*2.3. Fermented Discarded Durian Peel Preparation*

Discarded durian peel (Monthong-*Durio zibthinus Murray*) was obtained from Seahorse Intertrade Company Limited in Chana District, Songkhla Province, Thailand, and cut into 1 to 2 cm pieces. Then, discarded durian peel was fermented with the respective additives including molasses at 5% [5], cellulase at 2% [14], and *L. casei* TH14 at 1.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cfu/g fresh matter [4]. Cellulase (powder form, 5 <sup>×</sup> <sup>10</sup><sup>5</sup> U/g activity, CAS number: 9004-34-6, Sinobios Imp. & Exp., Thanghai, China) and *L. casei* TH14 as a silage starter (composed of 80% trehalose, 15% lactose, and 1.0 <sup>×</sup> <sup>10</sup><sup>11</sup> cfu/g *L. casei* TH14; Bio Ag Khon Kaen, Khon Kaen, Thailand) were used. Molasses was purchased from a local supplier located in Hat Yai District, Songkla Province, Thailand. Additives were dissolved in clean water, sprayed on discarded durian peel, mixed well, and fermented in 50 L plastic buckets (Changzhou Treering Plastics Co., Ltd., Changzhou, China) for 30 days. After fermenting for 30 days, fermented discarded durian peel samples were collected, dried at 60 ◦C for 72 h, and ground into 1 mm pieces to analyze the dry matter (DM), CP, and ash according to AOAC [15], and neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin according to Van Soest et al. [16]. The chemical composition of fermented discarded durian peel is provided under Table 1. Fermentation characteristics of fermented DP were assessed. pH was measured according to Chen et al. [14] using pH meter (HANNA instruments HI 98153 microcomputer pH meter, Kallang Avenue, Singapore); briefly, 20 g of fermented DP samples were taken and mixed with 80 mL of distilled water and kept at 10 ◦C for 24 h. Samples were prepared with ammonia nitrogen (NH3–N) using spectrophotometer (UV/VIS Spectrometer, PG Instruments Ltd., London, UK) and volatile fatty acids (lactic acid, acetate, and butyrate) using gas chromatography and analyzed according to So

digestible nutrient intake of growing goats.

Apparent total tract digestibility, %

Digestible nutrient intake, kg/d

Estimated energy intake ⸶

*Fermentation* **2022**, *8*, x FOR PEER REVIEW 6 of 14

**Table 3.** Effects of untreated and treated discarded durian peel in TMR on nutrient digestibility and

**Items Dietary Treatments SEM** *<sup>p</sup>***-**

DM 70.42 b 72.79 a 73.91 a 74.07 a 73.81 a 0.45 <0.01 OM 71.82 b 73.95 a 75.02 a 75.48 a 75.16 a 0.49 <0.01 CP 68.42 c 71.11 b 72.66 ab 73.07 ab 73.63 a 0.61 <0.01 NDF 63.83 b 70.48 a 70.23 a 70.96 a 71.03 a 0.47 <0.01

OM 0.543 0.515 0.534 0.562 0.536 0.02 0.47

ADF 0.098 0.102 0.102 0.102 0.092 <0.01 0.38

**FDP FDPM FDPC FDPL FDPML Value** 

et al. [8] The pH, NH3–N, lactate, acetate, and butyrate of fermented discarded durian peel are provided in Table 1. CP 0.087 0.084 0.086 0.090 0.087 0.01 0.68 NDF 0.338 0.332 0.307 0.325 0.307 0.01 0.21



between FDP and FDPML, at 32.93 g/d and 29.41 g/d, respectively, but FDP, FDPM, FDPC, and FDPL were comparable for CH4 production. FDP = untreated discarded durian peel in TMR; FDPM = treated discarded durian peel with molasses in TMR; FDPC = treated discarded durian peel with cellulase in TMR; FDPL = treated discarded durian peel with *L. casei* TH14 in TMR; FDPML = treated discarded durian peel with molasses and *L. casei* TH14 in TMR. TMR compositions contain 25% fermented discarded durian peel with or without additives, 15% rice straw, 35.8% ground corn, 7.9% soybean meal, 0.4% fish meal, 5.4% leucaena meal, 7.2% palm kernel cake, 2.2% molasses, 0.3% dicalcium phosphate, 0.2% salt, and 0.6% premix. Premix per kg contains vitamin A: 10,000,000 IU; vitamin E: 70,000 IU; vitamin D: 1,600,000 IU; Fe: 50 g; Zn: 40 g; Mn: 40 g; Co: 0.1 g; Cu: 10 g; Se: 0.1 g; and I: 0.5 g. DM = dry matter; OM = organic matter; CP = crude protein; NFC = non-fiber carbohydrate; NDF = neutral detergent fiber; ADF = acid detergent fiber; ADL = acid detergent lignin; GE = gross energy. ME Mcal/kg DM 2.55 c 2.62 b 2.65 ab 2.68 ab 2.69 a 0.02 <0.01 FDP = untreated discarded durian peel; FDPM = treated discarded durian peel with molasses; FDPC = treated discarded durian peel with cellulase; FDPL = treated discarded durian peel with *L. casei* TH14; FDPML = treated discarded durian peel with molasses and *L. casei* TH14; SEM = standard error of the mean; DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; ME = metabolizable energy. a–c Means in the same row with different letters differ (*p* < 0.05). ⸶ 1 kg DOM = 3.8 Mcal ME/kg [23]. NFC = 100 − (% NDF + % CP + % EE + % ash) [17].

#### *3.4. Rumen Fermentation Characteristics 2.4. Feeding, Sample Collection, and Analysis*

Dietary treatments significantly affected ruminal pH and NH3–N but not ruminal temperature (Table 4). Mean ruminal pH and NH3–N were significantly (*p* < 0.05) observed between FDP and FDPML but FDPM, FDPC, FDPL, and FDPML were comparable. Ruminal pH and NH3–N were 6.71 and 22.58 mg/dL in FDP and 6.44 and 17.29 mg/dL in FDPML, respectively. Dietary treatments significantly affected propionate concentration, the acetate-to-propionate ratio and acetate—butyrate-to-propionate ratio except total VFA, acetate, and butyrate concentration. The propionate concentration was significantly higher, while the acetate-to-propionate ratio and acetate—butyrate-to-propionate ratio were significantly lower in FDPML than FDP, FDPM, FDPC, and FDPL. FDPML showed the highest mean propionate concentration (20.31%) and lowest mean acetate-to-propio-Feeding trial consisted of five 21-day periods, in which 14 days were used for dietary treatment adaptation and 7 days were used for sample analysis. Goats were separately stored in pens (0.11 × 0.95 m) with free access to clean water and mineral lick and fed daily *ad libitum* total mixed rations at a 40:60 ratio (25% fermented discarded durian peel, 15% rice straw, and 60% concentrate) at 8:00 a.m. and 16:00 p.m. The diets were formulated to meet the nutrient requirements of goats according to NRC [18], and chemical composition of dietary treatments is provided in Table 1. Diets offered and refusal were recorded daily to calculate DM intake. Goats were weighed at the beginning of the trial and at the end of each period throughout the trial to adjust DM intake and calculate the BW change of goats at the end of the trial.

nate ratio (3.58) and acetate + butyrate-to-propionate ratio (3.94). CH4 production was affected significantly (*p* < 0.05) by dietary treatments. Mean CH4 production was significant between FDP and FDPML, at 32.93 g/d and 29.41 g/d, respectively, but FDP, FDPM, FDPC, and FDPL were comparable for CH4 production. During the last 7 days of each period, goats were kept in metabolism crate for sample collection and digestibility study. Diet and refusal data were collected throughout 7 days and divided into two portions. The first portion was used to analyze for DM content using oven drying at 100 ◦C, and second portion was deposited according to goat and period and stored at −20 ◦C for chemical composition analysis. Fecal and urine samples were gathered using total collection procedure. A total of 200 g of fecal sample was collected and oven-dried at 100 ◦C for DM analysis, and 5% of total feces was collected, deposited according to goat and period, and stored at −20 ◦C until analysis. Urine yield was collected using 5 L plastic tank consisting of 1 M H2SO<sup>4</sup> to prevent nitrogen loss, and 10% of total

urine was taken, deposited according to goat and period, and stored at −20 ◦C. Before analysis, diet, refusal, and fecal samples were thawed, oven-dried at 60 ◦C for 72 h, and ground through a 1 mm screen to analyze for DM, CP, and OM using AOAC [15]. The NDF, ADF and ADL content were analyzed using Ankom fiber analyzer according to Van Soest et al. [16]. Gross energy content in diet, refusal, and fecal samples was analyzed using Bomb calorimetry (LECO, Berrien, MI, USA). Urine samples were thawed and analyzed for nitrogen content using AOAC [15] method to study nitrogen balance.

On day 21 of each period, at 0 h before feeding and 4 h after feeding, approximately 100 mL of ruminal fluid was collected using vacuum pump attached with stomach tube. Ruminal pH measurement was conducted immediately using pH meter (HANNA instruments HI 98153 microcomputer pH meter, Kallang Avenue, Singapore). Then, 60 mL of ruminal fluid samples was kept in plastic bottle containing 1 M H2SO<sup>4</sup> at a ratio 1:10 (1 mL of H2SO4: 10 mL of ruminal fluid) and centrifuged at 3000× *g* for 15 min. Approximately 35 mL of supernatant was taken and stored at −20 ◦C to analyze NH3–N using Kjeltec Auto 2200 analyzer (Foss, Tecator, UK) according to Bremner and Keeney [19] and volatile fatty acid including acetate, propionate, and butyrate were analyzed using gas chromatography (model HP6890, Hewlett-Packard, Palo Alto, CA, USA; column: Restek 1207384, Stabilwax −60 ◦C−250 ◦C, 30 m × 250 µm × 0.25 µm) according to Osaki et al. [20] as described by So et al. [8] Methane (CH4) production was estimated using Moss et al. [21] equation, CH<sup>4</sup> (g/d) = 0.45 × acetate—0.275 × propionate + 0.4 × butyrate. Another 1 mL of ruminal fluid sample was kept in plastic bottle consisting of 9 mL of 10% formalin and stored in 4 ◦C in a refrigerator to count bacteria, protozoa, and fungi population using total direct count technique according to Galyean [22].

On day 21 of each period, samples of approximately 3 mL of blood were collected from the jugular vein at 0 h before feeding and 4 h after feeding and placed in heparinized tubes. Then, blood samples were centrifuged at 3000× *g* for 10 min, and plasma samples were taken and stored at −20 ◦C until analysis. Plasma samples were sent to Stanbio Laboratory (An EKF Diagnostics Company, Boerne, TX, USA) and used to analyze blood urea nitrogen (procedure no. 2020), glucose (procedure no. 1070), total protein (procedure no. 0250), and albumin (procedure no. 0285). Pack cell volume was measured using micro-hematocrit method, and mean corpuscular hemoglobin concentration, globulin, and albumin to globulin ratio were obtained by calculation. Hemoglobin was measured using commercial kits (Diamond Diagnostics, Egypt). Red blood cell count, mean corpuscular volume, and RBC distribution width were measured using hematological analyzer (ABX Micros 60, HORBA ABX, France).
