Kimchi cabbage (Brassica rapa L.) by-products treated with calcium oxide and alkaline hydrogen peroxide as feed ingredient for Holstein steers

The TMR was formulated based on the guideline of National Research Council (NRC) [17] for beef cattle and was prepared by mixing timothy hay, alfalfa hay, lupine seed, corn, corn gluten feed, whole cottonseed, tall fescue, molasses, salt, vitamin-mineral mix, protected fat, and limestone (Table 1). By-products of Kimchi cabbage were collected in Suncheon NongHyup Namdo Kimchi (Suncheon, Korea). Treatments used for the in vitro and in situ experiments include: TMR only (CON), TMR + 30% Kimchi cabbage by-products (TC), TMR + 30% Kimchi cabbage by-products + 5% CaO (TCC), and TMR + 30% Kimchi cabbage by-products + 5% CaO + 3.22% AHP (TCCA), fresh matter basis. The inclusion rate of alkali agents were based on Nuñez et al. [18]. For the in vivo experiments, CON and TC were used as treatments. The Kimchi cabbage by-products (4.68% of dry matter [DM] basis) were treated with powdered CaO (1% of DM basis) and/or AHP (0.64% of DM basis) by manual mixing in a plastic container for approximately 1 h. The treated Kimchi cabbage by-products were subsequently mixed with the TMR. The ingredients and chemical compositions of experimental TMRs were presented in Table 1. The chemical compositions evaluated were crude protein (CP), crude fiber (CF), ether extract (EE), calcium, phosphorus, neutral detergent fiber (NDF), and total digestible nutrients (TDN). Proximate analysis was performed according to the Association of Official Analytical Chemists [19] method. CF were analyzed using the method of Van Soest et al. [20].

Cannulated Holstein with body weight of 600 ± 47 kg, housed at animal facility in Suncheon National University located in Suncheon-si, Jeollanam-do, Korea, was used for the in vitro and in situ experiments. Rumen fluid was collected before morning feeding and was obtained by squeezing the ruminal contents of the animal through the four layers of a surgical gauze and collected in a stainless-steel vacuum bottle. After collection, the filtrates were subsequently sealed, stored at 39°C, and directly transported to the laboratory. The buffer used in this study consisted of (per L) 0.45 g dipotassium phosphate (K₂HPO₄), 0.45 g potassium dihydrogen phosphate (KH₂PO₄), 0.9 g ammonium sulfate ([NH₄]2SO₄), 0.12 g calcium chloride dihydrate (CaCl₂∙2H₂O), 0.9 g sodium chloride (NaCl), 0.19 g magnesium sulfate heptahydrate (MgSO₄∙7H₂O), 1.0 g trypticase peptone, 1.0 g yeast extract, and 0.6 g L-cysteine hydrochloride [21]. The prepared buffer was autoclaved for 15 min at 121°C and maintained in a 39°C water bath, adjusted to pH 6.9 using 10 N NaOH, and dispensed anaerobically by continuous bubbling of CO₂ gas. Meanwhile, the collected rumen fluid was strained again using a 4-layer surgical gauze placed in a funnel and the filtrate was allowed to flow into a 1-L graduated cylinder, with continuous bubbling of the CO₂ gas. The filtrated rumen fluid was then mixed with the prepared buffer medium at a ratio of 1:3 and allowed to bubble for 30 min. The pH was adjusted again to 6.9, before filling the serum bottle with the buffered rumen fluid. The serum bottle (160 mL capacity) was prepared by adding 1 g of weighed substrate of each treatment with three replications. One hundred milliliter of buffered rumen fluid was filled into individual serum bottles containing the substrate, under anaerobic conditions [22]. The bottles were subsequently sealed with rubber septum stoppers and aluminum caps and incubated at 39°C for 6, 12, and 24 h with shaking (100 rpm) [23].

Moreover, samples from each treatment were dried in an oven for 48 h at 80°C and ground to pass through a 1 mm screen [24]. The samples weighing 2 g (DM basis) were placed in nylon bags (5 × 10 cm; 45 μm pore size), and the bag openings were tied with nylon strings. Then, the bags were submerged in hot water (39°C–40°C) for approximately 30 min, and then deep fermentation in the rumen fistula of the cannulated animal immediately after morning feeding. The incubation times were 0, 3, 6, 9, 12, and 24 h, with six replications per treatment. After each incubation, the nylon bag was removed from the rumen cannula, immersed in clear water, and then washed without heating using a crude fiber analyzer (Ankom 220, Ankom Technology, Macedon, NY, USA). Samples for each incubation time were dried at 80°C for 48 h in a dry oven immediately after washing.

A press and sensor machine (EA-6, Laurel Electronics, Costa Mesa, CA, USA) was used to measure the total gas (TG). The pH was determined using the Schott ^® Instrumets Lab 860 (SI Analytics GmbH, Mainz, Germany) after opening each serum bottle. Samples of fermentation were also collected in 1.5 mL cryotubes and deep frozen at −80°C. These samples were later thawed at room temperature and then centrifuged at 13,000×g for 15 min at 4°C using a Micro 17TR centrifuge (Hanil Science Industrial, Incheon, Korea) and the supernatant was used for ammonia nitrogen (NH₃-N) and volatile fatty acids (VFA) analysis [22]. Using the Libra S22 spectrophotometer (Biochrom, Cambridge, UK) at an absorbance of 630 nm, NH₃-N concentration was measured according to the methods developed by Chaney and Marbach [25]. VFA were analyzed using high performance liquid chromatography (Agilent Technologies1200 series, Agilent Technologies, Santa Clara, CA, USA) with a UV detector (210 nm and 220 nm) and a Metacarb87H (Agilent Technologies) column using 0.0085 N H₂SO₄ as a buffer at a flow rate of 0.6 mL/min and temperature column of 35°C. The DM disappearance in the rumen was calculated according to the protocol of Van Emon et al. [26]. For NDF and acid detergent fiber (ADF) analysis, ANKOM 220 Fiber Analyzer (AnkomTechnology) based on the Van Soest et al. [20] method was used.

Thirty-four heads of Holstein steers (273 ± 45 kg) were used as experimental animals in this study for 150 d; animals were housed in the farm located in Gimje-si, Jeollanam-do, Korea. The animals were selected randomly and equally distributed into two groups (CON and TC). The steers were housed in steel-constructed pens (5 m × 10 m) with two or three steers per pen. The animals were fed twice a day (08:00 and 18:00) and water was made available ad libitum. The in vivo data included the body weight, average daily gain (ADG), and feed conversion ratio (FCR). We offered the same amount of TMR as group feeding (10 kg fresh matter basis/steer/d) for all steers throughout the feeding trial and no orts were observed. The ADG was calculated by subtracting the initial weight to the final weight and divided by the experimental period. The FCR was calculated by dividing the total input of the given feed by the total weight gain. At the end of the feeding trial, blood samples were collected 3 h after morning feeding from the jugular vein in 5 mL BD Vacutainer^® SST™ II Advance (BD Vacutainer Systems, Plymouth, UK), left at room temperature for 30 min, and subsequently centrifuged at 1,300×g for 10 min at 4°C to obtain the serum. Blood parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose, total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, creatinine, blood urea nitrogen (BUN), and total protein were analyzed using an automated blood biochemical analyzer (Hitachi 747, Hitachi, Tokyo, Japan).

All the data obtained in this experiment were collected for each incubation period, and statistical analysis was performed using the standard statistical analysis system (SAS) version 9.1 statistical package (SAS Institute, Cary, NC, USA). Using the general linear model (GLM), data were analyzed by analysis of variance (ANOVA). Duncan’s multiple range test was used to identify differences between and among control and treatment group. To indicate significance between mean, p < 0.05 was considered.

The effects of different dietary treatments on pH, TG, NH₃-N, and VFA during in vitro rumen fermentation are shown in Table 2. The in vitro pH was greater (p < 0.05) in TCCA and TCC groups at all the incubation times than the two other groups. The reason of high pH might be due to the influence of the alkaline substances such as CaO and AHP, which were added to the Kimchi cabbage by-products. Similarly, Boukila et al. [27] reported that feeding lambs with high dietary Ca(OH)₂ appears to delay a drop in ruminal pH. In addition, Schroeder et al. [28] stated that diets containing supplementary CaO had a greater pH than those that did not apply any additive. This is due to the fact that CaO and AHP are alkaline agents [27]. Nuñez et al. [18] stated that alkalizers can neutralize acidity as well as increase the pH. The greater pH of feed entering the gut may have buffered initial ruminal pH in cow supplied with supplementary CaO [28]. The amount of gas generated in all treatment groups increased with prolonged incubation time, and was greater (p < 0.05) in the CON, TC, and TCC groups with 220.33 mL, 217.00 mL, and 212.33 mL, respectively, during 24 h incubation, than in TCCA. In the in vitro fermentation, the amount of gas generated is used as an important indicator for determining fermentation properties in the rumen along with the pH [29]. This means that decomposition and fermentation of the substrate were effectively carried out in the course of fermentation [30]. Ammonia-nitrogen concentrations were lower in TCC and TCCA at 24 h of incubation with 15.90 mg/dL and 16.39 mg/dL, respectively (p > 0.05) than in CON and TC. Chaudhry [31] reported that concentration of NH₃-N in rumen was lower in straw treated with CaO, NaOH, and NaOH + AHP than untreated straw. Accordingly, the decrease in the NH₃-N was presumably a consequence of an increase in the available energy, and rumen microbes utilize ammonia during the enhanced degradation of straw components after chemical treatments [31]. It was reported that the NH₃-N concentration should be 5 mg/dL or more to ensure and maintain maximum microbial protein synthesis [32], while the intake and disappearance of the DM are maximized when the concentration is maintained at 20 mg/dL [33]. Acetate was greater in TCCA (47.66 mM) after 24 h followed by TCC, TC, and CON with 46.12 mM, 45.26 mM, and 44.93 mM, respectively (p > 0.05). In contrast, propionate was greater (p < 0.05) in CON and TC at 6 h incubation with 15.22 mM and 14.45 mM, respectively, than TCC (13.18 mM) and TCCA (13.04 mM). Schroeder et al. [28] reported that ruminal propionate concentration was decreased while acetate concentration was increased when CaO was added to the diet due to the improvement in forage fiber disappearance. On the other hand, butyrate was greater (p < 0.05) in TCC at 12 and 24 h of incubation with 17.26 mM and 23.54 mM, respectively, than the other treatments. Nuñez et al. [18], reported that butyrate increased linearly as CaO inclusion increased, indicating that the efficiency of dietary energy utilization was improved by the treatment. At 24 h of incubation, TCCA had the greatest acetate to propionate ratio (A:P ratio), numerically, compared to the other treatment but there were no significant differences. Nuñez et al. [18] reported that the A:P ratio increased linearly with increasing CaO inclusion due to the increased fiber disappearance. Total VFA concentration was greater (p < 0.05) in TC at 6 h than TCC and TCCA, but was not significantly different in CON. At 24 h of incubation, there was no significant difference among the treatments but numerically greater in TCC. This might be due to the improved degradation of fiber with the influenced of alkaline treatment [18].

The effect of different dietary treatments on DM, NDF, and ADF disappearance through in situ experiment is shown in Table 3. The DM disappearance gradually increased in all treatments after 3 h of incubation, and was greater (p < 0.05) in TCCA with 79.53% disappearance at 24 h than the other treatments. Wanapat et al. [34] reported that alkaline agent like CaO improves DM disappearance of roughage. Moreover, Chaudry [35] reported that AHP was the most effective treatment for improving the degradation of wheat straw. According to Khejornsart et al. [14] and Carvalho et al. [15], disappearance and feed intake of ruminants can be improved by alkaline treatment because it alters the structure of lignin removed from lignocellulose. In connection with Rezende et al. [36] stated that the removal of lignin from the inside parts of the cell wall damaged and weakened the cell morphology by separating the cell bundles, thus resulting in long cellular structures which are connected in the longitudinal direction. Wanapat et al. [34] stated that the application of alkaline agents to substrates can chemically break the ester bonds of lignin, hemicellulose, and cellulose, and physically swell the structural fibers. The NDF and ADF disappearance in the TCCA treatment was numerically greater than in the other treatments after 24 h of incubation with 40.77% and 35.57%, respectively, which indicates improvement in rumen fermentation and disappearance when CaO and AHP were added. Nuñez et al. [18] stated that the linear increase in NDF and ADF disappearance was due to the increase in CaO inclusion, which enhances ruminal fiber fermentation. Chaudhry [31] reported that when CaO and AHP were incorporated into rice straw, it effectively removes barriers that limit the digestion of structural carbohydrates. In addition, Castañón-Rodríguez et al. [16] revealed that supplementation with alkaline agents caused swelling of cellulose fibers that could increase the internal surface area, allowing cellulases to have direct contact with the substrates, allowing a greater level of hydrolysis in the amorphous region, and consequently increasing the proportion of crystalline cellulose. Thus, concentration of alkaline agents can effectively improve the breakdown of ester bonds between NDF and ADF and physically swollen structural fibers [37], which permits rumen microbes to breakdown the structural carbohydrates without difficulty [16].

The effect of dietary treatment on the growth performance of Holstein steers was shown in Table 4. The initial weight of the experimental animals was 273.06 kg in CON and 272.29 kg in TC. At the end of the 150-d feeding trial, the final body weight was 417.82 kg in CON and 413.88 kg in TC, but there was no significant difference. Results were the same with Song et al. [7], during their 12-wk feeding trial, the final body weight of steers were not significantly different between treatments. The ADG and FCR were not significantly different between treatments. It is determined by the number of factors, such as the composition and quality of the feed [38]; thus, the greater the feed quality, the greater the ADG and FCR [39]. The effect of dietary treatments on Holstein steers blood profiles was shown in Table 5. The blood ALT, AST, and HDL cholesterol were numerically greater (p > 0.05) in CON with 26.00 U/L, 70.00 U/L, and 112.71 mg/dL, respectively, than in the TC group. Glucose, total cholesterol, LDL cholesterol, and total protein were numerically greater (p > 0.05) in TC with 89.47 mg/dL, 135.94 mg/dL, 25.35 mg/dL, and 6.58 g/dL, respectively, than in CON. Briefly, ALT, AST, HDL, glucose, total cholesterol, LDL, and total protein were not affected by the change in TMR composition with the Kimchi cabbage by-product. Creatinine concentration was 0.92 mg/dL in TC, which was greater (p < 0.05) than CON group. Cao et al. [40] concluded that vegetable by-products such as Chinese cabbage have high nutritive value and in vitro DM disappearance and good potential as vegetable protein sources for ruminant, which might be the reason for the increase of creatinine concentration; since, increased protein intake contributes to greater muscle gains [41,42]. This creatinine, which is usually eliminated only via the kidneys, is produced as an end-product of muscle metabolism [43]. Costa e Silva et al. [44] reported that the increase in body weight is correlated with that of increase in creatine concentration. Thus, the relationship between muscle tissue or CP concentration and body weight could follow the similar relationship between the creatinine concentration and body weight [44]. Blood urea nitrogen was greater (p < 0.05) in the CON with 15.09 mg/dL than in the TC group. This BUN can be affected by different factors such as dietary N-to-energy ratio, level of forage intake, and protein degradability in the rumen [45]. Wanapat et al. [34] reported that the concentration of BUN is negatively correlated to the level of production of NH₃-N in the rumen, in which in the present study the production of NH₃-N was numerically greater in TC than in the CON group. Protein degradation occurs more rapidly than its synthesis or the imbalances of fermentable energy and available nitrogen, and thus ammonia accumulates in the rumen fluid and be absorbed into the blood where it is transported to the liver and converted to urea [46].

In conclusion, Kimchi cabbage by-products treated with CaO and AHP increased in vitro pH, and butyrate production. It also improved the in situ DM, NDF, and ADF disappearance. Moreover, NH₃-N, acetate, and propionate production were not hampered by CaO and AHP treatment of Kimchi cabbage by-products. ADG, FCR, and blood profiles during in vivo were not affected by the TMR substituted by Kimchi cabbage by-products, but it increased the creatinine production. Therefore, overall results suggest that Kimchi cabbage by-products either treated or untreated with CaO and AHP can be a potent substitutional ingredient of TMR for Holstein steers.