Effects of starch sugar by-product on rumen in vitro digestibility, in situ disappearance rate, and milking productivity of the lactating dairy cow

Starch sugar by-product

The SSB used in the experiment originated from the starch sugar factory of Daesang Industry (Fig. 1; 884, Oehang-ro, Gunsan-si, Republic of Korea). As the physical properties can be affected by variations in the production process, the SSB samples were collected 10 times for 5 weeks and stored at −20 °C until the experiment was conducted. The distribution, variation and chemical composition, fatty acid composition, and amino acid composition of SSB were shown in Table 1, A1 and A2, respectively.

Rumen in vitro digestibility and in situ disappearance rate

The experiment was carried out in the experimental farm at Pyeongchang-gun, Gangwon-do, South Korea (latitude 37.54° and longitude 128.44°). Two ruminally cannulated Holstein Friesian cows were assigned for rumen fluid collection and fed commercial concentrate pellet (dry matter [DM], 92.3%; crude protein [CP], 14.5% of DM; neutral detergent fiber [NDF], 31.8% of DM; acid detergent fiber [ADF], 11.6% of DM; ether extract [EE], 3.7% of DM; ash, 7.5% of DM) and rice straw (DM, 92.1%; CP, 5.5% of DM; NDF, 62.1% of DM; ADF, 36.6% of DM; EE, 1.4% of DM; ash, 5.2% of DM) ad libitum during the experiment. In the rumen in vitro digestibility test, McDougall’s buffer (McDougall, 1948), which is continuously purged with CO₂ at 39 °C before usage, was mixed with rumen fluid at a 4:1 ratio (v/v). The total mixed ration (TMR) with SSB (0, 2, 4, 6, and 8% DM) samples were dried and milled to pass through a 1-mm screen (Wiley Mill; Thomas Scientific, Swedesboro, NJ, USA) and chemical composition showed in Table 2. The 50 mL of buffer-rumen fluid mixture was randomly dispensed into a 125 mL serum bottle filled with 0.5 g DM of substrates and the experimental unit was each serum bottle. N₂ gas (99.9% pure) was flushed into headspace of the serum bottles and three replications were incubated at 0, 2, 4, 8, 16, 24, and 48 h.

Two ruminally cannulated Holstein cows were assigned for in situ disappearance rate measurements test. Dry matter, CP, EE, NDF, ADF, ash, and water-soluble carbohydrate (WSC) contents of SSB were 61.4, 14.4, 23.26, 35.7, 31.3, 32.6, and 9.96% of DM, respectively. The dried SSB samples were ground and screened using a 1-mm sieve prior to use in the experiment (Thomas Scientific, NJ, Swedesboro, NJ, USA). A total of 21 nylon bags (5 × 10 cm, 45 µm pore size; R510; ANKOM Inc.,NY, USA) containing 5 g of SSB have been placed in each rumen ventral sacs of two cannulated Holstein cows. These bags were incubated for 0, 2, 4, 8, 16, 24, and 48 h according to national research council (NRC) nutrient requirements of dairy cattle guidelines (NRC, 2001) in three replicate. After incubation, the nylon bags were washed in tap water, dried at 60 °C for 48 h, and then weighed for DM and CP analysis. Disappearance rate was assessed using the formula of Orskov (Menke et al., 1979) follow described in Choi et al. (2021). The following formula: ${\displaystyle P=a+b\left(1-e^{-\mathrm{ct}}\right),}$ where P is the actual degradation after time t; a is the intercept of the degradation curve at time zero; b is the potential degradability of the component of the protein which will, in time, be degraded; c is the rate constant for the degradation of b; and t is time. Regression analysis was performed using SAS PROC REG (Version 9.4; SAS Institute Inc., NC, USA) for estimation of the fraction b.

The effective degradability (ED) of DM and CP was calculated using the following equation described in Choi et al. (2021): ${\displaystyle ED=a+\left(b\times c\right)/\left(c+k\right),}$ where k is the estimated rate of outflow from the rumen and a, b, and c are the same parameters as described above. The ED was estimated as ED2, ED5, and ED8 assuming rumen solid outflow rates of 0.02, 0.05, and 0.08/h, which are representative of low, medium, and high feeding intake, respectively.

Animal study method and experimental design

Animal research protocols were approved by Konkuk University Animal Care and Use Committee (approval number: KU16139).

A total of sixteen Holstein Friesian cows (60.5 ± 20.4 months old, 706.8 ± 3.4 kg initial BW) fed experimental diets (Table 3) during the entire experimental periods. Experimental animals were the commercial breed and obtained from Konkuk University experimental farm (latitude, 37.06° and longitude,127.86°). The average temperature and relative humidity were 25.3 ± 4.2 °C and 45.6 ± 11.57% during the entire experimental periods, respectively. Cows were allocated according to milk yield, parity, and days in milk and then allotted into six sawdust-bedded pens (two or three head/pen; 5 m × 10 m) with an individual feeding gate. The treatments were basal diet (control) and 3.0% DM of SSB (experimental), with the diet formulated according to NRC (Table 3). The feeding trial was conducted as a randomized complete block design for six weeks, which included two weeks of individual feeding gate adaptation, four weeks of adaptation to the experimental diet, and two weeks for data collection. Experimental diets were fed twice a day at 0800 and 1600 h in form of a TMR. Experimental diets, water, and mineral block were fed ad-libitum. The experimental animals were not euthanized after the end of the experiment, they have continuously raised after moving to a commercial farm as healthy.

Physical and chemical analysis

Complex viscosity was analyzed using a rotational rheometer (DHR 1; TA instrument Ltd., DE, USA) at conditions of 0.1 to 100 Hz frequency and 20 °C. The particle size was determined by laser diffraction and scattering using particle size analyzer (LS 13-320; Beckman Coulter, Brea, CA, USA). Density of dried sample was analyzed using gas pycnometer (AccuPyc II 1340; Micromeritic Instrument Corporation, GA, USA).

All samples were dried in a drying oven (HB-503-LF; Hanbaek scientific technology, Buchun-si, Republic of Korea) at 60 °C for 48 h. Dry matter (DM; method No. 937.01), crude protein (CP; method No. 990.03), ether extract (EE, method No. 920.39), ash and silica (method No. 920.08) were analyzed according to AOAC method (AOAC, 2005). Neutral detergent fiber (NDF; method No. 2002.04) and acid detergent fiber (ADF; method No. 973.18) were analyzed with ANKOM Fiber Analyzer (A200; Ankom Inc., NY, USA) according to method of (Van Soest, Robertson & Lewis, 1991). Water soluble carbohydrate (WSC) was extracted using method of Kerepesi and Boross (Kerepesi, Toth & Boross, 1996) and was analyzed using phenol sulfuric acid method (Nielsen, 2017). Gross energy (GE) was determined using automatic bomb calorimeter (Parr 1261 bomb calorimeter; Parr Instruments Co., Moline, IL, USA). Gas production was measured with a 50 mL glass syringe (Hypodermic Glass Syringe; DHS Medical Co., Seoul, Republic of Korea). The pH values were measured using pH meter (Orion Dualster-F, Thermo fisher scientific, NJ, USA). Ammonia nitrogen was determined as previously described in Choi et al. (2019) according to a method of Chaney & Marbach (1962). The volatile fatty acid (VFA) was identified as previously described in Choi et al. (2019) using gas chromatography (HP 6890; Agilent Technologies, Santa Clara, CA, USA) equipped with an Omega Wax Fused Silica Capillary column (Length, 30 m 0.3 × 2 mm Df, 0.25 µm; Sigma-Aldrich Co, St. Louis, MO, USA) and flame ionization detector. The carrier gas was used He gas in gas chromatography.

Fatty acid composition analysis

For fatty acid (FA) analysis, SSB samples were extracted using a chloroform to methanol (2:1, v/v) solvent (Floch, 1957) and then methylated (Lepage & Roy, 1986). Methylated supernatant was injected into a gas chromatograph (Agilent 6890, NY, USA) equipped with a flame ionization detector and a capillary column (30 m × 0.25 mm × 0.25 µm; No. 122-3232; Agilent, Santa Clara, CA, USA) operated at 50 °C in the oven (Garces & Mancha, 1993). The inlet and detector temperatures were 180 and 250 °C, respectively. Helium was used as a carrier gas.

Amino acid composition analysis

To determine concentrations of amino acids (AAs) in SSB, each sample was placed in a volumetric flask to which was added 30 ml of 6N HCl. Then, the flask was hydrolyzed at 130 °C for 24 h min. The extracts were then passed through a 0.45 µm filter. For subsequent analysis, HPLC (Ultimate 3000; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used as described by method of Henderson et al. (2000).

Milk yield, milk composition, and blood profiles

Milk yield was collected previously described in Choi et al. (2019) using a tandem milking system (Milking Parlor Auto Tandem; GEA Co., Düsseldorf, Germany) twice a day at 0300 and 1500 during the entire experimental period. Milk samples were collected in 20 mL tubes using the sampling port of a milking machine every week and stored at 4 °C. Before milk sampling, Anti-corrosive agents (Broad spectrum micro tabs II; Advanced Instrument Inc., Norwood, MA, USA) were added to prevent any chemical changes until the analysis of the milk composition. The milk composition was evaluated previously described in Choi et al. (2019) using near-infrared spectrophotometer (Milko-scan FT 6000; Foss electric Co., Hilleroed, Denmark).

Blood samples were collected at d 28 and d 42 after the end of the adaptation period. Blood samples were collected as previously described in Choi et al. (2021) via the jugular vein using 18-gauge needles and transferred to silicon-coated serum tubes (15 mL Vacutainer; BD, Franklin Lakes, NJ, USA). The serum and plasma were obtained by centrifugation at 1,000 * g at 4 °C for 15 min. Serum was stored at −70 °C until analysis and chemical compositions of serum were analyzed using an chemical analyzer (Model 7180 Clinical Analyzer; Hitachi Ltd, Tokyo, Japan) following the manufacturer’s manual. Reagents were purchased from commercial products (JW Medical, Seoul, Korea) to determine glucose, blood urea nitrogen (BUN), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvate transaminase (GPT), and γ-glutamyltransferase (GGT). White blood cell, red blood cell, hematocrit, hemoglobin, and platelet were determined using hematology analyzer (VetScan HM2, Abaxix Inc., Holliston, MA, USA).

Statistical analysis

Data were analyzed using a MIXED procedure of SAS package program (SAS Inst. Inc., Cary, NC, USA) as a randomized completely block design. The model was, ${\displaystyle {\mathrm{Y}}_{\mathrm{i(t)}}=\mathrm{\mu }+{\mathrm{T}}_{\mathrm{i}}+{\mathrm{E}}_{\mathrm{i(t)}},}$ where µ is average value, T_i is treatment value and E_i(t) is the error value. The fixed effect SSB concentration, and random effects were not considered. Polynomial orthogonal contrasts were used to determine SSB supplementation effect using the CONTRAST option. The crossing point of quadratic broken-line and the quadratic line was determined using NLIN code in order to determine proper SSB concentration in feed. Pairwise comparison was performed to determine SSB supplementation effect using the TTEST option. Outlier was excluded using the method of interquartile range (IQR). Least squares mean between treatments were assessed using a pairwise comparison method. Statistical difference and tendency were accepted at p-value less than 0.05 and 0.10, respectively. All means are presented as least square means.