 10⁴/mL; P = 0.011).

Table 3: Effect of feed supplementation with Gelidium sp. extract on protease and CMCase activity and protozoa population.

Variable	Treatment
Variable	GE0	GE1	GE2	GE3	SEM	P Value
Protease (U/g)	7.56b	7.54b	7.54b	7.07a	0.08	0.050
CMCase (U/g)	8.74	9.07	9.09	9.22	0.20	0.120
Protozoa (×104)	4.81b	4.58b	4.44b	3.68a	0.15	0.011

a.b: Different superscripts within the same row indicate significant differences (P<0.05). SEM: standard error of means; GE0 = Control/0% Gelidium sp. Extract; GE1 = Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2 = Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

Table 4: Nutrient digestibility of feed supplemented with various levels of Gelidium sp. extract.

Variable	Treatment
Variable	GE0	GE1	GE2	GE3	SEM	P value
Rumen digestibility
IVDMD (%)	56.90	56.97	57.24	57.56	0.66	0.989
IVOMD (%)	55.97	56.68	56.18	57.67	0.46	0.633
IVCPD (%)	46.87b	47.83b	44.88ab	39.23a	1.26	0.037
IVCFD (%)	49.00	48.72	46.27	45.92	0.58	0.096
Total digestibility
IVDMD (%)	71.44	71.27	72.50	72.73	0.94	0.947
IVOMD (%)	66.99	67.56	67.91	69.03	0.93	0.918
IVCPD (%)	63.54a	66.47a	64.19a	74.67b	1.52	0.010
IVCFD (%)	52.62	52.68	51.41	51.23	0.40	0.466

a.b: Different superscripts within the same row indicate significant differences (P<0.05). SEM: standard error of means. IVDMD: in vitro dry matter digestibility; IVOMD: in vitro organic matter digestibility; IVCPD: in vitro crude protein digestibility; IVCFD: in vitro crude fiber digestibility. GE0 = Control/0% Gelidium sp. Extract; GE1 = Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2 = Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

Supplementation of Gelidium sp. extract did not affect ruminal or total dry matter (IVDMD), organic matter (IVOMD), or crude fiber digestibility (IVCFD; P > 0.05; Table 4). Ruminal crude protein digestibility (IVCPD) was decreased at the highest inclusion level (GE3; 39.23%) compared with GE0 and GE1 (P < 0.05), whereas GE2 did not differ from either group. In contrast, total IVCPD was significantly greater at GE3 (74.67%) than in all other treatments (P = 0.010). These findings indicate that Gelidium sp. extract reduced protein degradation in the rumen while enhancing post-ruminal protein availability.

The effects of Gelidium sp. extract supplementation on gas production and fermentation kinetics are shown in Table 5. Total gas production and kinetic parameters (fraction a, fraction b, a+b, and c) were not affected by treatments (P > 0.05). In contrast, methane production was significantly reduced at GE3 (8.38 mL/300 mg DM) compared with the other treatments (9.99–10.53 mL/300 mg DM; P = 0.019). The effects of Gelidium sp. extract supplementation on the ruminal fatty acid profile are shown in Table 6. Increasing levels of extract supplementation decreased (P < 0.01) the concentrations of several saturated fatty acids (SFA), including stearic acid (C18:0). Conversely, oleic acid (C18:1n-9c), linoleic acid (C18:2n-6c), and total unsaturated fatty acids (UFA), including both MUFA and PUFA, increased significantly (P < 0.01).

Table 5: Gas production and kinetics of feed supplemented with Gelidium sp. extract.

Variable	Treatment
Variable	GE0	GE1	GE2	GE3	SEM	P value
Total gas production (ml/300 mgDM)	76.28	76.77	76.98	76.57	0.53	0.965
Methane emission (ml/300 mgDM)	10.53a	10.16a	9.99a	8.38b	0.30	0.019
a (ml/300 mgDM)	3.33	3.98	3.93	3.40	0.13	0.145
b (ml/300 mgDM)	74.33	75.44	77.18	77.02	0.53	0.174
a+b (ml/300 mgDM)	77.66	79.42	81.11	80.42	0.51	0.063
c (ml/h)	0.06	0.06	0.06	0.06	0.00	0.874

a.b: Different superscripts within the same row indicate significant differences (P<0.05). SEM: standard error of means. GE0 = Control/0% Gelidium sp. Extract; GE1 = Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2 = Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

DISCUSSION

Supplementation of Gelidium sp. extract modified ruminal microbial activity, protein metabolism, gas emissions, and fatty acid profiles without impairing overall feed digestibility. The observed reduction in protease activity at the highest supplementation level (GE3) is consistent with the inhibitory effects of phenolic compounds on microbial enzymes. Tannins and related phenolics can bind to proteins through hydrophobic and hydrogen bonds, forming complexes that inhibit proteolytic activity and reduce substrate accessibility (Santos-Buelga and Freitas, 2009; Setiawan et al., 2023; Song et al., 2020). These complexes are resistant to hydrolysis at ruminal pH but dissociate in the acidic abomasum, releasing protein for post-ruminal digestion (Getachew et al., 2000; Jayanegara and Sofyan, 2008). The lack of effect on CMCase activity indicates that cellulolytic enzymes were less sensitive to

Table 6: Rumen fatty acid profile following feed supplementation with Gelidium sp. extract in several rations.

Fatty acid (% of Total Fatty Acids)	Treatment
Fatty acid (% of Total Fatty Acids)	GE0	GE1	GE2	GE3	SEM	P value
Laurate (C12:0)	3.11d	3.08c	3.05b	3.03a	0.01	0.000
Myristate (C14:0)	3.94d	3.91c	3.88b	3.85 a	0.01	0.000
Pentadecanoate (C15:0)	0.98 d	0.95c	0.93b	0.90a	0.01	0.000
Palmitate (C16:0)	0.15b	0.15b	0.14a	0.13a	0.00	0.010
Heptadecanoate (C17:0)	3.97d	3.95c	3.91b	3.89a	0.01	0.000
Stearate (C18:0)	0.49d	0.47c	0.46b	0.38a	0.01	0.000
Arachidate (C20:0)	8.07c	8.06c	8.03b	7.98a	0.01	0.000
Tricosanoate (C23:0)	7.08c	7.07c	7.03b	7.00a	0.01	0.000
Lignocerate (C24:0)	0.84d	0.82c	0.80b	0.78a	0.01	0.000
Myristoleic acid methyl ester (C15:1)	0.11	0.11	0.11	0.11	0.00	0.703
Cis-10 Pentadecanoate (C15:1)	0.11	0.11	0.11	0.11	0.00	0.695
Palmitoleate (C16:1)	27.26a.b	27.25a	27.28b.c	27.29c	0.01	0.019
Cis-9 Oleate (C18:1n-9c)	12.89a	12.90a	12.93b	12.94b	0.01	0.000
Nervonate (C24:1)	0.67a	0.70b	0.73c	0.77d	0.01	0.000
Cis-4.7.10.13.16.19-docosahexaenoate (C22:6n-3)	0.77d	0.80c	0.82b	0.84a	0.01	0.000
Linolelaidate (C18:2n-6t)	29.09a	29.15b	29.13b	29.28c	0.02	0.000
Linoleate (C18:2n-6c)	0.11a	0.17b	0.31c	0.40d	0.03	0.000
Gamma-linolenic acid methyl (C18:3n-6)	0.11	0.11	0.11	0.11	0.00	0.674
Linolenate (C18:3n-3)	0.11	0.11	0.11	0.11	0.00	0.791
Cis-11.14-eicosadienoate (C20:2)	0.11	0.11	0.11	0.11	0.00	0.441
SFA	28.63d	28.46c	28.21b	27.91a	0.08	0.000
UFA	71.37a	71.54b	71.79c	72.09d	0.08	0.000
MUFA	41.05a	41.09b	41.18c	41.14d	0.02	0.000
PUFA	30.31a	30.46b	30.61c	30.86d	0.06	0.000

a.b.c.d: Different superscripts within the same row indicate significant differences (P<0.05). SEM: standard error of means; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; UFA: unsaturated fatty acids. GE0 = Control/0% Gelidium sp. Extract; GE1 = Supplementation of 1% of Gelidium sp. extract (diet DM based); GE2 = Supplementation of 2% of Gelidium sp. extract (diet DM based); and GE3 = Supplementation of 3% of Gelidium sp. extract (diet DM based).

the levels and types of phenolics in Gelidium, a finding consistent with the unchanged fiber digestibility.

The decrease in protozoa population observed with increasing supplementation further supports the antimicrobial effects of phenolic compounds. Protozoa contribute to ruminal proteolysis and fiber degradation through the secretion of hydrolases (Bera-Maillet et al., 2005), and their reduction is often associated with lower methane emissions due to their symbiotic relationship with methanogens (Bhatta et al., 2009; Choudhory et al., 2015). Phenolic compounds, particularly tannins and saponins, disrupt protozoal membranes and impair nutrient availability, leading to population declines (Francis et al., 2002; Abarghuei et al., 2021). These findings suggest that Gelidium phenolics selectively suppressed proteolytic and protozoal activity while sparing cellulolytic microbes, resulting in altered nitrogen metabolism without compromising fiber digestion.

The effects on nutrient digestibility align with the microbial changes. Ruminal crude protein digestibility decreased at GE3, likely due to protein–phenol complex formation and reduced protease activity. However, total crude protein digestibility increased, reflecting greater bypass of dietary protein to the post-ruminal tract, where it could be digested under abomasal conditions. This shift is advantageous, as it improves amino acid supply to the host animal and enhances nitrogen utilization efficiency (Wang et al., 2018). In contrast, digestibility of DM, OM, and fiber was unaffected, in agreement with previous reports showing that moderate tannin supplementation does not impair overall digestibility (Maulana et al., 2022; Jayanegara et al., 2009). The consistency between digestibility outcomes and CMCase activity further indicates that fibrolytic activity was maintained in the presence of Gelidium phenolics.

Gas production kinetics were similarly unaffected, demonstrating that supplementation did not alter carbohydrate fermentation dynamics. Parameters including soluble (a), potentially degradable (b), and total degradable (a+b) fractions, as well as the fractional degradation rate (c), were stable across treatments. This stability suggests that phenolics did not interfere with amylolytic or fibrolytic pathways responsible for VFA and gas production (Groot et al., 1996; Tang et al., 2006). However, methane production decreased significantly at GE3, a response consistent with both the reduction in protozoa population and the hydrogen-shifting properties of phenolics. Tannins can reduce methane either by limiting hydrogen release from fiber fermentation or by inhibiting methanogens directly (Tevendale et al., 2005). Moreover, phenolics may redirect metabolic hydrogen toward propionate synthesis, reducing its availability for methanogenesis (Ungerfeld, 2020; Moss et al., 2000).

The most pronounced effect of Gelidium sp. extract was observed in the ruminal fatty acid profile. Supplementation decreased stearic acid concentration while increasing oleic, linoleic, and total unsaturated fatty acids. These shifts reflect inhibition of complete biohydrogenation by phenolic compounds. Biohydrogenating bacteria such as Butyrivibrio fibrisolvens and Butyrivibrio proteoclasticus normally convert linoleic acid into vaccenic acid and subsequently into stearic acid (Paillard et al., 2007; Vasta et al., 2019). Phenolics likely suppressed these pathways by inhibiting lipase activity and interfering with isomerization and reduction steps (Morales and Ungerfeld, 2015; Makmur et al., 2020). Similar findings have been reported where phenolic-rich extracts enhanced the accumulation of conjugated fatty acids (CLA) and vaccenic acid while lowering C18:0 (Mannelli et al., 2018). Importantly, reduced biohydrogenation not only preserved beneficial unsaturated fatty acids but also coincided with lower methane production, suggesting a shared mechanism whereby phenolics serve as hydrogen sinks (Jayanegara et al., 2011; Lourenco et al., 2010).

In summary, the results demonstrate that Gelidium sp. extract supplementation can beneficially modulate ruminal fermentation by reducing proteolysis and protozoa populations, increasing post-ruminal protein availability, decreasing methane emissions, and shifting lipid metabolism toward greater preservation of unsaturated fatty acids. These outcomes support the potential of Gelidium extracts as a natural strategy to improve the nutritional quality of ruminant products while mitigating environmental impacts.

CONCLUSION

Supplementation with Gelidium sp. extract modified ruminal fermentation without impairing overall nutrient digestibility. The extract reduced protease activity and protozoa populations, thereby decreasing ruminal crude protein degradation and enhancing post-ruminal protein availability. Methane emissions were lowered at the highest inclusion level, consistent with shifts in hydrogen utilization and reduced protozoal activity. In addition, supplementation altered the ruminal fatty acid profile by suppressing complete biohydrogenation, leading to reduced stearic acid and increased concentrations of beneficial unsaturated fatty acids. These findings indicate that Gelidium sp. extract has potential as a natural feed additive to improve protein utilization efficiency, enhance the nutritional quality of ruminant products, and contribute to methane mitigation strategies.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Laboratory of Tropical Animal Research Center and Laboratory of Nutritional Biochemistry, Faculty of Animal Science, Universitas Gadjah Mada, for providing facilities and technical support during this study. This work was supported in part by the Faculty of Animal Science under the Research Grant Scheme 2255/UN1/PT/PT.01.03/2024.

Novelty Statement

This study provides novel mechanistic evidence that Gelidium sp. extract acts as a selective rumen modulator, improving protein utilization efficiency and lipid metabolism (including a targeted inhibition of bio-hydrogenation) without compromising nutrient digestibility.

AUTHOR’S CONTRIBUTION

Muhlisin conceptualized the study, administered the project, acquired funding, supervised the research, and contributed to manuscript writing, review, and editing. Salma Dani Hidayati conducted the investigation, performed laboratory analyses, curated and validated the data, and drafted parts of research reports. Chusnul Hanim supervised sample preparation, contributed to data analysis and interpretation, and reviewed the manuscript. All authors read and approved the final version of the manuscript.

Generative AI and AI-assisted technology statement

Generative AI and AI-assisted technologies were used to support the editing and refinement of scientific language, improve clarity and conciseness, and ensure consistency of terminology and style throughout the manuscript. All scientific concepts, data interpretation, and conclusions were developed, verified, and approved by the authors, who take full responsibility for the content of this work.

Conflict of interest

The authors have declared no conflict of interest.

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