Effects of Dietary Cauliflower Leaf Meal Supplementation on Growth Performance, Nutrient Utilization, Rumen Fermentation, and Methane Emission in Goats

Abstract

Feed stress is a very critical factor impacting livestock health and productivity. One of the major contributors to quantitative feed deficiency is the continued adherence to conventional diets and feeding practices, which renders livestock populations vulnerable to environment-induced scarcity events as well as shortages arising from supply-chain bottlenecks. These challenges occur in the face of the ever-expanding demand from a continuously growing livestock population. In a world increasingly experiencing qualitative and quantitative resource constraints due to rising demand and increasing pollutant concentrations in the environment, conventional dietary compositions require timely modification and supplementation with alternative feed ingredients. These may include the hitherto unutilized by-products of agricultural production, which are often discarded as agricultural waste, in order to mitigate the stress induced by feed availability shortfalls. Cauliflower leaf meal is one such by-product whose suitability as a feed supplement was evaluated in the present study, with results that can be reliably described as promising. The present study assessed the impact of dried cauliflower leaf meal (CLM) on growth performance, nutrient utilization, rumen fermentation, and methane emission in goats. Fifteen non-descript male goats, aged 6–8 months, were randomly allocated into three groups of five animals each and housed separately in identical pens within the same shed for the duration of the experiment. Three dietary treatments were administered: T0 (control; concentrate, hybrid Napier, and wheat straw); T20 (20% replacement of wheat bran with CLM in the concentrate, along with hybrid Napier and wheat straw); and T30 (30% replacement of wheat bran with CLM in the concentrate, along with hybrid Napier and wheat straw). The results indicated that the goats in all groups achieved a similar body-weight gain with a comparable dry-matter intake (DMI). The feed conversion ratio (FCR), nutrient digestibility, and mineral balance were also comparable across treatments. However, the methane emission rate was significantly lower (p < 0.05) in the T30 group compared with the other groups. CLM supplementation did not cause deviations in rumen pH, NH₃-N concentration, volatile fatty acid production, or bacterial and protozoal populations. The hematological parameters remained unaffected by the increased dietary inclusion of CLM, while both cell-mediated and humoral immune responses showed an improvement in the CLM-fed groups. Notable reductions in methane emission were observed in goats fed diets containing 20–30% dried CLM, highlighting the positive environmental implications of such a dietary inclusion.

1. Introduction

Anthropogenic activities have been a major contributor to environmental pollution over the past two centuries, including the emission of greenhouse gases such as carbon dioxide and methane. Although anthropogenic greenhouse gas emissions have been dominated by carbon dioxide, with methane contributing a smaller share in terms of the total emission volume, methane is approximately 80 times more potent in its warming effect [1]. Organic waste materials decomposing under anaerobic conditions, such as those prevailing in landfills, along with enteric fermentation in ruminant animals, constitute the major sources of methane production. In India, methane emissions arising from the disposal of vegetable waste in landfills account for approximately 29% of overall greenhouse gas emissions [2]. Globally, an estimated 10–20% of horticultural waste is disposed of in landfills, thereby perpetuating environmental degradation [3]. Curbing methane emissions has therefore assumed considerable significance in the context of a warming climate [4]. India produces large quantities of vegetables annually to meet the needs of its population; however, when coupled with the limited agrologistical infrastructure, this results in a high vegetable waste index [5]. The proper disposal and utilization of vegetable waste are essential to prevent methane generation and its release into the environment. The primary biogenic sources of methane (CH₄) include enteric fermentation in ruminant animals (16%) and rice cultivation (11%) [6], both of which are particularly significant in the Indian context [7].

According to the 20th Livestock Census, India has approximately 535.82 million heads of livestock, including a goat population of 148.88 million, representing a significant increase from the 135.17 million recorded in the previous census [8]. A substantial proportion of this large livestock population suffers from feed availability constraints, which adversely affect animal health and productivity. The estimated net deficiencies in India stand at 10.95% for dry fodder, 35.6% for green fodder, and 44% for concentrate feed materials [9]. In light of this considerable gap between the demand and availability of conventional feed resources, the search for alternative feed materials that are easily accessible, affordable, and nutritionally comparable becomes increasingly important. One such readily available alternative, subject to a conclusive evaluation of its suitability, is vegetable waste, the utilization of which would also help address challenges associated with its disposal in landfills [10]. Cauliflower is a major winter vegetable cultivated in India for human consumption, with annual production reaching approximately 88 MT [11]. Of this total, nearly 30% is discarded as waste that cannot be recycled or salvaged for human consumption [12]. This vegetative waste releases methane during decomposition and contributes to environmental pollution. In cauliflower, the crown is the primary edible portion, while the surrounding tissues, including leaves and stalks, are typically discarded as waste. Notably, these discarded components are rich in energy, protein, phytochemicals, antioxidants, vitamins, and minerals [13,14,15]. Cauliflower waste contains more than 20% crude protein, which can substantially reduce the dependence on costly protein supplements in livestock feed [15]. This cost-effective feed resource not only supplies essential nutrients but also serves as a reservoir of health-promoting phytochemicals that enhance immune function and exhibit anti-methanogenic properties [13,16].

This study represents one of the first attempts to assess the nutritional value of cauliflower leaf meal (CLM) and to evaluate its suitability as an alternative feed resource in the context of a large livestock population, a scarcity of conventional feed materials, and the wide availability, affordability, and favorable nutritional and non-methanogenic properties of CLM. The use of cauliflower leaf meal as a protein-rich feed for goat production addresses both feed shortages and the environmentally sound disposal of an agricultural by-product.

2. Materials and Methods

2.1. Cauliflower Leaf Meal Preparation

Cauliflower (Brassica oleracea var. botrytis) leaves were collected from a local vegetable market during January 2022 (Supplementary Figure S1A,B). The leaves were thoroughly washed and chopped into 1–2 cm pieces using a chaff cutter to facilitate drying, followed by sun drying for 8 h to minimize the loss of phytonutrients (Supplementary Figure S1C). After wilting under sunlight, the leaves were transferred to a hot-air oven maintained at 50 °C and dried for 7–8 h to achieve more than 90% dry matter and to prevent fungal infestation. The dried leaves were then coarsely ground to a particle size of approximately 5 mm using a hammer mill (Agro Mech Hindustan Engineering, India), located at ICAR-IVRI, Izatnagar, Uttar Pradesh, India. Particle size distribution of the cauliflower leaf meal was determined using the sieve method with a set of sieves of 3, 5, and 10 mm mesh sizes [17]. The processed samples were subjected to chemical and proximate analyses and subsequently stored in airtight containers for further use in concentrate formulation. The durations of sun drying and oven drying, as well as the particle size required to maintain the proximate and chemical composition of cauliflower leaves, were standardized at the Unconventional Feed Laboratory, Division of Animal Nutrition, ICAR–Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.

2.2. Analysis of Proximate and Chemical Compositions of Cauliflower Leaf Meal (CLM), Feed, Fecal, and Urine Samples

2.2.1. Proximate Analysis

The proximate composition—moisture, crude fiber (CF), ether extract (EE), crude protein (CP), nitrogen-free extract (NFE), and total ash (TA)—of cauliflower leaf meal, offered and leftover feed from the trial, and fecal samples was analyzed according to the guidelines of the Association of Official Analytical Chemists (AOAC, 2012) [18]. Nitrogen content in urine samples was determined using the Kjeldahl method in accordance with AOAC (2012) guidelines [18].

2.2.2. Analysis of Fiber Fractions

The fiber fractions—acid detergent lignin (ADL), neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose, and cellulose—of cauliflower leaf meal, both offered and leftover feed, from the trial and fecal sample were determined using the Van Soest fiber fraction method given by Van Soest et al. (1991) [19].

2.2.3. Estimation of Minerals

Estimation of Calcium

The calcium (Ca) content in the CLM, offered and leftover feed, and fecal and urine samples were determined by the method of Talapatra et al. (1940) [20]. Calcium was estimated by precipitating Ca as insoluble calcium oxalate in an acidic medium maintained at pH 6.3 through the addition of a saturated ammonium oxalate solution. The precipitate was subsequently dissolved in dilute sulfuric acid, heated to liberate oxalic acid, and titrated against a standard N/10 potassium permanganate (KMnO₄) solution at 60 °C.

Estimation of Phosphorus

The phosphorus (P) content in CLM, offered and leftover feed, and fecal and urine samples were determined by the method of AOAC (2000) [21]. The phosphorus content was estimated from the mineral extract using UV–Visible spectrophotometer. The macroelement in mineral extract (aliquots prepared after total ash estimation was used for P analysis) reacts with molybdovanadate reagent to form a colored phopsphomolybdovanadate compound. The intensity of the color formed was directly proportional to the amount of phosphorus in the sample, which was measured at 400 nm.

2.2.4. Estimation of Vitamins

The vitamins A and E content of CLM were estimated with slight modifications [22]. The saponification and extraction of vitamins A and E were carried out and measured by reverse-phase HPLC equipped with Model-Shimadzu. For the estimation, samples were prepared by drying for 24 h at 60 °C and the dried samples were pulverized before extraction using a Wiley mill (fitted with 1 mm mesh sieve). Feed sample weighing 3 g was taken in a plastic bottle (sample vial), containing 20 mL of 6% pyrogallol (in ethanol) and 5 mL of 60% KOH, and heated on a water bath for 15 min at 70 °C. After cooling down, 45 mL hexane and 45 mL water were added in it and the bottle was gently shaken for 30 sec. Later, it was allowed to settle down for 30 min and centrifuged. The hexane layer was transferred to 150 mL flask. Then, the sample was re-extracted with additional 45 mL hexane by shaking it gently for 30 sec. After that, the sample was again allowed to stand for 30 min and the hexane layer was mixed with those from the previous extraction and allowed to evaporate on rotary evaporator to bring the volume down to 5 mL. This concentrated extract was used for HPLC analysis after being filtered through a 0.22 μm filter paper. The column 18 was used for vitamins A and E estimation. The identification and quantification of the peaks were estimated by comparing with vitamin A (β-carotene) and vitamin E (α-tocopherol) standards. The total vitamin A and E concentration was expressed in mg/g.

2.2.5. Total Phenolic Estimation

The TPC estimation of cauliflower leaf meal was carried out using Folin–Ciocalteu reagent [23]. The principle involves 0.1 mg/mL tannic acid, which reacts with Folin–Ciocalteu reagent at alkaline pH and gives maximum absorbance (λ_max) at 725 nm. TPC is estimated on dry matter basis.

2.2.6. Estimation of Glucosinolate

For glucosinate estimation [24], grounded sample (10 g) was deactivated by boiling for 5 min in 250 mL boiling water, following which the contents were filtered using buncher funnel and the resulting residue washed with warm water and made into 500 mL solution. Aliquot (25 mL) was taken into beaker and to it was added 10 mL of 0.1 N AgNO₃ solution and 25 mL of ethanol. The contents were refluxed for about 45 min in a water bath and left to cool. After cooling down to room temperature, 100 mL volume was prepared with distilled water (DW) and filtered through Whatman’s filter paper no. 40. An aliquot of 25 mL of supernatant was taken in a flask containing 2 mL of 6 N HNO₃ and 8% W/V ferric ammonium sulfate. The homogenous mixture was titrated against 0.01 N KSCN until a salmon color was obtained. A blank was also run with each determination. The percentage of glucosinolate (%) was calculated by following formula:

$$\mathrm{G}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{s}(\mathrm{\%})={\displaystyle \frac{\left(\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}-\mathrm{t}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\right)\times 4\times 0.01\times \mathrm{m}\mathrm{o}\mathrm{l}.\mathrm{w}\mathrm{t}.\mathrm{G}\mathrm{S}\mathrm{L}=411)\times (\mathrm{t}\mathrm{o}\mathrm{t}.\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}=500)}{1000\times 2\times \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} \mathrm{w}\mathrm{t}\times 25}}$$

(1)

2.2.7. Estimation of Gross Energy

The gross energy (GE) of CLM was determined by adiabatic-type bomb calorimeter (Gallenkamp auto bomb). For this procedure, a 5 g of sample was electrically ignited and burnt in excess of oxygen in the bomb. The maximum temperature rise was measured with the thermometer in a controlled system. By comparing this rise with the temperature when a sample of a known calorific value was burnt, the calorific value of the sample material was estimated. The bomb equivalent of the bomb calorimeter is determined with calorimetric-grade benzoic acid (gross energy content 6318 cal/g).

$$\mathrm{G}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{s} \mathrm{e}\mathrm{n}\mathrm{e}\mathrm{r}\mathrm{g}\mathrm{y}(\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}/\mathrm{g})={\displaystyle \frac{\left(\mathrm{B}\mathrm{o}\mathrm{m}\mathrm{b} \mathrm{e}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{e}\mathrm{n}\mathrm{t}\times \mathrm{T}\right)-\mathrm{A}}{\mathrm{T}\mathrm{h}\mathrm{e} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{h}\mathrm{e} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\left(\mathrm{g}\right)}}$$

(2)

where,

Rise in temperature (°C) = T

Correction factors for wire and thread = A

The metabolizable energy (ME) is calculated by multiplying the GE value by a factor of 0.82 (NRC, 1994) [25].

2.3. Animal Ethics Statement

The protocol of the present study was approved by the Institutional Animal Ethics Committee (IAEC) and carried out as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Fisheries, Animal Husbandry and Dairying (MoFAH&D), Government of India (Protocol No. IAEC/27.11.2021/L2, dated 10 February 2022).

2.4. Experimental Design and Animal Feeding

A total of 15 non-descript male goats aged 6–8 months with a starting average body weight (BW) of 10.96 ± 2.21 kg were procured from the Sheep and Goat Farm, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh and used for the 120-days feeding trial from 15 February 2022 to 15 June 2022. The animals were housed in the Animal Isolation Facility, ICAR-IVRI for 28 days for quarantine and acclimatization. The animals were tested for peste des petits ruminants virus (PPRV) and hemoparasites prior to the study, and found to be negative. Animals were dewormed and vaccinated against PPR. All the goats selected for the experimental trial were healthy. Three identical pens having same size, feeding, ventilation, lighting, etc. in the Animal House Facility of the Institute were allotted to three groups comprising five goats each for distinct dietary treatments. The cauliflower leaf meal was mixed in a concentrate mixture manually and the 20 and 30% inclusion levels were decided based on glucosinolate tolerance level and the references from other study [16]. For the experiment, dietary treatments were as follows: T0, control group [concentrate mixture consisted of maize, soybean meal (SBM), wheat bran, mineral mixture, and salt], hybrid Napier, and wheat straw; T20, concentrate mixture containing 20% CLM, hybrid Napier, and wheat straw; and T30, concentrate mixture containing 30% CLM, hybrid Napier, and wheat straw. The level of CLM in feed was decided on the basis of weight of individual goats. The experimental goats were fed with a concentrate mixture, wheat straw, and hybrid Napier to fulfill the nutrient requirements as per the ICAR (2013) feeding standard [26]. The concentrate was iso-nitrogenous. A roughage and concentrate ratio of 50:50 was maintained for experimental trial. Clean drinking water was ensured for the experimental goats ad libitum. The goat shed was maintained in strict hygienic condition with regular cleaning to ensure optimal sanitation to minimize infection risks. Humidity and temperature were maintained at 60% and 24–26 °C, respectively, by providing air inlet fan systems and heater.

2.5. Measurement of the Growth Performance

2.5.1. Body Weight Changes

Growth performance was measured by recording the individual weights of goats at two weeks interval using an electronic weighing balance during the entire course of the study spanning 120 days. Weighing was conducted before the provision of feed and water throughout the experimental period.

2.5.2. Estimation of Dry Matter Intake (DMI), Average Daily Gain (ADG) and FCR

The dry matter intake (DMI), comprising concentrate mixture, wheat straw, and hybrid Napier, was recorded daily for each individual goat. The average daily gain (ADG) was calculated. From dry matter intake and weight gain, the feed conversion ratio (FCR) was calculated:

ADG = final body weight − Initial Body weight/15

(3)

FCR = DMI/Weight gain

(4)

2.6. Estimation of Methane Emission

The methane estimation study was conducted midway through the growth and feeding trial, after the goats had adequately adapted to the test feed. During the final month of the trial, cell-mediated and humoral immunity studies were initiated to check the immunity status of goats. Following the completion of the immunity study, metabolic trials were conducted. For methane estimation study, the goats were weighed using an electronic weighing balance and then shifted to an open-circuit respiration chamber maintained at 25 °C and 65% relative humidity. Average daily dry matter intake (ADDMI) was calculated based on the intake of concentrate mixture, hybrid Napier, and wheat straw. The daily organic matter intake (DOMI) of each goat was estimated by subtracting total ash content of feed from 100. The dimensions of the respiratory chamber used for goats were 146 × 91 × 172 cm (2.285 m³). Prior to measurements, goats were acclimatized to the chamber for 48 h, followed by observations recorded over three consecutive days. The air flow rate within the chamber was maintained at 50 L/min and the total volume (L/d) of air passing through the chamber was measured. Methane concentrations in air flowing in and out of the chamber was measured using an automatic infrared methane analyzer (Model 300; Analytical Development Co. Ltd., Hoddesdon, UK).

The total volume of CH₄ production was computed by formula as given below:

CH₄ (L) = V_STP (M_f − M_i)/10

(5)

V_STP = Volume at standard temperature and pressure

M_f = CH₄ existing in outgoing air from chamber

M_i = CH₄e existing in incoming air into the chamber

2.7. Rumen Fermentation Study

Rumen fermentation study was carried out to study the effects of the various bioactive compounds such as TPC, vitamins A and E, minerals such as Ca and P, and secondary plant metabolites such as saponin, tannin, glucosinolates, etc. contained in cauliflower leaf meal. The rumen fermentation study was conducted following the conclusion of the methane emission study. For the study, 50 mL rumen liquor was collected from all goats using PVC stomach tubes (1 m length and 8 mm external diameter). The extracted rumen liquor was transferred to a thermo-flask (prewarmed to be maintained at 39 °C) which was sealed immediately to maintain anaerobic conditions. The rumen liquor thus collected was filtered through two layers of muslin cloth prior to analysis.

2.7.1. Measurement of Rumen Liquor pH

The pH of rumen liquor was measured using pocket-sized portable pH meter immediately following the transfer of rumen liquor to laboratory as any delay may alter the rumen liquor quality due to ongoing microbial activity.

2.7.2. Measurement of Protozoa Count

Ciliate protozoa were counted using a microscope (Olympus CX-23 LED binocular microscope, Tokyo, Japan). For counting protozoa, one volume of methyl green formal saline solution was mixed with one volume of rumen liquor. For calculation, two drops of stained rumen fluid were placed on a clean hemocytometer and the coverslip was carefully placed to avoid the formation of air bubbles under the cover slip. The total protozoal count was determined by counting 30 microscopic fields under 10× magnification, leaving the corners. The number of protozoa/mL of rumen liquor was calculated by the following formula:

N = n × A × D/a × V

(6)

where, N—number of protozoa/mL of rumen liquor, n—average number of cells per microscopic field,

Area of the haemocytometer chamber—A,

Dilution of rumen liquor—D,

Area of microscopic field—a, and

A volume of diluted rumen liquor in the cavity—V.

2.7.3. Measurement of Bacterial Count

The total bacterial count in rumen liquor was estimated using the most probable number (MPN) technique. The tubes were incubated for 15 days at 39 °C and changes in pH were monitored. The tubes of different media were inoculated as detailed below. Dilutions ranging from 10⁸ to 10¹³ were prepared for the estimation of total bacterial count. The tubes that presented a fall in pH of 0.3 units were marked and selected from three consecutive dilutions. This was calculated as follows:

No. of cells/g = MPN × dilution

(7)

2.7.4. Assessment of Total Volatile Fatty Acids (VFA)

The measurement of total volatile fatty acids (VFAs) and its fractions viz. acetic, propionic, and butyric acids in the rumen liquor was carried out using gas liquid chromatography (GLC). The concentration of various VFAs in rumen liquor was determined using Nucon-5765 gas chromatograph (AMIL, New Delhi, India) equipped with a flame ionization detector and a glass column (4-foot length and 1/8-inch diameter) packed with Chromosorb 101. In GLC, the mobile phase was carrier gas and the stationary phase was liquid, which was adsorbed onto an inert solid matrix.

2.7.5. Estimation of Ammonia-Nitrogen

For the estimation of ammonia-nitrogen, one drop of 20% sulfuric acid was added to each 5 mL sample of strained rumen liquor and stored at –20 °C. Ammonia-nitrogen in rumen liquor was estimated by the method of Weatherburn (1967) [27]. Then, 0.9 mL of Distilled Water (DW) was added to 0.1 mL of rumen liquor and mixed in 5.0 mL of solution A (6 g phenol was dissolved in 50 mL of DW, 5.0 mg of sodium nitroprusside was added, and volume was made up to 100 mL with DW) and 5.0 mL of solution B (0.5 g of sodium hydroxide was dissolved in 50 mL of DW). Afterwards, 0.84 mL of sodium hypochlorite was added and the volume was made up to 100 mL with DW. The solution thus obtained was thoroughly mixed. The tubes were then incubated for 5 min at 39 °C for color development. Samples were read spectrophotometrically at 625 nm against a blank. In a similar way, standard samples (ammonia-nitrogen concentration ranging from 0.5 to 10.0 μg) were analyzed and a calibration curve was plotted. From the standard curve, the concentration of the unknown sample was calculated.

2.8. Hematological Analysis

For hematological analysis, blood samples of 5 mL each were collected from the jugular veins of all the goats at 0, 60, and 120 days of research trial. White blood cells (WBCs), Mean Corpuscular Hemoglobin (MCH), red blood cells (RBCs), and Mean Corpuscular Volume (MCV) were estimated using hematocrit analyzer [28].

2.9. Assessment of Immunity Status of Goats

At the last month of trial, cell-mediated and humoral immunity studies were conducted to check the immunity status of goats. Humoral immunity status in goats was checked through qualitative measurement of production of antibody in response to intravenous injection of 0.5 mL of 20% chicken red blood cells (CRBCs). The antibody production against CRBCs was estimated from serum collected on 7, 14, 21, and 28 days post-injection. After humoral immune response study, the cell-mediated immune response was assessed through a delayed type hypersensitivity (DTH) reaction against phytohemagglutinin-P (PHA-P) [29].

2.10. Metabolic Trial

For the study of the nutrient digestibility and nutrient balance, an 8-day metabolic trial was carried out at the end of the trial involving the quantitative collection of fecal pellets and urine for six days, including the 2-day suitable adaptation period in metabolic cages. The goats were weighed before and after the metabolic trial. During the collection period of 6 days, the samples of feed offered, leftover feed residues, fecal pellets, and urine were collected at 24 h interval for further sampling in laboratory and analysis to determine nutrient digestibility and nitrogen, calcium, and phosphorus balance. The urine was collected in plastic cans which were kept underneath the metabolic cages of experimental goats containing 20 mL of 10% sulfuric acid to maintain the pH below 3. The cumulative samples of individual goats were collected over the collection period, and, following the completion of metabolic trials, the pooled samples were pulverized to pass through 2 mm screen and preserved in airtight plastic containers for evaluation.

2.11. Statistical Analysis

All the data used for completely randomized design were assessed using Excel version, 2021 and analyzed using SPSS version 26.0 [30,31]. Duncan multiple-range tests were performed to determine the group differences. Treatment groups were considered as a unit for the evaluation of growth performance, nutrient digestibility, methane emission, rumen fermentation, metabolic trial, and economics of production. A significant difference was measured at the p-value < 0.05.

3. Results

3.1. Proximate and Chemical Compositions of CLM and Composition and Nutrients Level of Experimental Diet

The proximate and chemical compositions of CLM are depicted in

Table 1. Proximate and chemical compositions of cauliflower leaf meal (CLM).

Attributes	Cauliflower Leaf Meal (CLM)
Dry matter (DM, %)	97.5
Organic matter (OM, %)	88.68
Crude protein (CP, %)	21.5
Ether extract (EE, %)	7.33
Crude fibre (CF, %)	11.47
Total ash (TA, %)	11.32
Nitrogen-free extract (NFE, %)	48.38
Neutral detergent fibre (NDF, %)	40
Acid detergent fibre (ADF, %)	28
Lignin (%)	11.78
Hemicellulose (%)	12
Cellulose (%)	16.22
Calcium (%)	0.23
Phosphorous (%)	0.20
Vitamin A (mg/g)	0.2
Vitamin E (mg/g)	2.5
Glucosinolate (%)	0.04
Total phenolic content (TPC) (mg of GAE/100 g)	8.80
Gross Energy (kcal/kg)	2560
Metabolizable energy (kcal/kg) (Calculated)	2099

. The CLM contained 21.5% CP, 11.17% CF, 7.33% EE, and 11.32% TA. The metabolizable energy level was calculated as 2099 kcal/kg. The glucosinolate content was 0.04%. The CLM was rich in Ca (0.23%), P (0.20%), Vit. A (0.2 mg/g), Vit. E (2.5 mg/g), and TPC [8.80 mg of gallic acid equivalent (GAE)/100 g].

The ingredient composition and nutrient levels of experimental concentrate feed is presented in

Table 2. Ingredients composition and nutrient levels of experimental concentrate feed on dry matter basis (%).

Ingredients (kg)	T0	T20	T30
Maize	41	45	42.5
Soybean meal (SBM)	17	16	12.5
Wheat bran	39	19	15
Cauliflower leaf meal (CLM)	0	20	30
Mineral mixture	2	2	2
Salt	1	1	1
Total	100	100	100
Nutrient levels (%)
Crude protein (CP, %)	19.13	18.69	18.56
Crude fibre (CF, %)	8.43	8.51	5.56
Neutral detergent fibre (NDF, %)	32.56	33.60	34.12
Acid detergent fibre (ADF, %)	19.55	17.11	16.25
Acid detergent lignin (ADL, %)	10.11	10.18	11.25
Hemicellulose (%)	10.12	10.48	11.02
Cellulose (%)	13.25	14.75	15.82
Total ash (%)	4.33	5.28	5.97
Calcium (%)	0.78	0.82	0.87
Phosphorous (%)	0.68	0.74	0.79

Note: T0: Control group; T20: 20% CLM fed group; T30: 30% CLM fed group.

. Goats in T0 group were fed with basal feed composed of maize, SBM, wheat bran, salt, and mineral mixture. In the T20 and T30 groups, CLM was added to the basal feed and constituted 20% and 30% of the whole, respectively. Crude protein levels were estimated at 19.13, 18.69, and 18.56%, respectively, in the diets of the T0, T20, and T30 groups, which fully satisfy the protein requirement of goats under ICAR (2013) [26] feeding standards. The concentrate supplementing CLM was rich in minerals in comparison to the control feed. The CP, NDF, ADF, cellulose, and hemicellulose contents of the feed of all three diets were similar.

3.2. Growth Performance

3.2.1. Body Weight Changes (BW), DMI and FCR

Throughout the experimental period, the fortnightly body weight changes, DMI, and FCR of the experimental goats were closely monitored and no significant (p > 0.05) effects of dietary CLM feeding were observed on the final body weight changes, average daily gain, dry matter intake, and feed conversion ratio (

Table 3. Effect of feeding cauliflower leaf meal (CLM) on fortnightly body weight changes, dry matter intake (DMI) and feed conversion ratio (FCR) of goats.

Days Post Experimental Body Weight	Dietary Treatment			Standard Error of the Mean (SEM)	p-Value
	T0	T20	T30
0	10.79	10.92	11.18	0.66	0.842
15	11.02	11.66	11.77	0.27	0.05
30	11.67	11.86	12.09	0.36	0.525
45	12.78	13.13	13.39	0.29	0.163
60	13.49	14.17	14.39	0.28	0.210
15	14.04	14.10	14.53	0.44	0.501
90	13.92	14.96	15.02	0.38	0.023
105	14.89	15.38	15.70	0.52	0.092
120	15.22	15.41	16.40	0.69	0.226
Total body weight gain (kg)	4.43	4.46	5.22	6.20	0.75
Average daily gain (ADG, g)	48.27	50.13	52.42	5.24	0.71
Feed conversion ratio (FCR)	8.67	8.31	8.04	0.62	0.60
Dry matter intake (DMI, g/d)	419.73	421.06	424.12	50.83	0.99

Note: T0: control group; T20: 20% CLM-fed group; T30: 30% CLM-fed group. Duncan multiple-range test was performed to determine the group differences. A significant difference was considered when the p-value < 0.05.

). No linear and quadratic effects were observed in these parameters (p > 0.05).

3.2.2. Nutrients Digestibility and Balance

The effect of dietary CLM inclusion on the intake and digestibility of nutrients is presented in

Table 4. Effect of feeding cauliflower leaf meal (CLM) on the nutrient intake and digestibility of nutrients in goats.

Attributes (%)	Dietary Treatments			Standard Error of the Mean (SEM)	p-Value
	T0	T20	T30
Dry matter (DM) digestibility	77.20	78.61	78.65	2.05	0.77
Organic matter (OM) digestibility	87.95	87.47	89.40	2.42	0.24
Crude protein (CP) digestibility	80.64	81.00	81.96	1.62	0.70
Ether extract (EE) digestibility	82.35	81.59	80.02	2.09	0.54
NFE digestibility	87.40	89.45	89.52	0.66	0.10
Neutral detergent fibre (NDF) digestibility	46.69	47.55	45.78	2.41	0.77
Acid detergent fibre (ADF) digestibility	34.66	34.97	35.26	2.56	0.05
Acid detergent lignin (ADL) digestibility	21.67	20.45	20.21	3.01	0.67
Hemicellulose digestibility	25.73	27.52	27.05	2.02	0.46
Cellulose digestibility	25.36	24.55	23.60	2.18	0.72

Note: T0: Control group; T20: 20% CLM fed group; T30: 30% CLM fed group.

. The intake (g/day) and digestibility (%) of DM, CF, OM, EE, CP, NFE, ADF, NDF, hemicellulose, and cellulose showed non-significant changes (p > 0.05) in all three groups. The nutrient balance for the experimental goats is presented in

Table 5. Effect of feeding cauliflower leaf meal (CLM) on nutrient balance of experimental goats.

Treatments	Nitrogen (N) Balance			Calcium (Ca) Balance			Phosphorous (P) Balance
	N Intake (g/d)	N Retension (g/d)	N Retension as Intake (%)	Ca Intake (g/d)	Caretension (g/d)	Caretension as Intake (%)	P Intake (g/d)	P Retension (g/d)	P Retension as Intake (%)
T0	12.40	3.34	26.86	5.59	2.03	60.62	3.17	1.09	67.59
T20	12.42	4.08	32.62	5.33	1.94	60.43	3.25	1.13	67.49
T30	12.25	4.05	32.81	5.64	2.24	58.48	3.20	1.27	67.88
Standard error of the mean (SEM)	0.55	0.55	3.09	0.47	0.61	8.65	0.43	0.01	0.44
p-value	0.95	0.35	0.14	0.70	0.89	0.96	0.98	0.85	0.51

Note: T0: control group; T20: 20% CLM-fed group; T30: 30% CLM-fed group. Duncan multiple-range test was performed to determine group differences. A significant difference was considered when the p-value < 0.05.

. There were no significant changes (p > 0.05) in calcium, phosphorous, and nitrogen intake, excretion, and retention among experimental groups. All the experimental goats maintained positive nitrogen, calcium, and phosphorous balances.

3.3. Methane Emission Study

The CLM inclusion in the feed did not affect the average DMI (kg/day) of the goats in the respiration chamber. The methane emission rate was significantly lower (p < 0.05) in the T30 group of goats as compared to the T0 and T20 groups. Among the treatment groups, a significant reduction (p < 0.05) in the methane emission rate in terms of the g/kg daily organic matter intake (DOMI) and ADDMI were the maximum in the T30 group of goats fed 30% CLM, followed by the T20 and T0 groups (

Table 6. Effect of feeding cauliflower leaf meal (CLM) on methane emission rate in goats.

Attributes	Dietary Treatments			Standard Error of the Mean (SEM)	p-Value
	T0	T20	T30
Body weight (kg)	14.05	14.50	15.7280	0.86	0.175
dry matter intake (DMI, g/d)	415.80	420.40	422.6000	3.44	0.174
Methane (g/day)	12.43 c	10.98 b	9.11 a	0.55	0.001
Methane (g/kg DMI)	35.36 c	29.09 b	24.89 a	0.70	0.001
Methane (g/kg DDMI)	41.44 c	39.48 b	33.49 a	1.10	0.001
Methane (g/kg OMI)	34.57 c	32.15 b	28.43 a	0.99	0.001
Methane(g/kg DOMI)	43.43 c	42.62 b	35.32 a	1.31	0.001

Note: T0: control group; T20: 20% CLM-fed group; T30: 30% CLM-fed group. Duncan multiple-range test was performed to determine the group differences. A significant difference was considered when the p-value < 0.05. Values with a,b,c superscripts in a row differ significantly. Values without superscripts in a row differ non-significantly.

).

3.4. Rumen Fermentation Study

The effect of CLM on rumen fermentation was presented in

Table 7. Effect of feeding cauliflower leaf meal (CLM) on rumen fermentation in goats.

Attributes	Dietary Treatment			Standard Error of the Mean (SEM)	p-Value
	T0	T20	T30
pH	6.46	6.52	6.4	0.23	0.69
Ammonia-Nitrogen (µg/mL)	6.14	6.09	6.11	0.03	0.56
Fractions of VFA
Acetate (%)	64.93	63.79	65.27	5.73	0.96
Propionate (%)	23.47	23.82	21.67	2.71	0.76
Butyrate (%)	11.61	12.02	12.94	1.59	0.75
Total volatile fatty acids (TVFA, mmol/dL)	15.56	16.88	16.18	1.15	0.53
Protozoa (106/mL)
Oligotrichs	5.16	5.13	5.1	0.04	0.78
Holotrichs	4.39	4.36	4.31	0.06	0.79
Total bacteria (109/mL)	10.16	10.91	10.87	0.4	0.14

Note: T0: control group; T20: 20% CLM-fed group; T30: 30% CLM-fed group. Duncan multiple-range test was performed to determine the group differences. A significant difference was considered when the p-value < 0.05.

. It was observed that the rumen pH was in the range of 6.40 to 6.52, with non-significant differences (p > 0.05) among all the experimental groups. The concentration (µg/mL) of ammonia, total volatile fatty acids such as acetate, propionate, and butyrate, and the total bacterial count in rumen liquor were affected by the dietary inclusion of CLM in the T20 and T30 groups compared to the T0 group. The oligotrichs and holotrichs protozoa population were also found to be similar (p > 0.05) among the experimental groups of goats.

3.5. Hematology Study

The mean RBC (10⁶/µL) values were 9.97, 10.44, and 10.89 in the T0, T20, and T30 groups, respectively. The mean WBC (10³/µL) values were 11.97, 12.12, and 12.99 in the T0, T20, and T30 groups, respectively. The mean MCV (fl) values were 14.01, 13.18, and 12.53 in the T0, T20, and T30 groups, respectively. The mean MCH (pg) values were 7.12, 7.19, and 7.24 in the T0, T20, and T30 groups, respectively. The RBC, WBC, MCV, and MCH values remained (p > 0.05) unaffected by CLM feeding in all the experimental groups.

3.6. Immunity Status of Goats

The humoral immune response of the experimental kids under different dietary groups was measured towards the end of the trial. The antibody titer (in log ₂) was significantly higher (p < 0.05) up to 21 days post inoculation. In the humoral immunity assessment, the skin thickness absolute value was the highest at 24 h, and then was gradually reduced until 72 h post-injection, which was the typical immunity pattern. The mean absolute and relative response showed a significant result among the CLM-fed treatment groups and for different periods.

4. Discussion

Feedstuff availability remains a major challenge confronting the livestock sector in India, with future projections appearing even less promising when the anticipated impacts of climate change are taken into account. Demand–supply mismatches of this nature push up the prices of conventional feed ingredients to unsustainable levels. The current market prices of soybean (₹75/kg), maize (₹35/kg), and wheat bran (₹40/kg), among others, underscore the strain the sector is likely to experience in the coming years if alternative feed resources comparable in nutritional value and free from adverse effects on animal health are not identified and introduced without delay.

Vegetable waste, or portions of vegetable produce traditionally discarded after the economically valuable parts are removed, represents one of the most viable alternatives due to its substantial nutritional composition [3]. If proven safe and productive, such utilization can simultaneously address feedstuff shortages and mitigate the environmental degradation resulting from the accumulation and decomposition of organic waste in open environments or landfills. Discarded fruit and vegetable residues are rich in bioactive compounds that confer health benefits to livestock [32]. Their repurposing as feed resources also reduces feed costs for farmers while alleviating the environmental burden.

Nutritional evaluation studies of vegetable waste have revealed that cauliflower leaves are rich in essential nutrients. In addition to their nutrient content, cauliflower leaf meal (CLM) contains tannins and saponins, which exhibit antibacterial [33], antiviral, and antiparasitic activities [34]. These bioactive compounds also contribute to reduced methane emission and improved animal performance. The present study aimed to evaluate the effects of CLM on growth performance, rumen fermentation, methane emission, and hematological and immune status, and to quantify its impact on production economics. In doing so, the study seeks to bridge the research gaps that have hitherto existed across these parameters.

The cauliflower leaf meal used in the study was prepared by sun-drying chopped cauliflower leaves. The resulting CLM was found to be high in crude protein (21.5%) and crude fiber (11.47%) and rich in total phenolic content. Glucosinolate levels were reduced through thermal degradation during chopping and drying, rendering the feed safe for animal consumption. Owing to its protein content, mineral composition (Ca and P), bioactive compounds, vitamin content, and total phenolics, CLM was considered safer than several other brassica-based feedstuffs.

The number of animals per dietary treatment was fixed at five, in accordance with the guidelines for large-animal experimental research, to generate statistically meaningful data [31,35,36]. Experimental diets were formulated into three treatments: a control group (T0) fed a conventional diet, and two treatment groups (T20 and T30) receiving diets containing 20% and 30% CLM, respectively, on a total feed weight basis.

In the present study, the dietary inclusion of cauliflower leaf meal (CLM) at 20% and 30% levels in the concentrate mixture had no adverse effects on the final body weight, dry matter intake (DMI), average daily gain (ADG), or feed conversion ratio (FCR). These findings are in agreement with those of Partovi et al. (2020), who reported that lambs fed a mixture of broccoli (Brassica oleracea) by-product (100 or 200 g/kg diet DM) and wheat straw silage did not exhibit significant differences (p > 0.05) in body weight or FCR [37]. Similarly, no adverse effects on productive performance were observed with the incorporation of 9% broccoli stem and leaf powder in the diets of laying hens [38]. The present findings are also consistent with studies involving the inclusion of vegetable waste in goat diets, wherein body weight gain remained unaffected (p > 0.05) [39]. In contrast, some studies have reported differing outcomes. The dietary inclusion of kale (Brassica oleracea) and grass silage at ratios of 0:100 (K0), 60:40 (K60), 85:15 (K85), and 100:0 (K100) resulted in a reduced dry matter intake and lower live weight gain in dairy cows [40]. Nevertheless, in the present study, feed consumption remained unaffected by CLM supplementation, possibly due to the comparable nutrient digestibility among diets, particularly neutral detergent fiber (NDF) digestibility [41]. Notably, NDF digestibility is considered a reliable predictor of dry matter intake [42]. The improved growth performance observed may be attributed to the presence of phytonutrients in CLM, including nitrogen, calcium, and phosphorus, which promote favorable digestion under varying gastrointestinal conditions influenced by chemical factors, enzyme activity, pH, and temperature [14]. Nutrient intake and digestibility remained unaffected by the inclusion of CLM in goat diets. These findings align with studies conducted on South African Dorper lambs fed cabbage waste [43] and lambs fed wheat straw–broccoli by-product silage [37]. However, increasing levels of discarded cabbage in lamb diets were associated with reductions in organic matter digestibility (from 73% to 65%) and NDF digestibility (from 56% to 47%) [43]. The digestibility of most nutrients, except NDF, was reported to be similar for cabbage and cauliflower leaves in buck diets [44]. Conversely, significant differences in nutrient digestibility were observed in goats fed diets containing 20% broccoli [45], while an increased digestibility of crude protein (CP) and nitrogen-free extract (NFE) was reported in sheep fed diets of varying levels of broccoli plant waste [46].

The nitrogen, calcium, and phosphorus intake, excretion, and retention in the experimental groups were comparable to those reported in studies on South African Dorper lambs fed cabbage waste [43] and lambs fed broccoli by-product–wheat straw silage [37], with no significant effects on the nitrogen balance. Similarly, goats fed diets containing mustard (Brassica juncea) cake, with or without iodine supplementation, exhibited no significant differences in calcium and phosphorus intake and retention, or fecal and urinary excretion [47].

Methane is generated in the rumen through the microbial fermentation of feeds that are rich in fiber. Diets with a high fiber content are strongly associated with reduced digestibility and increased methane emissions [48]. In the present study, the methane emission was observed to decrease with the inclusion of cauliflower leaf meal in the diet. This reduction can be attributed to the presence of plant secondary metabolites, such as saponins and tannins, which are known to suppress methanogenesis.

Giller et al. (2022) conducted an in vitro study evaluating the effects of aronia, orange, pomegranate, apple, red grape, white grape, and three vegetables (beetroot, carrot, and tomato), and reported that the inclusion of pomegranate at 500 g/kg significantly (p < 0.05) reduced methane production by 28% without adversely affecting nutrient digestibility [49]. This effect was attributed to the high polyphenol content, highlighting pomegranate as a promising unconventional feed resource [49]. Further reports have shown that the use of processed vegetable waste as feed reduced annual methane emissions by 0.43 Gg while also lowering food–feed competition for cultivable land [50]. Sahoo et al. (2021) reported that the inclusion of fruit and vegetable waste biomass in sheep diets significantly (p < 0.05) reduced annual methane emissions by 3.12% and nitrous oxide (N₂O) emissions by 15.18% [3].

Certain plant secondary metabolites (PSMs), including phenolic monomers, tannins, and saponins, have been reported to be toxic to several rumen microorganisms, particularly ciliate protozoa, fiber-degrading bacteria, and methanogenic archaea. This antimicrobial activity can inhibit methanogenesis in the rumen [50]. It can therefore be reliably inferred that the presence of these compounds contributed to methane mitigation in the present study. Additionally, the physical and chemical processing of forage has been shown to improve feed intake, nutrient utilization, and animal performance [51]. Accordingly, the processing of cauliflower leaves for feed use appears to have positively influenced both animal health and methane mitigation in this study.

Overall, the rumen fermentation parameters remained comparable across all experimental groups. The rumen pH was unaffected (p > 0.05) among the three groups of goats. Similarly, the total volatile fatty acid concentration, individual volatile fatty acid fractions (acetate, propionate, and butyrate), ammonia-nitrogen levels, protozoal counts, and total bacterial counts did not differ significantly among groups. Importantly, the inclusion of cauliflower leaf meal did not adversely affect the rumen fermentation patterns, likely due to the similarities in nutrient digestibility and ruminal fermentation characteristics among the diets.

These findings are consistent with studies conducted in sheep fed diets containing 15% cabbage (Brassica oleracea var. capitata) waste [52]. In another study, goats fed diets containing 0%, 5%, and 10% Moringa oleifera leaves showed no significant effects on rumen fermentation, which was attributed to the presence of saponins [53]. Similar observations were reported in dairy cows fed kale-based diets, which showed no significant changes in the acetate-to-propionate ratio [40]. Likewise, no significant effects (p > 0.05) on the volatile fatty acid fractions were observed in goats fed grass silage diets [54]. In contrast, Mukodiningsih et al. (2018) reported that a diet containing 6% fermented cabbage waste resulted in significantly lower (p < 0.05) ruminal NH₃-N and volatile fatty acid concentrations in cows when compared with the standard diet [55].

In the present study, red blood cell (RBC), white blood cell (WBC), Mean Corpuscular Volume (MCV), and Mean Corpuscular Hemoglobin (MCH) values were not affected in goats fed diets containing 20% and 30% cauliflower leaf meal (CLM) [28]. These findings were in agreement with previous studies, which reported no significant differences (p > 0.05) in goats fed diets containing 25% and 50% unconventional protein-rich karanj (Pongamia pinnata) cake [56]. The results of the present study are further supported by Kumar et al. (2019), who reported an improved cell-mediated and humoral immunity in lambs fed sugarcane press mud [57]. Similarly, Jadhav (2017) observed enhanced cell-mediated and humoral immune responses in kids fed Moringa oleifera leaves [53]. Improved cell-mediated and humoral immunity was also observed in the CLM-fed groups in the present study. However, these findings differ from those of Natarajan (2020), who reported only non-significant (p > 0.05) changes in humoral immunity in lambs fed cabbage waste [52]. Likewise, studies in broilers fed vegetable waste reported no significant changes in immunity status [58].

Cauliflower leaves are an agricultural by-product that is widely discarded across farms and markets. The cost of processing this waste material into feed is approximately Rs. 10/kg. Given the health and nutritional benefits of cauliflower-leaf-based feed and its affordability compared with conventional feeds, which typically cost Rs. 35–40/kg, CLM represents a suitable alternative feed resource. These findings are consistent with studies in sheep fed carrot and cabbage waste, which reported reduced rearing costs [59]. Similar reductions in feeding costs were also reported in lambs fed diets containing 15% cabbage waste [52] and in pigs fed diets containing 15% cauliflower leaves [60]. Additionally, a 9% reduction in feeding cost was observed in rabbits fed diets containing 30% dates and apricot kernel meal [61].

Cauliflower leaves contain both nutritional and antinutritional compounds. Glucosinolates present in cauliflower leaves can interfere with iodine uptake and thyroid hormone synthesis, leading to goitrogenic effects. This limitation can be mitigated through processing methods such as chopping [62], followed by sun drying and hot-air oven drying [63], which reduce glucosinolate concentrations to tolerable levels. Furthermore, ruminants possess some capacity to tolerate glucosinolates due to the microbial activity in the rumen. Dietary glucosinolate levels below 10 µmol g⁻¹ have been reported to have no adverse effects on nutrient intake or digestibility in young lambs, whereas concentrations above this threshold may reduce the growth rate [63]. In the present study, CLM inclusion was therefore limited to 30% to maintain the glucosinolate levels within safe limits. Accordingly, diets containing up to 30% CLM can be considered safe for goats.

Future research may focus on improving CLM through the addition of probiotics and enzymes, as well as exploring methods to further reduce antinutritional factors that currently limit CLM inclusion to 30%. Additionally, advanced approaches such as nutrigenomics could be employed to study the effects of CLM on gene expression related to nutrient metabolism and immune function.

5. Conclusions

The inclusion of cauliflower leaf meal (CLM) in the diet had no adverse effects on dry matter intake (DMI), feed conversion ratio, growth performance, nutrient digestibility, or rumen fermentation in goats. Furthermore, dietary supplementation with CLM significantly reduced methane emissions, indicating positive environmental implications. Cauliflower leaf meal is considerably cheaper than conventional protein-rich feeds and is safe for use when its inclusion level is limited to 30% of the diet on a weight basis, making it an affordable alternative for farmers.