AAVS_13_10_2188-2194

Introduction

The success of increasing sheep populations largely depends on the quality of feed, particularly its nutritional value, which is critical for maintaining animal health, growth, and reproduction (Patience et al., 2015). One of the major challenges in sheep nutrition is the limited availability and high cost of energy-rich feed ingredients. To address this issue, dietary energy can be enhanced by supplementing with fats, while protein requirements may be met through the inclusion of protected proteins that resist degradation in the rumen (Pramono et al., 2013).

Lemuru fish oil, derived from the Sardinella longiceps species, presents a promising and cost-effective alternative energy source due to its high content of unsaturated fatty acids (Ibrahim et al., 2018). This oil, a by-product of the canning industry in Banyuwangi Regency, contains 38.15% saturated fatty acids (SFA), 32.18% monounsaturated fatty acids (MUFA), and 28.44% polyunsaturated fatty acids (PUFA), including substantial amounts of 14.36% eicosapentaenoic acid (EPA) and 4.60% docosahexaenoic acid (DHA) (Suseno et al., 2014). Supplementing ruminant diets with PUFA-rich fish oil can improve energy utilization, support protein synthesis, and promote better overall performance, as essential fatty acids cannot be synthesized endogenously by ruminants (Hatami et al., 2022; Hernandez-Casner et al., 2017).

However, incorporating high levels of unsaturated fat into ruminant diets must be approached cautiously, as it can disrupt rumen fermentation, inhibit fiber digestion, and impair microbial activity (Dewi et al., 2021). To overcome these challenges, protective treatments are necessary to stabilize the fat and preserve rumen function. One effective strategy is the use of calcium hydroxide (Ca(OH)₂), which helps prevent lipid oxidation, maintains fatty acid integrity, and supports normal rumen activity (Dewi et al., 2022). The protective mechanism involves the reaction between Ca(OH)₂ and free fatty acids to form calcium soaps, which are insoluble in the rumen’s aqueous environment. These calcium soaps are less susceptible to microbial lipolysis and biohydrogenation, allowing unsaturated fatty acids to bypass rumen fermentation and reach the small intestine for absorption.

Calcium hydroxide treatment, through a saponification process, enables safe and effective inclusion of fish oil in ruminant rations without compromising rumen microbial ecosystems (Pramono et al., 2018; Pena et al., 2023). Studies have demonstrated that protected fish oil improves digestibility of organic and dry matter, enhances animal health, and contributes to better meat quality (Sudibya et al., 2016; Dewi et al., 2022). Therefore, this study aims to evaluate the impact of calcium hydroxide-protected Lemuru fish oil on in vitro digestibility in sheep.

Materials and Methods

Study location and research design

This research was conducted at the Animal Nutrition and Feed Laboratory, Faculty of Animal Husbandry, Universitas Brawijaya, for the in vitro analysis. Rumen fluid was obtained from fistulated sheep at the Field Laboratory of Animal Nutrition and Feed, Faculty of Animal Husbandry, Universitas Brawijaya. The experimental design used in this study was a laboratory-based trial employing a 2 × 5 factorial randomized block design (RBD) with three replicates per treatment, as shown in Figure 1. The first factor was the type of fish oil treatment, comprising unprotected Lemuru fish oil (MUP) and Lemuru fish oil protected with calcium hydroxide (MP). The second factor was the level of Lemuru fish oil inclusion in the diet, which included 0% (no supplementation), 5%, 7.5%, 10%, and 12.5%. The feed used in this study was formulated to meet the standard nutritional requirements of sheep, with its composition and nutrient profile shown in Table 1.

Preparation of calcium-protected lemuru fish oil

Lemuru fish oil was obtained as a byproduct from the sardine canning industry at PT Sumber Yalasamudra, Muncar, Banyuwangi, East Java, and calcium hydroxide (Ca(OH)₂) was used as the reacting agent. The formation of the calcium–oil complex is based on the hydrolysis of triglycerides by a basic compound, producing glycerol and fatty acid salts. In this reaction, the carboxyl (-COOH) groups of the fatty acids bind to calcium cations. The stoichiometric ratio involves two molecules of triglycerides reacting with three molecules of calcium hydroxide.

Table 1: Composition and nutrient content of experimental diets.

Feed ingredients	Treatment
	MUP					MP
	0	5	7.5	10	12.5	0	5	7.5	10	12.5
Odot grass (%)	50	50	50	50	50	50	50	50	50	50
Concentrates (%)	50	50	50	50	50	50	50	50	50	50
Fish oil (%)	0	5	7.5	10	12.5	0	0	0	0	0
Ca-fish oil (%)	0	0	0	0	0	0	5	7,5	10	12,5
Nutrient content (%)
CP	11.15	11.19	11.43	11.52	11.61	11.15	11.29	11.36	11.43	11.50
CFa	4.09	7.60	9.36	11.12	12.87	4.09	6.73	8.04	9.36	10.68
CFi	20.91	20.95	20.97	20.99	21.00	20.91	20.94	20.91	20.97	20.98
Ash	11.15	11.15	11.46	11.56	11.67	11.15	11.42	11.56	11.70	11.84
NFE	59.07	59.87	60.27	60.67	61.07	59.07	59.67	59.97	60.27	60.57

Note: MUP = unprotected Lemuru fish oil; MP = Lemuru fish oil protected with calcium hydroxide; CP = crude protein; CFa = crude fat; CFi = crude fiber; NFE = Nitrogen-Free Extract

Based on the molecular weights of the fatty acid components and calcium hydroxide, the optimal ratio of fish oil to calcium hydroxide was determined to be 4:1. The preparation of calcium-protected Lemuru fish oil was carried out by first placing 1,000 g of Lemuru fish oil into a 1,000 mL Erlenmeyer flask. Calcium hydroxide was dissolved in distilled water at a 1:1 weight-to-volume (w/v) ratio and added to the fish oil, followed by thorough mixing until a homogeneous mixture was formed. The resulting calcium–fish oil complex was then aerated at room temperature to allow crystallization. Once hardened, the complex was ground using a blender or mortar and subsequently analyzed. A diagram of the saponification process in preparation of calcium-protected fish oil is shown in Figure 2.

Preparation of rumen fluid and in vitro fermentation

Rumen fluid was collected directly from sheep using a stomach tube connected to a vacuum pump. The fluid was immediately filtered through layers of gauze, and the resulting filtrate was transferred into a pre-warmed crucible maintained at 40 °C, while being continuously flushed with CO₂ to preserve anaerobic conditions. To maintain microbial viability, the rumen fluid was kept at 40°C, and the time between collection and use did not exceed two hours.

A 500 mg feed sample was placed into a fermentation tube, followed by the addition of 40 mL of McDougall’s buffer solution and 10 mL of rumen fluid. To establish anaerobic conditions, the mixture was flushed with CO₂ gas for 30 seconds before the tube was sealed with a ventilated rubber stopper. The tubes were then incubated at 39°C in a shaking water bath for 48 hours, with pH monitored at the 4-hour mark. After 48 hours, the rubber stopper was removed, and 1 mL of 5% HgCl₂ was added to halt microbial activity. The fermentation mixture was centrifuged for 15 minutes, after which 50 mL of a pepsin-HCl solution was added. The tubes were subsequently incubated for another 48 hours at 39°C in a water bath shaker.

At the end of the incubation period, the mixture was filtered to separate the supernatant and the sediment. The sediment, representing the undigested residue, was then analyzed to determine nutrient digestibility.

Statistical analysis

The variables observed in this study included the digestibility of dry matter, organic matter, crude fat, crude fiber, and crude protein in both the rumen and post-rumen regions. The obtained data were analyzed using Analysis of Variance (ANOVA) following a factorial pattern in a Randomized Group Design. When significant differences between treatments were detected, further analysis was conducted using Duncan’s Multiple Range Test (DMRT) with the Genstat version 12.0 software. The mathematical model used for analysis is as follows:

Where, Yijk is observations of fish oil protection, μ is the mean, αi is the effect of fish oil protection, βj is the effect of the Lemuru fish oil’s level, (αβ)ij is the interaction between fish oil protection and level of Lemuru fish oil factors, and εijk is the random error.

Results and Discussion

Fish oil, rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), supports immune function, reduces oxidative stress, optimizes rumen fermentation, and enhances livestock performance (Nurfatahilah et al., 2022). The findings of this study revealed that the different methods (unprotected and protected) of feeding have different effects in rumen and post-rumen digestion (Figure 3). Our present study showed that increasing Lemuru fish oil inclusion significantly (P < 0.01) reduced the digestibility of dry matter (DM), organic matter (OM), crude fat (CFa), crude fiber (CFi), and crude protein (CP) in both rumen and post-rumen phases (Figure 4). The 0% inclusion consistently yielded the highest digestibility, whereas the 12.5% treatment resulted in the lowest values for DM (63.33% and 68.16%), OM (65.73% and 66.37%), CFa (62.00% and 59.59%), CFi (55.95% and 57.92%), and CP (63.11% and 68.04%). The reduction may be attributed to oil coating feed particles, limiting microbial access, and altering rumen pH and volatile fatty acid (VFA) profiles, which impair fermentation and nutrient absorption (Abdillah et al., 2024; Vargas et al., 2017). High fat inclusion can also suppress cellulolytic and proteolytic bacteria (Roy et al., 2017), consistent with previous findings of reduced digestibility and feed intake at elevated fish oil levels (Brelaz et al., 2024; Fadhilah et al., 2019).

Protection of fish oil with calcium hydroxide (Ca(OH)₂) effectively mitigated these negative effects by preventing microbial biohydrogenation and preserving fatty acid integrity, thereby supporting fiber-digesting microbes (Handojo et al., 2018; Pedroso et al., 2018). In the rumen, protected fish oil significantly (P < 0.01) improved DM (69.95%), OM (71.99%), CFi (61.86%), and CP (67.46%) digestibility compared to the unprotected form (Table 2). Post-rumen digestibility was also significantly higher (P < 0.01) for protected oil diets, with averages of 71.05% for DM, 71.81% for OM, 65.41% for CFi, 63.39% for CFa, and 69.29% for CP, reflecting the release of bound fatty acids during enzymatic digestion (Hakim et al., 2022).

Crude fat digestibility, however, was significantly lower in the rumen for protected oil (57.92%) compared to unprotected oil (66.12%) (Table 2), due to reduced microbial access to protected fat (Putri et al., 2021). This limitation was offset post-ruminally, where calcium soaps dissociate under acidic conditions, enabling efficient emulsification and hydrolysis; as a result, post-rumen crude fat digestibility was higher in the protected group (65.41%) than in the unprotected group (59.52%) (Table 2).

A significant interaction (P < 0.01) between protection method and inclusion level was observed for rumen digestibility. The 12.5% unprotected oil treatment produced the lowest DM (58.29%), OM (62.72%), CFi (50.84%), and CP (58.78%) values, whereas crude fat digestibility was lowest (54.27%) in the 12.5% protected oil group. Excessive fat inclusion can form a lipid barrier that reduces microbial and enzymatic activity, increases digesta viscosity, and slows nutrient absorption (Ponnampalam et al., 2024). These effects were most pronounced at the 12.5% inclusion level, indicating that while protection alleviates some negative impacts, it cannot fully counteract the drawbacks of high-fat diets in ruminants.

Table 2: In vitro digestibility evaluation of feed supplemented with Lemuru fish oil (Rumen)

Parameters	Method (M)	Level fish oil (L)						SEM	P-value
Parameters	Method (M)	0	5	7.5	10	12.5	Mean	SEM	M	L	M*L
Lemuru fish oil (Rumen)
Dry matter	MUP	69.45de	70.25e	63.26c	61.44b	58.29a	64.54A	0.81	<0.01	<0.01	<0.01
	MP	69.45de	69.84de	70.88e	71.20e	68.37d	69.95B
	Mean	69.45C	70.05C	67.07B	66.32A	63.33A
Organic matter	MUP	72.69d	70.71c	68.92b	68.17b	62.72a	68.64A	0.59	<0.01	<0.01	<0.01
	MP	72.69d	72.82d	72.87d	72.83d	68.75b	71.99B
	Mean	72.69D	71.77CD	70.89BC	70.50B	65.73A
Crude fat	MUP	62.90d	63.15d	66.11e	68.74f	69.72f	66.12A	0.93	<0.01	<0.01	<0.01
	MP	62.90d	58.54c	58.13c	55.75b	54.27a	57.92B
	Mean	62.90B	60.84A	62.12B	62.24B	62.00B
Crude fiber	MUP	62.22d	58.67c	54.45b	52.35ab	50.84a	55.71A	0.81	<0.01	<0.01	<0.01
	MP	62.22d	61.46d	62.20d	62.37d	61.07d	61.86B
	Mean	62.22D	60.06C	58.33B	57.36AB	55.95A
Crude Protein	MUP	67.73d	65.14bc	64.88b	59.52a	58.74a	63.20A	0.62	<0.01	<0.01	<0.01
	MP	67.73d	66.74cd	67.88d	67.47d	67.48d	67.46B
	Mean	67.73C	65.94B	66.38B	63.49A	63.11A
Lemuru fish oil (After rumen)
Dry matter	MUP	70.89d	69.53bc	68.90b	66.93a	66.05a	68.46A	0.36	<0.01	<0.01	<0.01
	MP	70.89d	72.48e	71.34d	70.28cd	70.27cd	71.05B
	Mean	70.89C	71.01C	70.12B	68.61A	68.16A
Organic matter	MUP	73.63e	72.33d	70.98c	68.79b	62.90a	69.73A	0.57	<0.01	<0.01	<0.01
	MP	73.63e	72.43de	72.71de	70.46c	69.84bc	71.81B
	Mean	73.63D	72.38C	71.84C	69.63B	66.37A
Crude Fat	MUP	62.87b	64.01cd	58.81b	58.10b	53.82a	59.52A	0.76	<0.01	<0.01	<0.01
	MP	62.87b	65.04de	66.06e	67.71f	65.36e	65.41B
	Mean	62.87B	64.52C	62.43B	62.91B	59.59A
Crude fiber	MUP	58.57d	57.40c	54.47b	51.42a	50.70a	54.51A	1.05	<0.01	<0.01	<0.01
	MP	58.74d	59.17d	65.66e	68.26f	65.14e	63.39B
	Mean	58.65a	58.29a	60.07b	59.84b	57.92a
Crude Protein	MUP	66.55	68.78	70.23	67.77	68.27	68.44A	0.31	0.029	<0.01	0.658
	MP	66.55	70.48	70.90	68.55	68.31	69.26B
	Mean	66.55A	69.63BC	70.57C	68.16B	68.04BC

Note: SEM= standard error of mean; M= methode; L= level fish oil; M*L = interaction methode with level fish oil. A,B: Superscripts within in the same row or column show significant differences between treatment (P<0.01); a,b,c,d: Superscripts showed significant differences between the interaction of treatments (P<0.01).

Protein digestibility was similarly influenced by both factors. Protection improved CP digestibility by reducing ruminal degradation and increasing bypass protein available for post-rumen enzymatic digestion (Hakim et al., 2022). However, higher inclusion levels still reduced CP digestibility due to fermentation disruption, lower protease activity, and delayed protein release from the feed matrix (Mohammed et al., 2014).

Overall, Lemuru fish oil can enhance ruminant diets through its polyunsaturated fatty acids, but excessive inclusion, particularly above 7.5%, adversely affects nutrient utilization. Calcium hydroxide protection improves digestibility by safeguarding fatty acids from rumen microbial degradation and enhancing post-rumen absorption. These findings emphasize the need to optimize both inclusion level and protection method to maximize feed efficiency and animal productivity.

Conclusion

The supplementation of Lemuru fish oil in sheep feed significantly influences nutrient digestibility, with calcium hydroxide protection proving effective in mitigating the adverse effects of unsaturated fatty acids on rumen microbial activity. While higher inclusion levels of fish oil can impair fermentation and reduce nutrient availability, the use of protected oil enhances post-rumen digestibility by facilitating enzymatic breakdown and absorption. Overall, calcium hydroxide-protected Lemuru fish oil presents a viable approach to improve feed utilization and energy efficiency in ruminants, provided its inclusion level is optimized to avoid excessive fat interference with digestion.

Acknowledgement

The authors are grateful to the Beasiswa Pendidikan Indonesia (BPI) program from the Indonesia Endowment Fund for Education (LPDP) through the Center for Higher Education Funding and Assessment (PPAPT), Ministry of Higher Education, Science, and Technology of Republic Indonesia.

Novelty Statement

This study is the first to evaluate the use of calcium hydroxide as a protective agent for Lemuru fish oil in ruminant feed, focusing on its impact on both rumen and post-rumen in vitro digestibility. The findings demonstrate that calcium hydroxide protection effectively enhances nutrient utilization while minimizing the negative effects of high unsaturated fat levels on rumen fermentation.

Author’s Contribution

TIWK, MM, and HH designed and coordinated the study. HH and MM supervised the experiment. TIWK and RBL performed the experiment, analysed the data, and drafted the manuscript. LER and MM took part in preparing and critically checking this manuscript. All authors read and approved the final manuscript.

Generative AI and AI-assisted technology statement

The authors declare that they did not use generative artificial intelligence (AI) and AI-assisted technologies in the writing process.

Conflict of interest

The authors have declared no conflict of interest.

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