Agroecological Zone-Specific Diet Optimization for Water Buffalo (Bubalus bubalis) through Nutritional and In Vitro Fermentation Studies

Simple Summary

This study involves the formulation of distinct diets for water buffalo based on locally available feed resources to specific agroecological zones. The diets were categorized into three groups addressing the maintenance, growth, and lactation/production requirements of buffaloes. This study assessed the chemical composition and in vitro gas and methane emissions of each diet. The implication of this work suggests a promising future for buffalo feeding systems, as it focuses on need-based formulations using specific regional ingredients. This approach may enhance the efficiency and sustainability of buffalo farming in specific zones.

Abstract

The water buffalo faces challenges in optimizing nutrition due to varying local feed resources. In response to this challenge, the current study introduces originality by addressing the lack of region-specific feeding strategies for water buffaloes. This is achieved through the formulation of 30 different diets based on locally available resources, offering a tailored approach to enhance nutritional optimization in diverse agroecological contexts. These diets were segmented into three groups of ten, each catering to the maintenance (MD₁ to MD₁₀), growth (GD₁ to GD₁₀), and lactation/production (PD₁ to PD₁₀) needs of buffaloes. Utilizing local feed ingredients, each diet was assessed for its chemical composition, in vitro gas and methane emissions, and dry matter (DM) disappearance using buffalo rumen liquor. The production diets (127 and 32.2 g/kg DM) had more protein and fats than the maintenance diets (82.0 and 21.0 g/kg DM). There was less (p < 0.05) fiber in the production diets compared to the maintenance ones. Different protein components (P_B1, P_B2) were lower (p < 0.05) in the maintenance diets compared to the growth and production ones, but other protein fractions (P_B3, P_c) were higher (p < 0.05) in the maintenance diet. Furthermore, the growth diets had the highest amount of other protein components (P_A), while the maintenance diets had the highest amount of soluble carbohydrates (586 g/kg DM), whereas the carbohydrate fraction (C_B1) was highest (p < 0.05) in the production diets (187 g/kg DM), followed by the growth (129 g/kg DM) and maintenance diets (96.1 g/kg DM). On the contrary, the carbohydrate C_A fraction was (p < 0.05) higher in the maintenance diets (107 g/kg DM) than in the growth (70.4 g/kg DM) and production diets (44.7 g/kg DM). The in vitro gas production over time (12, 24, and 48 h) was roughly the same for all the diets. Interestingly, certain components (ether extract, lignin, NDIN, ADIN, and P_B3 and C_C) of the diets seemed to reduce methane production, while others (OM, NPN, SP, P_A and P_B1, tCHO and C_B2) increased it. In simple words, this study reveals that different diets affect gas production during digestion, signifying a significant step towards a promising future for buffalo farming through tailored, region-specific formulations.

1. Introduction

India’s vast agroecological diversity offers a surplus of locally available feed resources. These diverse regions provide an opportunity to create diets specific to the needs of buffaloes, depending on their lifecycle stage, be it maintenance, growth, or lactation. The country’s agricultural backbone stands not just on its crops but, significantly, also on its livestock, with buffaloes playing a pivotal role [1]. The buffalo, often deemed the ‘Black Gold’ of India and Pakistan, is central to the rural economy [2,3]. This is not surprising given that India boasts a multitude of buffalo breeds, each with its unique attributes, suiting the varied climatic and topographical conditions of the country. From the Murrah, known for its high milk yield, to the Bhadawari, appreciated for its adaptability, the diversity is truly expansive [4]. In India, buffalo and cattle farming face challenges with limited feed resources and insufficient farmer knowledge on animal nutrition, impacting dairy animal productivity, which varies across regions due to differences in feed availability, types, and adherence to scientific feeding practices [5]. Livestock is the primary contributor of 50% of the 14.17 Tg methane emission total that comes from the Indian agricultural sector [6]. Methane, an important GHG (greenhouse gas) about 22–25 times more potent than carbon dioxide [7], is produced by ruminants as an end product of microbial digestion [8]. During the fermentation process (digestion and metabolism) of diets in the gastrointestinal tract, between 2 and 12% of dietary gross energy is lost as methane [7]. Several factors, viz., animal species and size, animal physiological stage, feed intake, digestibility, diet composition, etc., influence enteric methane production [9,10]. Diet composition (chemical and physical qualities) and its intake level (quantity consumed) influence methane production due to their effect on the rate of digestion and the rate of passage [11]. Animal species and diet composition play an important role in methane production [7,12,13]. Animals have three main nutritional needs: staying healthy (maintenance), growing, and producing things like milk or offsprings (production). To meet these needs, we created three different diets for each region. These diets were made by combining different amounts of locally available food resources, like dry and green roughages, along with concentrated mixtures. Protein in vitro fermentation has been shown to be associated with lower CH₄ production than carbohydrates fermentation [14,15]. Dietary nitrogen (N) concentrations play an important role in influencing rumen methanogenesis [16], specifically where feed N is low [17], leading to the reduced microbial growth of methanogens, which face difficulty competing under low N conditions [18]. The type of carbohydrate being digested, such as cellulose, hemicelluloses, and soluble residue, holds a notable influence over methane production [19,20,21]. Moreover, a strong relationship is observed between CH₄ production and digestible neutral detergent fiber (NDF) for cows and calves [22].

The main goal of this study was to develop and evaluate 30 water buffalo diets tailored for various life stages and agroecological zones in India. The assessment involved scrutinizing their nutritional compositions and in vitro methane production. The ultimate aim was to redirect methane emissions into a valuable energy source, thereby improving livestock productivity and simultaneously addressing global environmental concerns.

2. Materials and Methods

2.1. Formulation of Concentrate Mixtures

Local feed ingredients and their use in feeding livestock according to their suitable agroecological regions were considered for the formulation of concentrate mixtures (CM) for different regions of the country. A total of nine CM were prepared using protein and energy sources in different proportion for use in different diets as described in

Table 1. Proportion (%) and composition of ingredients in different concentrate mixtures.

Ingredients	CM1	CM2	CM3	CM4	CM5	CM6	CM7	CM8	CM9
Mustard seed cake	35	40	-	-	-	-	40	45	-
Wheat bran	25	-	25	-	25	-	-	-	-
Maize grain	40	-	-	60	-	-	20	-	40
Cotton seed cake	-	-	35	40	-	-	-	-	45
Oat grain	-	-	40	-	-	60	-	-	-
Barley grain	-	60	-	-	40	-	-	-	-
Groundnut cake	-	-	-	-	35	40	-	-	-
Rice bran	-	-	-	-	-	-	40	55	15

CM: Concentrate mixture.

.

2.2. Preparation Diets/Rations

The nutritional requirements of livestock were classified into three categories based on animals’ functional needs, viz., maintenance, growth, and production. For each category, ten diets/rations were prepared, and, hence, a total of thirty diets were formulated via the uniform mixing of dry and green fodder with concentrate mixtures in different proportions (

Table 2. Composition of diets (ingredients and their proportions).

AER No.	Region	Maintenance			Growth			Production
		Diet	Composition	Proportions	Diet	Composition	Proportions	Diet	Composition	Proportions
1	Western Himalayan region	MD1	Grass: GOL	65:35	GD1	SST:L:CM2	60:30:10	PD1	WS:B:CM2	30:40:30
2	Eastern Himalayan region	MD2	Grass:LL	75:25	GD2	RS:LL:CM1	50:35:15	PD2	Grass: LL:CM1	35:40:25
3	Eastern plateau and plains region	MD3	RS:MG	20:80	GD3	RS:NG:CM7	30:50: 20	PD3	MST:NG:CM7	20:45:35
4	Middle Gangetic plain	MD4	WS:MG	50:50	GD4	RS:B	40:60	PD4	MST:CM6	60:40
5	Trans and Upper Gangetic plain	MD5	WS:B	70:30	GD5	SST:B:CM2	60:25:15	PD5	WS:B:CM3	30:40:30
6	Central plateau and hills	MD6	LS	100	GD6	GS:CM2	80:20	PD6	LS:CM5	60:40
7	Western plateau and hills	MD7	WS:SG	50:50	GD7	SST/L/B	55:45	PD7	WS:B:CM4	35:35:30
8	Southern plateau and hills region	MD8	RS:L	65:35	GD8	SST:ST:CM7	40:40:20	PD8	SST:CM8	60:40
9	Western dry zone	MD9	PST:LL	75:25	GD9	PST: LL:CM2	55:30:15	PD9	PST:CM2	60:40
10	Coastal and island region	MD10	RS:LL	65:35	GD10	RS:LL:CM9	45:40:15	PD10	RS:LL:CM9	30:35:35

AER: Agroecological regions; CM: Concentrate mixture; MD: Maintenance diet; GD: Growth diet; PD: Production diet; GOL: Grewia optiva leaves; LL: Leucaena leucocephala leaves; MG: Maize green; RS: Rice straw; SST: Sorghum stover; L: Lucerne; WS: Wheat straw; B: Berseem; LS: Lentil straw; SG: sorghum green; PST: Pearl millet stover; NG: Napier grass; MST: Maize stover.

).

2.3. Determination of Chemical Composition

The dry matter (930.15), ash (932.05), N (976.05), and ether extract (EE, 920.39) contents of the diets’ samples were estimated following the standard method of the Association of Official Analytical Chemists (AOAC) [23]. The nitrogen values were multiplied by 6.25 to convert them into crude protein (CP) values. Neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose, and lignin (sa) were estimated as per the sequential method [24] using a fiber analyzer (Fibra Plus FES 6, Pelican, Chennai, India). Both the NDF and the ADF were expressed inclusive of their residual ash. There was no complex plant matrix included in our diet compositions; therefore, heat-stable α-amylase and sodium sulfite were not used in NDF determination. The lignin (sa) was determined by solubilizing cellulose with 72% of sulfuric acid in the ADF residue [24]. The cellulose was calculated as the difference between the ADF and the lignin (sa) in the sequential analysis. The hemicellulose was calculated as the difference between the NDF and ADF contents.

2.4. Estimation of Carbohydrate Fractions

The carbohydrate (CHO) fractions of the different diets samples were estimated as per the Cornell Net Carbohydrate and Protein (CNCP) system [25]. This system classifies CHO fractions into four fractions, as follows: C_A indicates rapidly degradable sugars; C_B1 classifies intermediately degradable starch and pectin; C_B2 includes slowly degradable cell walls; and C_C comprises unavailable/lignin-bound cell walls based on their degradation rate. The diets’ total CHO (tCHO; g/kg DM) was determined by subtracting the CP, ether extract (EE), and ash contents from 1000. The structural carbohydrates (SC) were calculated as the difference between the NDF and the neutral detergent-insoluble protein (NDIP), and the non-structural CHO were estimated as the difference between the tCHO and the SC [26]. For the starch estimation, samples were extracted with ethyl alcohol to solubilize free sugars, lipids, pigments, and waxes. The residue rich in starch was solubilized with perchloric acid, and the extract was treated with anthrone–sulfuric acid to determine glucose colorimetrically using a UV spectrophotometer (LABINDIA3000) at 630 nm [27].

2.5. Estimation of Protein Fractions

The CP fractions of the diets were partitioned into five fractions according to the CNCPS, [25] as modified previously [28]. These are the following: fraction P_A, indicating non-protein N, estimated as the difference between the total N and the true CP N precipitated with sodium tungstate (0.30 M) and 0.5 M of sulfuric acid; P_B1, the buffer-soluble protein calculated as the difference between the true protein and the buffer-insoluble protein, estimated with a boratephosphate buffer (pH 6.7–6.8) and a freshly prepared 0.10 M sodium azide solution; fraction P_B2, the neutral detergent-soluble protein, estimated as the buffer-insoluble protein minus the ND-insoluble protein; fraction P_B3, the acid detergent-soluble CP, estimated as the difference between the ND-insoluble protein and the acid detergent-insoluble CP; and fraction P_C, assumed to be indigestible (protein associated with lignin, tannin–protein complexes, and Maillard products which are unavailable to animals).

2.6. In Vitro Incubation

The in vitro gas production was determined using the pressure transducer technique [29]. Ruminal fluid was collected before feeding from two fistulated adult male Murrah water buffaloes (Bubalus bubalis) fed a wheat straw-concentrate diet (65:35 DM basis). The rumen fluid was filtered through a double layer of cheese cloth and bubbled with CO₂ before the commencement of incubation. The incubation medium was prepared by means of the sequential mixing of a buffer solution (NH₄HCO₃ and NaHCO₃), a macro-mineral solution, a micro-mineral solution, and resazurin solution [30]. Samples (1 g) of air-dry green forages were weighed into three serum bottles (150 mL capacity). Three serum bottles without substrate were used as blank cultures. The sample and control serum bottles were gassed briefly with CO₂ before adding 65 mL of medium. The bottles were continuously fluxed with CO₂, and then 3 mL of reducing solution were added in each bottle. The gassing of bottles with CO₂ continued until the pink color turned colorless. Before inoculation, the gas pressure transducer was used to adjust the head-space gas pressure in each bottle (to adjust the zero reading on the LED display). The serum bottles were inoculated with 8 mL of ruminal fluid inoculum using a 10 mL syringe. The inoculated bottles were sealed and incubated at 39 °C. The samples were incubated in triplicates, and the gas production (mL) was measured at 12, 24, and 48 h of incubation. The whole process was repeated on a different day.

2.7. Methane Measurements

The methane (CH₄) in the total gas was measured from three bottles incubated for each of the thirty diets at the 12, 24, and 48 h timepoints using gas chromatography (Nucon 5765 microprocessor-controlled gas chromatograph (GC), Okhla, New Delhi, India) equipped with a stainless-steel column packed with Porapak-Q and a flame ionization detector. Gas (1 mL) was sampled from the gas produced using a Hamilton syringe and injected manually (pull and push method of sample injection) into a GC. The GC was calibrated using standard methane and CO₂. The methane level was additionally measured in blank samples at different fermentation stages, and these measurements were used to correct for methane produced by the inoculum. The methane measured was related to the total gas to estimate its concentration [31] and converted into energy and mass values using 39.54 kJ/L CH₄ and 0.716 mg/mL CH₄ factors, respectively [32].

2.8. In Vitro Dry Matter Digestibility (IVDMD) and Energy of Diets

For the determination of the IVDMD for the evaluated diets a standard method was followed [33], wherein a 0.5 g sample was incubated for 48 h and then digested with 0.1 g of pepsin (1:3000 Sisco Research Laboratories, Mumbai, India) and 2 mL of 6N HCl at 39 °C for 24 h. The samples were incubated in triplicate with ruminal inoculum from the two fistulated buffaloes described previously. A provision was also made for the blanks, as described for the in vitro gas production. The digestibility was estimated as the difference between the DM incubated and the residual DM at the end of the second stage of digestion. The gross energy (GE) of the forages was measured with a bomb calorimeter (Toshniwal Brothers CLOI/M2, Bangalore, India) using benzoic acid as the standard.

2.9. Statistical Analysis

The data were subjected to an analysis of variance using the GLM procedure of SAS (2002). The model was the following: Yij = [1] + Fi + Eij, where Yij represents the individual observation of the variable, and Fi is the fixed effect of the ith diet (i = 1–30). The overall mean is expressed as [1], and Eij is the random error associated with Yij, not accounted in the fixed effect. The means were separated using Fisher’s LSD and all the statistical tests were at the p = 0.05 level of significance. The means of cereals, grasses, and legumes were compared using orthogonal contrasts (i.e., cereals vs. grasses, cereals vs. legumes, and grasses vs. legumes). The differences among forage means with p < 0.05 were accepted as statistically significant. A correlation analysis was used to establish relationships between chemical constituents, carbohydrate fractions, protein fractions, and CH₄ production. Pearson’s correlation analysis was performed to establish the relationship of chemical composition with methane production, carbohydrate fractions, and protein fractions at level p < 0.05.

3. Results

3.1. Chemical Composition

The crude protein (CP) and ether extract (EE) values were significantly higher (p < 0.05) for the production diets (127 and 32.2) than the maintenance diets (82.0 and 21.0 g/kgDM), respectively. The CP values of all three diets including the MD, the GD, and the PD varied, measuring 69.8–96.1, 106–130, and 103–153 g/kg DM, respectively (

Table 3. Chemical composition of the maintenance diets (g/kg DM) *.

Diet	CP	OM	EE	NDF	ADF	Cellulose	H cellulose	Lignin
MD1	76.0 cd	876 cd	32.1 a	646 c	453 a	298 c	193 de	93.4 b
MD2	93.3 a	871 c	27.5 b	678 b	456 a	274 d	222 c	109 a
MD3	84.3 b	923 a	17.8 d	668 bc	361 d	309 b	306 a	49.6 de
MD4	69.8 de	903 b	14.4 e	651 c	379 c	318 b	271 b	45.7 ef
MD5	96.1 a	884 c	18.1 d	573 e	386 c	301 c	187 e	49.7 de
MD6	76.9 cd	914 a	13.4 e	537 f	386 c	283 d	151 f	93.9 b
MD7	68.0 e	920 a	20.7 cd	713 a	406 b	345 a	307 a	42.6 f
MD8	77.8 bc	857 e	26.4 b	591 d	391 bc	272 d	200 d	55.8 cd
MD9	81.8 bc	920 a	21.5 c	545 f	344 e	256 e	201 d	62.5 c
MD10	95.5 a	852 e	17.9 d	575 e	392 bc	274 d	184 e	53.7 d
LSD	7.12	10.20	3.17	12.96	15.62	15.07	9.17	6.84
Diet	CP	OM	EE	NDF	ADF	Cellulose	H cellulose	Lignin
GD1	121 ab	917 ab	19.7 de	610 b	411 b	311 b	199 cde	80.7 b
GD2	116 bcd	874 d	32.4 a	527 e	341 e	231 c	186 e	55.4 fg
GD3	88.7 e	856 e	24.7 bc	676 a	392 cd	334 a	284 a	55.9 fg
GD4	111 bcd	864 de	18.6 e	619 b	411 bc	311 b	209 cd	52.0 g
GD5	117 bc	922 a	26.7 b	610 b	391 d	303 b	219 c	64.2 de
GD6	110 cd	909 bc	18.4 e	546 d	412 b	306 b	134 f	92.5 a
GD7	106 d	899 c	17.7 e	584 c	389 d	306 b	195 de	71.6 cd
GD8	111 bcd	916 ab	18.1 e	690 a	438 a	329 a	252 b	75.5 bc
GD9	111 bcd	917 ab	23.1 e	493 f	300 f	213 d	193 de	62.5 ef
GD10	130 a	872 d	35.5 a	512 e	328 e	220 d	183 e	52.0 g
LSD	10.87	10.29	3.35	18.25	18.32	11.30	22.70	7.90
Diet	CP	OM	EE	NDF	ADF	Cellulose	H cellulose	Lignin
PD1	130 bc	901 c	18.9 de	491 e	345 b	262 c	146 f	54.4 d
PD2	153 a	899 c	39.4 b	537 c	292 d	177 g	245 b	79.4 b
PD3	103 e	882 d	33.6 cd	633 a	360 b	311 a	273 a	45.0 e
PD4	116 d	912 ab	30.7 e	589 b	350 b	249 de	239 bc	68.1 c
PD5	137 b	904 bc	26.3 f	529 cd	326 c	253 cd	203 d	49.7 de
PD6	121 cd	918 a	32.8 de	453 f	288 d	203 f	165 e	74.3 b
PD7	121 cd	917 a	33.4 cd	549 c	318 c	251 d	231 bc	51.1 d
PD8	121 cd	902 c	35.7 c	634 a	455 a	301 b	179 e	98.0 a
PD9	116 d	916 a	21.7 g	539 c	310 c	240 e	229 bc	49.3 de
PD10	149 a	886 d	49.3 a	508 de	283 d	200 f	225 c	50.9 d
LSD	9.82	7.95	2.68	22.11	16.41	10.41	18.67	5.68

MD, maintenance diets; GD, growth diets; PD, production diets; CP, crude protein; OM, organic matter; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; H cellulose, hemi cellulose; LSD, least significant difference at p value < 0.0001; different superscript letters within a column in the table signify statistical differences among the corresponding values; *, each value is a mean of four observations.

), whereas, the concentrations of NDF, ADF, and cellulose were lower (p < 0.05) in the production diets (546, 333 and 245) than in the maintenance diets (618, 395 and 293 g/kg DM). Interestingly, no significant difference was observed in the lignin contents among all three diets.

3.2. Nitrogen Fractions

The protein fraction values (P_B1 and P_B2; g/kg DM) followed a lower to higher (p < 0.05) order from the MD (150.2 and 357.3) to the GD (204.7 and 409.7), followed by the PD (217.4 and 412.3), while the mean concentration of the slowly degradable protein fraction (P_B3) and the lignin-bound protein fraction (Pc) were higher (p < 0.05) in the maintenance diets (205.6 and 181.3) than in the growth diets (113.9 and 114.8), followed by the production diets (151.5 and 104.0 g/kg DM) (

Table 4. Protein fractions of the diets (g/kg CP) *.

Maintenance						Growth						Production
Diet	PA	PB1	PB2	PB3	PC	Diet	PA	PB1	PB2	PB3	PC	Diet	PA	PB1	PB2	PB3	PC
MD1	35.6 de	101 f	358 cd	237 cd	268 a	GD1	310 a	213 b	228 f	125 cd	123 abc	PD1	124 d	242 bcd	423 c	95.2 ef	116 b
MD2	18.1 e	95.3 f	287 de	301 b	299 a	GD2	27.8 fg	85.4 d	547 a	194 a	146 a	PD2	49.4 g	146 fg	364 d	291 b	148 a
MD3	194 a	195 ab	233 e	157 e	220 b	GD3	171 c	213 b	359 de	138 bcd	118 abcd	PD3	249 a	279 ab	175 f	187 c	110 bc
MD4	187 a	185 bc	327 d	88.8 f	211 bc	GD4	172 c	202 b	385 cde	155 b	85.7 e	PD4	158 b	296 a	304 e	133 d	108 bc
MD5	173 a	213 a	429 bc	48.9 f	135 def	GD5	167 c	295 a	336 e	114 de	87.2 de	PD5	134 cd	259 abc	464 e	55.5 h	87.4 cd
MD6	123 b	130 e	576 a	70.9 f	99.6 f	GD6	141 d	292 a	429 bc	28.4 f	110 bcde	PD6	96.9 e	208 ed	508 bc	113 de	73.4 d
MD7	188 a	218 a	24.3 f	425 a	145 de	GD7	203 b	192 b	357 de	100 e	147 a	PD7	71.3 f	171 ef	595 a	72.1 fgh	90.4 cd
MD8	14.9 e	67.8 g	485 b	257 bc	175 cd	GD8	109 e	218 b	416 cd	146 bc	110 bcde	PD8	101 e	223 cd	492 b	65.6 gh	117 b
MD9	74.4 c	159 cd	412 bc	209 d	145 de	GD9	50.8 f	188 b	490 ab	142 bc	129 ab	PD9	143 c	228 cd	431 c	88.3 efg	108 bc
MD10	47.7 d	138 de	440 b	261 bc	114 ef	GD10	15.9 g	147 c	550 a	195 a	91.4 cde	PD10	20.2 h	120 g	364 d	413 a	82.3 d
LSD	22.84	27.36	78.36	47.84	40.64	LSD	23.03	31.46	64.18	24.93	32.45	LSD	14.29	39.28	57.32	26.78	22.65

MD, maintenance diets; GD, growth diets; PD, production diets; P_A, non-protein nitrogen; P_B1, buffer-soluble protein; P_B2, neutral detergent-soluble protein; P_B3, acid detergent-soluble protein; P_C, indigestible protein; LSD, least significant difference at p value < 0.0001; different superscript letters within a column in the table signify statistical differences among the corresponding values; *, each value is a mean of four observations.

). The average value of P_A (g/kg DM) was significantly higher (p < 0.05) for the growth diets (136.9) than in the production and maintenance diets, which had values of 114.8 and 105.6, respectively. In the maintenance diets, the affinity of protein binding to ADF was observed to be higher than in the growth and production diets, whereas the SP concentration was in the reverse order, meaning that the concentration in the maintenance diets was lower than in the two other diets.

3.3. Carbohydrate Fractions

Among the carbohydrate fractions, no significant difference was observed among all three diets for the total carbohydrate levels (tCHO), while the SC contents were (p < 0.05) higher in the maintenance diets (586.2) than in the production diets (513.0 g/kg DM), respectively. The average value of the rapidly degradable carbohydrate fraction (C_B1) differed (p < 0.05) among the diets, being highest in the production diets (187.2), followed by the growth (129.5) and maintenance diets (96.1 g/kg DM;

Table 5. Carbohydrate fractions of maintenance diets (g/kg DM) *.

Diet	tCHO	NSC	SC	CC	CB2	CB1	CA
MD1	768 c	161 d	607 c	224 b	383 e	20.3 g	140 b
MD2	751 d	128 e	622 bc	262 a	360 f	95.6 cde	32.9 d
MD3	821 ab	185 c	636 b	119 de	517 b	74.5 ef	110 b
MD4	818 ab	189 c	630 b	110 ef	520 b	87.6 def	101 c
MD5	770 c	215 b	555 d	119 de	436 c	120 bc	94.8 c
MD6	824 ab	300 a	523 f	225 b	298 g	178 a	122 b
MD7	832 a	157 d	674 a	102 f	572 a	114 bcd	43 d
MD8	753 d	196 c	557 d	134 cd	423 cd	135 b	60.4 d
MD9	817 b	300 a	516 f	150 c	366 ef	58.4 f	242 a
MD10	739 d	199 c	540 e	129 d	411 d	76.8 ef	123 b
LSD	14.23	15.14	14.70	16.43	18.56	30.53	31.46
Diet	tCHO	NSC	SC	CC	CB2	CB1	CA
GD1	776 a	196 d	580 b	194 b	386 d	144 cd	51.9 def
GD2	726 c	238 b	488 e	133 fg	355 e	175 bc	62.9 cde
GD3	743 b	88.9 f	654 a	134 fg	520 a	59.2 g	29.8 fg
GD4	735 bc	142 e	593 b	125 g	468 b	59.4 g	82.5 c
GD5	779 a	192 d	586 b	154 de	433 c	155 bc	37.3 efg
GD6	780 a	248 b	532 d	222 a	310 f	179 ab	68.7 cd
GD7	776 a	218 c	558 c	172 cd	386 d	86.1 fg	132 b
GD8	786 a	125 e	662 a	181 bc	480 b	108 ef	16.6 g
GD9	783 a	320 a	463 f	150 ef	313 f	121 de	199 a
GD10	707 d	232 bc	475 ef	125 g	350 e	208 a	23.5 fg
LSD	15.52	19.72	17.94	18.97	28.14	31.11	29.1
Diet	tCHO	NSC	SC	CC	CB2	CB1	CA
PD1	752 bc	288 b	464 d	131 d	333 d	239 b	49.4 bc
PD2	707 d	237 d	470 d	190 b	280 f	194 d	43.1 c
PD3	746 c	143 f	602 a	108 e	494 a	103 g	40.2 c
PD4	765 ab	203 e	562 b	163 c	398 b	169 e	34.1 cd
PD5	741 c	231 e	509 c	119 de	390 bc	133 f	98.0 a
PD6	764 b	333 a	431 e	178 b	252 g	302 a	31.7 cd
PD7	762 b	233 d	529 c	122 d	407 b	217 bcd	16.1 d
PD8	745 c	133 f	612 a	235 a	376 c	97.8 g	35.2 cd
PD9	779 a	262 c	516 c	118 de	398 b	197 cd	65.4 b
PD10	687 e	253 cd	434 e	122 d	312 e	219 bc	34.1 cd
LSD	13.55	22.04	22.31	13.64	19.94	24.31	19.49

MD, maintenance diets; GD, growth diets; PD, production diets; tCHO, total carbohydrates; NSC, non-structural carbohydrates; SC, structural carbohydrates; C_C, unavailable/lignin-bound cell wall; C_B2, slowly degradable cell wall; C_B1, intermediately degradable starch and pectin; C_A, rapidly degradable CHO, including sugars; LSD, least significant difference at p value < 0.0001; different superscript letters within a column in the table signify statistical differences among the corresponding values; *, each value is a mean of four observations.

). The contrary carbohydrate C_A fraction was (p < 0.05) higher in the maintenance diets (107.1) than in the growth (70.4) and production diets (944.7 g/kg DM). The carbohydrate fractions C_B2 and C_C were relatively lower in the production diets than in the maintenance and growth diets.

3.4. Gas and Methane Production Kinetics

The average values (mL/g DM) for the diets’ in vitro gas production were found to have a consistent pattern at 0–12, 12–24, and 24–48 h. The observed values for the maintenance diets were 63.0, 52.0, and 48.15; for the growth diets, they were 63.8, 52.7, and 48.2, and, for the production diets, they were 63.5, 52.5, and 47.2. The cumulative gas production values (48 h) were close and similar 163, 165, and 163 mL/g DM for the maintenance, growth, and production diets, respectively (

Table 6. Gas and methane production kinetics from the maintenance diets fermented in buffalo inoculums *.

Diets/Rations	Gas (mL/g)				Methane (mL/g)
	0–12 h	12–24 h	24–48 h	Cumulative	0–12 h	12–24 h	24–48 h	Cumulative
MD1	64.3 c	50.0 e	50.0 b	164 c	9.83 d	5.77 f	5.08 h	20.7 g
MD2	58.5 h	51.0 d	44.3 f	154 e	6.57 f	5.56 f	11.3 b	23.4 e
MD3	59.5 gh	55.8 b	47.6 d	163 c	11.2 c	12.3 a	16.3 a	39.8 a
MD4	63.6 cd	54.3 c	50.0 b	168 b	12.6 b	11.2 b	17.0 a	40.4 a
MD5	66.0 b	53.7 c	45.3 e	164 c	11.2 c	8.77 c	5.66 g	25.7 d
MD6	62.3 de	49.2 e	47.9 d	160 d	15.1 a	8.63 c	9.56 d	33.3 b
MD7	69.8 a	58.5 a	48.3 cd	178 a	12.1 b	7.89 d	8.32 e	28.3 c
MD8	63.8 cd	54.5 c	46.1 e	164 c	8.72 e	6.68 e	7.02 f	22.4 f
MD9	61.8 ef	44.5 g	49.3 c	156 e	9.03 de	6.67 e	8.21 e	23.9 e
MD10	60.5 fg	47.8 f	52.6 a	160 d	7.12 f	7.98 d	10.8 c	25.9 d
LSD	1.535	1.038	0.960	2.245	0.829	0.528	0.359	1.348
Diets/Rations	Gas (mL/g)				Methane (mL/g)
	0–12 h	12–24 h	24–48 h	Cumulative	0–12 h	12–24 h	24–48 h	Cumulative
GD1	65.0 b	50.3 f	48.8 cd	164 c	12.8 c	5.74 h	3.95 i	22.5 gh
GD2	59.5 d	55.8 b	46.8 e	162 de	8.20 ef	10.1 b	15.3 c	33.6 c
GD3	62.4 c	55.2 bc	49.2 bc	167 b	10.5 d	10.6 a	15.8 b	36.9 b
GD4	64.8 b	52.6 e	48.8 cd	166 b	15.5 a	10.9 a	16.2 a	42.6 a
GD5	65.8 b	51.8 e	45.6 f	163 cd	13.2 c	7.39 f	6.40 g	26.96 f
GD6	67.3 a	54.5 cd	44.3 g	166 b	10.9 d	7.96 e	4.73 h	23.6 g
GD7	67.8 a	57.0 a	48.3 d	173 a	14.5 b	9.49 c	8.22 f	32.2 d
GD8	62.8 c	54.0 d	49.8 b	167 b	9.09 e	6.07 g	6.40 g	21.6 h
GD9	63.5 c	46.3 g	49.6 b	159 f	11.1 d	8.48 d	9.67 e	29.2 e
GD10	59.5 d	50.0 f	51.0 a	161 ef	7.87 f	8.50 d	10.27 d	26.6 f
LSD	1.136	0.844	0.729	1.836	0.906	0.300	0.357	1.175
Diets/Rations	Gas (mL/g)				Methane (mL/g)
	0–12 h	12–24 h	24–48 h	Cumulative	0–12 h	12–24 h	24–48 h	Cumulative
PD1	62.8 e	54.3 cd	44.0 g	161 d	13.9 b	12.7 a	14.5 b	41.1 a
PD2	62.6 e	53.0 e	46.8 e	162 d	11.1 d	9.02 de	14.2 b	34.3 cd
PD3	61.0 f	54.6 bc	48.8 cd	164 c	10.9 d	11.2 bc	16.0 a	38.1 b
PD4	64.3 d	54.0 d	50.0 ab	168 b	14.0 b	11.0 bc	15.9 a	40.9 a
PD5	67.6 a	55.0 b	45.0 f	168 b	15.6 a	11.1 bc	8.03 d	34.7 c
PD6	66.3 b	54.8 bc	44.1 g	165 c	15.0 a	11.4 b	6.94 e	33.4 cd
PD7	66.0 b	58.6 a	45.3 f	170 a	13.0 c	10.5 c	6.94 e	30.4 e
PD8	60.5 f	43.8 h	48.3 d	153 f	6.42 f	5.68 f	6.56 e	18.6 g
PD9	65.0 c	47.5 g	49.4 bc	162 d	13.5 bc	9.41 d	9.75 c	32.7 d
PD10	59.0 g	49.7 f	50.4 a	159 e	8.28 e	8.63 e	10.11 c	27.0 f
LSD	0.663	0.557	0.796	1.278	0.883	0.741	0.786	1.970

MD, maintenance diets; GD, growth diets; PD, production diets; LSD, least significant difference at p value < 0.0001; different superscript letters within a column in the table signify statistical differences among the corresponding values; *, each value is a mean of four observations.

). The in vitro methane production mean values at 0–12, 12–24, and 24–48 h and the cumulative values of the maintenance diets tended (p > 0.05) to be lower than those of the growth and maintenance diets, whereas, the cumulative methane production was lower in the maintenance diets (28.4) than in the production diets (33.1 mL/g DM).

3.5. Methane Production and Percentage Loss of Dietary Energy as Methane

The mean values of the in vitro methane production (mL/g DDM, g/kg DM and g/kg DDM) were similar in the diets formulated for maintenance (41.2, 13.3 and 29.5;

Table 7. Methane production and loss of dietary energy as methane from the maintenance diets *.

Diets/Rations	IVDMD g/kg DM	CH4 mL/g DDM 24h	CH4 g/kg DM	CH4 g/kg DDM	GE kJ/g	GE in CH4 g DDM	CH4 %GE DDM
MD1	422 bc	37.2 f	11.2 c	26.7 ef	16.9 cd	1.42 ef	8.45 ef
MD2	402 c	30.4 g	8.70 d	21.7 g	18.0 ab	1.16 g	6.46 g
MD3	482 b	48.9 a	16.9 a	35.0 a	17.5 bc	1.87 a	10.7 ab
MD4	468 b	49.4 a	17.1 a	35.4 a	16.3 d	1.89 a	11.6 a
MD5	468 b	42.5 bcd	14.3 b	30.5 bcd	17.5 bc	1.63 bcd	9.31 cde
MD6	584 a	43.2 bc	17.0 a	31.0 bc	18.7 a	1.66 bc	8.87 de
MD7	417 bc	47.0 ab	14.4 b	33.7 ab	17.4 bc	1.80 ab	10.3 bc
MD8	367 e	41.5 cde	11.0 c	29.8 cde	16.2 d	1.59 cde	9.8 bcd
MD9	474 bc	33.5 fg	11.3 c	24.1 fg	17.6 bc	1.28 fg	7.31 fg
MD10	389 de	38.5 de	10.8 c	27.6 de	17.2 c	1.47 de	8.56 e
LSD	43.58	4.64	0.81	3.33	0.768	0.178	1.14
Diets/Rations	IVDMD g/kg DM	CH4 mL/g DDM 24 h	CH4 g/kg DM	CH4 g/kg DDM	GE kJ/g	GE in CH4 g DDM	CH4 %GE DDM
GD1	450 d	41.3 bc	13.27 e	29.6 bc	16.9 cd	1.57 bc	9.29 bc
GD2	496 bc	37.0 de	13.14 e	26.5 de	17.5 bc	1.41 de	8.06 e
GD3	530 b	39.9 cd	15.13 c	28.6 cd	16.9 cd	1.53 cd	9.07 cd
GD4	628 a	42.1 bc	18.94 a	30.2 bc	17.0 cd	1.61 bc	9.49 bc
GD5	466 cd	44.3 b	14.74 c	31.8 b	16.9 cd	1.7 b	10.0 b
GD6	530 b	35.7 e	13.55 de	25.6 e	16.6 d	1.37 e	8.21 de
GD7	409 e	58.7 a	17.21 b	42.1 a	17.9 ab	2.24 a	12.5 a
GD8	372 f	40.9 bc	10.87 g	29.3 bc	16.7 d	1.57 bc	9.43 bc
GD9	469 cd	41.9 bc	14.01 d	30.0 bc	18.3 a	1.61 bc	8.82 cde
GD10	408 e	40.1 cd	11.74 f	28.8 cd	17.4 bc	1.53 cd	8.83 cde
LSD	35.36	3.88	0.686	2.78	0.614	0.148	0.865
Diets/Rations	IVDMD g/kg DM	CH4 mL/g DDM 24h	CH4 g/kg DM	CH4 g/kg DDM	GE kJ/g	GE in CH4 g DDM	CH4 %GE DDM
PD1	621 a	42.9 c	19.06 a	30.7 c	17.5 bcd	1.66 c	9.48 cd
PD2	605 a	33.2 de	14.39 d	23.8 de	17.7 abc	1.28 de	7.24 e
PD3	600 ab	36.9 d	15.82 c	26.5 d	16.8 ef	1.41 d	8.40 d
PD4	523 cd	47.9 ab	17.92 b	34.3 ab	16.9 def	1.82 ab	10.8 ab
PD5	520 d	51.5 a	19.11 a	36.9 a	17.4 bcd	1.99 a	11.4 a
PD6	563 bc	47.0 bc	18.96 a	33.7 ab	16.7 f	1.78 bc	10.7 ab
PD7	496 d	47.4 ab	16.82 c	34.0 ab	18.1 a	1.82 ab	10.1 bc
PD8	391 e	31.1 e	8.68 f	22.3 e	17.2 cdef	1.20 e	6.92 e
PD9	534 cd	43.1 c	16.4 c	30.9 c	17.3 bcde	1.66 c	9.59 c
PD10	533 cd	31.8 e	12.1 e	22.8 e	17.8 ab	1.20 e	6.84 e
LSD	40.91	4.21	1.003	3.02	0.575	0.161	1.029

MD, maintenance diets; GD, growth diets; PD, production diets; IVDM, in vitro dry matter digestibility; GE, gross energy; LSD, least significant difference at p value < 0.0001; different superscript letters within a column in the table signify statistical differences among the corresponding values; *, each value is a mean of four observations.

), for growth (42.2, 14.3 and 30.2; Table 7), and for production (41.3, 15.9 and 29.6; Table 7). Furthermore, a similar trend was observed for the gross energy from each diet being lost as methane, with comparable values in the maintenance (1.57), growth (1.61), and production diets (1.58 kJ/g DDM), equivalent to 9.09, 9.37, and 9.14% of dietary energy lost as methane, respectively.

3.6. Correlation between Chemical Constituents and Methane Production

Among the proximate constituents, the EE and lignin were significantly (p < 0.05) negatively associated (r = −0.422 ** and r = −0.365 **) with dietary methane production, while the OM contents of the diets were positively (p < 0.05 r = 0.266 *) correlated with methane production (

Table 8. Correlation between in vitro methane production and chemical constituents of the diets/rations.

Chemical Constituents	CH4 g/g DDM	Protein Fractions	CH4 g/g DDM	CHO Fractions	CH4 g/g DDM
CP	−0.134	NDIP	−0.448 (**)	tCHO	0.353 (**)
OM	0.266 (**)	ADIP	−0.272 (**)	NSC	0.115
EE	−0.422 (**)	SP	0.387 (**)	SC	0.083
NDF	−0.009	NPN	0.450 (**)	Starch % NSC	−0.104
ADF	−0.127	PA	0.412 (**)	CC DM	−0.365 (**)
Cellulose	−0.073	PB1	0.284 (**)	CB2DM	0.278 (**)
Hemi cellulose	0.130	PB2	−0.053	CB1DM	0.031
Lignin	−0.365 (**)	PB3	−0.341 (**)	CADM	0.091
Energy	−0.032	PC	−0.145

CP, crude protein; OM, organic matter; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; NDIP, neutral detergent-insoluble protein; ADIP, acid detergent-insoluble protein; SP, soluble protein; P_A, non-protein nitrogen; P_B1, buffer-soluble protein; P_B2, neutral detergent-soluble protein; P_B3, acid detergent-soluble protein; P_C, indigestible protein; tCHO, total carbohydrates; NSC, non-structural carbohydrates; SC, structural carbohydrates; C_C, unavailable/lignin-bound cell wall; C_B2, slowly degradable cell wall; C_B1, intermediately degradable starch and pectin; C_A, rapidly degradable CHO, including sugars; DM, dry matter; DDM, digestible dry matter; **, statistically significant.

). The protein fractions NDIP, ADIP, and P_B3 of the diets were negatively associated with CH₄ production (r = −0.448 **, r = −0.272 **, and r = −0.341 **). On the other hand, the N fraction, the NPN, the SP, the P_A, and the P_B1 fraction of the diets were positively associated (r = 0.450 **, 0.387 **, r = 0.412 **, and r = 0.284 **) with the in vitro CH₄ production. Among the diets, the tCHO and carbohydrate fraction C_B2 contents were positively associated (r = 0.353 ** and 0.278 **) with the in vitro CH₄ production, while the carbohydrate fraction C_C DM was negatively associated (r = −0.365 **) with CH₄ production.

4. Discussion

4.1. Chemical Composition

All three diet categories including maintenance, growth, and production showed crude protein (CP) levels equal to or exceeding the minimum required for microbial growth. A minimum of 7.0% CP is essential to optimize the growth and functionality of rumen microbes [34]. The reason for higher CP contents in the production diets (127) than in the growth (112) and maintenance diets (82.0 g/kg DM) may be due to the inclusion of a protein-rich concentrate mixture in the production diets. These values of CP were within the range (64.4 to 150.4 g/kg DM) of values reported for 45 rations [35]. Additionally, the higher values of NDF, ADF, and cellulose contents in the maintenance diets may be due to the sole roughage ingredients in its compositions. Further, the variability in the cell wall constituents of the maintenance, growth, and production diets may be attributed to the composition and level of diverse sources of dry, green, and concentrate mixtures. The average value of the CP and hemicelluloses obtained from the growth and production diets were in similar to the value reported in a combined mixed-diet ration containing low-protein and high-protein rations [36]. The OM contents of the maintenance, growth, and production diets evaluated in the present study were within the range of OM values observed in an experiment involving seven diets [37] and utilizing local-based feed resources and tropical grass pastures [38,39].

4.2. Carbohydrate and Protein Fractions

Carbohydrates and proteins are the two most important constituents of diets required for the different physiological functions of animals, viz., maintenance, growth, and production. The total carbohydrate (tCHO) and NSC content of the maintenance, growth, and production diets recorded in the present study were aligned within the range (773.3–859.4 and 95.1–335.6 g/kg DM) of values reported for 45 rations of different roughage and concentrate feed ratios formulated using different roughage and concentrate feed types [35]. A previous study conducted on top foliage [40] and concentrate feed [10] observed values within a similar range and following a comparable trend. The maintenance, growth, and production diets had the highest contents of carbohydrate fraction C_B2 (429, 400, and 364 g/kg DM, respectively), following a similar pattern to that of the 45 rations reported by Dong and Zhao [35]; also, our diets’ C_B2 contents were within the range (344.8–588.2 g/kg DM) of values reported in the above-mentioned study. The variations in the concentration of the C_A, C_B1, C_B2, and C_C carbohydrate fractions of the maintenance, growth, and production diet are similar to those observed for the 45 rations reported in the earlier study mentioned previously [35]. The carbohydrate fractions C_B2 and C_B1’s contents in most of our growth and production diets were within the range of values reported for six farm diets in a previous study [41]: the observed lower content of C_C fraction in the above-mentioned study can likely be attributed to the elevated lignin levels in the diets analyzed in our study. The higher lignin content may hinder the release or accessibility of cellulose and hemicellulose, resulting in reduced C_C fraction values. This phenomenon suggests a potential influence of diet composition on the structural components of plant material, with a higher lignin content acting as a limiting factor for the measured C_C fraction. Further, the tCHO values of the growth and production diets were similar to the tCHO values of the diets reported in the above-mentioned study [41], while their NSC contents were relatively higher than our values. The difference in the protein/nitrogen fractions of the maintenance, growth, and production/lactation diets may be attributed to the differences in the proportion of different dietary ingredients and their chemical constituents.

4.3. Gas and Methane Production and Loss of Energy as Methane

The average values of the gas production kinetics at three time intervals (12, 24, and 48 h) and the cumulative gas production (mL/g DM) from the high-protein and low-protein diets showed values higher than the gas production values in the maintenance, growth, and production diets in our study [36]. The gas production from the total mixed rations collected from seven dairies ranged between 211 and 256 mL/g DM after 48 h, which was higher than our gas production values. This variation in gas production may be due to differences in the chemical constituents, mainly the cell wall fractions and the carbohydrate and protein fractions, and their degradability. The availability of nutrients to microbes influences gas production from any feed/diet [42,43]. The total gas production (mL/g DM) from 45 rations of various concentrate to roughage ratios ranged between 165 and 281 [35], which partially agrees with our gas production values. The relatively higher cumulative methane production (mL/g DM) at the 48 h timepoint in the lactation diets (33.1) compared to the maintenance diets (28.4) may be due to the higher digestibility of the production diets, as the degradability of a substrate influences both gas and methane production. In a previous study conducted on lactating cows’ diets, the methane production (mL/200 g) reported was higher in the lactating ration (8.85) than in the dry ration (7.24), which substantiates our observations [44]. Further, in same study [44], they recorded higher gas production values in the lactation ration (54.4) than in the dry ration (43.0 mL/200 g). The methane production of 45 rations with varied roughage was the following: the concentrate ration ranged from 30 to 51 mL/g DM after 48 h of fermentation [35], which partially agreed with our results. A similar trend of methane production was observed in a study [45] where goats were fed three diets of different roughage to concentrate ratios (25:75, 50:50 and 73:27), and the values were 37.1, 36.4, and 34.5 g/kg DDM, respectively. Further, the CH₄ (%GE) for these three diets (8.6, 7.3, and 6.0%) differed significantly (p < 0.05), which agreed with our observations that the level of concentrate and the dietary ingredients’ composition influences methane production. The percentages of CH₄ and GE were lower in the above-mentioned study than our average values in the maintenance, growth, and production diets.

4.4. Correlation between Methane Production and Chemical Constituents (Proximate Constituents, Carbohydrate Fractions, and Protein Fractions)

The correlations studies between chemical constituents and CH₄ production of forages and concentrate feeds are crucial for optimizing animal nutrition, reducing environmental impact, and improving overall feed efficiency in livestock production [34,46,47]. However, the information on the correlation between methane production and diets/rations’ chemical constituents is limited. In this study, the EE and lignin from the proximate constituents and the NDIP and ADIP protein fractions were negatively associated with methane production. Similar to our observations, earlier studies [48,49,50] reported that EE, lignin, NDIP, and ADIP were negatively associated with methane production. Contrary, a positive correlation between EE and methane production was recorded by Ellis et al. [51]. Information on diets/rations’ carbohydrate and/or protein fractions’ relationship with in vitro methane production is scarce. In a study of 45 rations, a relationship between CNCPS carbohydrate fractions and methane production was reported [35]. They also reported that the carbohydrate fractions C_A, C_B1, and C_B2 were positively related to methane production, and this agrees of our correlation results. In our study, C_C was negatively related to methane production, and this could be due to the unavailability of lignin-bound carbohydrates for digestion. The evaluated diets’ soluble protein, NPN, P_A, and P_B1 were positively associated with methane production. This is probably due to the ready availability of these more degradable protein fractions to microbial fermentation.

This study effectively created diets for water buffaloes, but it has limitations. It mainly looks at diet composition and gas emissions; therefore, future research should explore the diets’ long-term effects on buffalo health, practical use on farms, and economic factors to get a fuller picture of their feasibility in real-world farming.

5. Conclusions

This study highlighted key findings on three categories (maintenance, growth, and production/lactation) of thirty different diet compositions for water buffaloes based on local resources, addressing the need for region-specific feeding strategies. The production diets exhibited higher crude protein contents, while the maintenance diets had more fiber. The soluble protein fractions (P_B1 and P_B2) were more present in the production and growth diets and the indigestible fraction (P_C) in the maintenance diets. The higher levels of non-structural carbohydrates in the production diets suggest dietary optimization possibilities. The loss of energy as CH₄ from the diets/feeding systems varied from 6.48% to 12.56 for the buffalos observed. Amongst the agroecological regions studied (AERs), the livestock from the AER-2 and AER-10 regions emitted the lowest CH₄. The diets in the AER-2 and AER-10 regions consisting of tree leaves as the green fodder source produced less CH₄, with lower losses of dietary energy as methane. The AER-10 diets supplemented with coconut cake as the protein source emitted less CH₄. These findings emphasize the importance of tailoring diets to meet the nutritional needs of buffaloes, marking a significant step forward in optimizing buffalo farming practices. Future implications involve refining agroecological regional feeding practices and considering correlations for a targeted and sustainable diet selection process, promoting both livestock health and environmental stewardship.