2. Materials and Methods
2.1. Animals, Experimental Design and Diets
The experimental procedures were reviewed and approved by the Animal Ethics Committee of Khon Kaen University, based on the Ethics of Animal Experimentation of the National Research Council of Thailand (Record No. IACC-KKU-10/63, Reference No. 660201.2.11/17).
Four multiparous Holstein-Friesian crossbred lactating dairy cows (88.6% HF), non-pregnant, with a mean initial body weight (mean ± SD) of 438 ± 16 kg, milk production of 16.9 ± 3.5 kg/d, and 70 ± 31 days in milk at the beginning of the experiment, were used in this study. Cows had continuous access to concrete feed bunks and automatic water troughs. Cows were milked in pen twice daily at 06:30 am and 15:30 pm and fed fermented total mixed rations ad libitum twice daily at 08:00 and 16:00 h.
The experimental design was a 4 × 4 Latin square design, in which each cow was randomly assigned to 1 of 4 dietary treatments that alternated over four periods. Each experimental period lasted for 21 days, beginning with 14 days of adaptation, and followed by daily measurements of milk, feces, urine, and respiratory gas exchange from day 17 through 21 (5 days). Throughout the experiment, cows were housed individually in adjacent holding pens (2.5 m × 4.5 m) with free access to feed and clean drinking water.
The four dietary treatments (
Table 1) included one basal diet (0% DM of broken rice) and three test diets. For the three test diets, rice straw, cassava pulp, wet brewery waste, rice bran, palm kernel cake, and soybean meal in the basal diet were substituted by broken rice at the ratios of 12%, 24%, and 36% DM, respectively (
Table 1). The substitution and regression methods were used to determine the metabolizable and net energy value of diets, according to Wei et al. [4]. Substitution and regression methods are required for evaluating the energy content of individual feedstuffs, and a regression method is based on multiple-point substitution. Therefore, this study involves different rations of broken substitution into basal diets.
Fermented total mixed ration preparation, ingredients (
Table 1) mixed in a horizontal mixer with a 5,000 kg capacity (Celikel TMR feed mixer, 108 Agriculture Machine and Equipment Co., Ltd, Lop Buri, Thailand). Approximately 2,000 kg of each batch of the treatment mixture was mixed, 35 kg packed per bag in high-density polyethylene bags (25 in width x 44 in-length, Kwankhawpanich, Nakhon Ratchasima, Thailand), vacuum compressed (Hitachi 1600 W model CV-930F, Hitachi Consumer Products Co., Ltd., Prachinburi, Thailand), covered with a polypropylene woven bag (23 in width x 37 in length, Thailand), and stored at ambient temperature (26 to 39 °C) until feeding.
2.2. Feed Intake and Digestibility
The dairy cows were weighed and recorded at 07:30 on the first and last day of each experimental period to determine their dry matter intake as a percentage of body weight and metabolic body weight. Throughout the feeding period, the feed offered, and the feed refused for each animal were weighed and recorded, and the individual daily feed intake was calculated as the difference between the amount of feed offered and feed refused.
The total collection technique was conducted in an indoor digestion trial pen (165x375 cm) installed with a head cage respiratory gas system (width 105 cm x depth 80 cm x height 173 cm) for each animal over 5 consecutive days using Suzuki et al. method [19]. The animals have relocated to a digestion trial pen on days 17 to 21 of each experimental period phase. Feces and urine amounts were measured daily. The excreted feces were immediately collected using pans placed behind the animal and weighed daily, and fecal samples were taken for digestion analysis. A tube urine cup collected the total urine volume in plastic tanks containing 6 normal hydrochloric acid to maintain the pH below 3.0. Feces (1 kg) and urine (120 mL) were recorded and sampled daily in the morning for 5 days and stored at 4 °C. After the metabolic data collection period was completed, 1 kg daily aliquot samples of offered feed, refusals, and excreted feces and 120 mL of excreted urine were well mixed and then stored at -18 °C until analysis.
2.3. Animal Calorimetry
During the digestion trial, the oxygen consumption (O2), carbon dioxide (CO2), and methane (CH4) production of each animal were measured using a head cage respiratory gas measurement chamber system [19]. The system consisted of a digestion trial pen, a head cage, a gas sampling and analysis unit, and a data acquisition and processing unit. A head cage is installed in front of the digestion trial pen and designed to be airtight, except for an air-inlet adjustable collar. Cows kept their heads in the hood section and had access to feed trays and automatic water for the day. Cows can lie down on rubber mate floor. A flow meter (NFHY-R-O-U, Nippon Flow Cell, Tokyo, Japan) was used to measure the respiration chamber's flow rate and total air volume flow. The temperature and humidity of outflowing air were recorded electronically (ESPEC MIC CORP, Japan, model RS-12). The oxygen concentration in the inflow and outflow lines was measured using a dual-chamber paramagnetic oxygen analyzer (4100 Gas Purity Analyzer, Servomex Group, East Sussex, UK). Carbon dioxide and methane were also measured using an infrared gas analyzer (IR200 Infrared Gas Analyzer, Yokogawa Electric Co., Tokyo, Japan). The gas analyzers were calibrated every day against certified gases (Takachiho Chemical Industrial Co., Tokyo, JP); reference gases included two oxygen concentrations (19.0% and 20.6%), 1.89% carbon dioxide, and 1960 ppm methane. Calorimetric system recovery tests (98% to 104%) were conducted using the carbon dioxide injection method, by which a weighed amount of carbon dioxide gas was released into the system. The respiratory gas exchange measurements were taken at intervals of 7.5 min for 23.30 hours per day from the initial day at 08:00 am to 07:30 am the next day to determine energy partitioning and consumption over the last three days of the respiration collection period. Ambient temperature and relative humidity were not conditioned; their average was 27.7°C (23.3 to 36.6°C) and 82.7% (41.0 to 99.0), respectively. The metabolizable energy intake was calculated by deducting the urine and methane energy outputs from the digestible energy (DE) intake. Heat production was estimated according to the Brouwer method [20], and the energy balance was calculated [20].
2.4. Sample Collection and Chemical Analysis
Feed samples were collected each period to determine the nutritive value and fermentation quality. The DM content of feeds and feces was analyzed in a fan forced oven at 105 °C, reaching a constant weight. Each subsample (800 g wet weight) was dried in an oven at 60 °C for 72 h and then ground to pass through a 1-mm screen. Then, feeds and feces were analyzed using the Association of Official Analytical Chemists (AOAC) methods [21] for DM, ash, crude protein (CP), and ether extract (EE) determination (method numbers 967.03, 942.05, 984.13, and 920.39, respectively). The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were analyzed using a fiber analyzer (ANKOM 200/220, ANKOM Technology, Macedon, NY, USA), NDF treated with thermostable alpha-amylase, and sodium sulfite [22]. The non-fiber carbohydrate (NFC) content was calculated according to the equation NFC (%) = 100 − (%CP + %NDF + %EE + %Ash). The gross energy content was determined using a bomb calorimeter (IKA C2000 Basic, IKA-Werke, Staufen, Germany).
To measure the fermentation quality of the silage, a 20 g fresh sample was blended with 180 mL of distilled water and stored in a refrigerator at 4 °C for 24 h. Thereafter, the extracts were passed through a nylon filter funnel. The silage juice was used to determine pH, ammonia nitrogen (NH3-N), lactic acid, and volatile fatty acid (VFA) concentrations. The pH was measured immediately with a bench pH meter (Eutech pH 700, Eutech Instruments Pte Ltd., Ayer Rajah Crescent, Singapore). Lactic acid and volatile fatty acid concentrations were analyzed using gas chromatography (GC2014, Shimadzu, Tokyo, Japan) [23].
The milk yield was recorded daily during the morning (06:30 h) and afternoon (15:30 h) milking, and milk samples were collected for 5 consecutive days from day 17 to 21 of each period. One aliquot (110 mL) was refrigerated at 4 °C and sent to the Veterinary Research and Development Center, Upper Northeastern Region (Khon Kaen, Thailand) to determine the milk fat, protein, solid not fat (SNF), and lactose concentrations using MilkoScan™ 7RM (Foss Electric, Denmark) and somatic cell count using the Milk Analyzer Fossomatic™ 7 DC (Fossomatic DC, Denmark). The second aliquot was frozen at -20 °C for gross energy measurement using a bomb calorimeter (IKA C2000 Basic, IKA-Werke, Staufen, Germany).
2.5. Calculation
Fat and protein-corrected milk (FPCM, kg/day) was calculated according to Gerber et al. [24] as in Equation (1):
Energy-corrected milk (ECM, kg/day) was calculated according to Cabezas-Garcia et al. [25] as in Equation (2):
Milk energy (MJ/kg) was calculated according to Cabezas-Garcia et al. [25] units for fat, protein, and lactose in milk are g/kg, as in Equation (3):
Heat production (HP, kJ/day) was calculated according to Brower [20] as in Equation (4):
using volumes of O
2 consumption (L/day), CO
2 production (L/day), CH
4 production (L/day), and urinary nitrogen (UN) excretion (g/day). Methane energy (kJ/day) was calculated [26] as CH
4 = 39.54 kJ/L × CH
4 (L/day). Energy balance (EB; kJ/kg BW
0.75) was calculated using Equation (5).
The efficiency of ME use for lactation (
kl) was calculated according to a previous report [2,27] using Equation (6).
where E
l(0) is the milk energy output (E
l) adjusted to zero energy balance (MJ/day) and calculated from Equations (7) and (8). ME intake was calculated as follows: ME intake = gross energy intake − fecal energy − urinary energy − methane energy. ME
m is the ME requirement for maintenance (MJ/day).
The DE, ME and NE
L values of the test ingredients were calculated by the substitution method, as described by Wei et al. [4] in Equation (9).
where E
ti is the energy value of the test ingredient, E
td (MJ/kg DM) is the energy value of the test diet, E
bd (MJ/kg DM) is the energy value of the basal diet, and P
ti is the test ingredient substitution ratio in the basal diet.
Alternatively, the DE, ME and NEL values in test ingredients can be estimated by the regression of the test ingredient-associated energy intake (MJ/day) against kilograms of the test ingredient substitution amount intake (kg/day), with the slope of the regression equation representing the energy concentration in MJ/kg DM. The product of Eti at each level of test ingredient broken rice substitution rate energy concentration (MJ/kg) and kilograms of broken rice intake (0.12, 0.24, or 0.36 kg) is the broken rice-associated DE, ME, or NEL intake in kilocalories [4].
2.6. Statistical Analysis
Regression equations to estimate the energy value of broken rice were generated using the REG procedure of SAS [29]. The dependent variables in the prediction equation were broken rice-associated energy intake, and the independent variable was testing ingredient intake.
The experimental data were analyzed using the general linear model (GLM) of SAS version 9.0 for a 4 × 4 Latin square design using the following statistical model:
Yijk = μ + ρi + γj + τk+ εijk,
where Yijk is a dependent variable, μ is the mean for all observations, ρi is the fixed effect of period (i = 1 to 4), γj is the fixed effect of cow (j = 1 to 4), τk is the fixed effect of dietary treatment (k = 1 to 4), and εijk is the residual error. Within treatments, linear, quadratic, and cubic contrasts were evaluated using contrast statements in SAS to determine the effect of increasing proportions of broken rice in the diet. The effects of dietary treatment were also compared using Duncan’s new multiple-range test, and differences were considered significant at p < 0.05.