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Yuko Kamiya, Tomoyuki Suzuki, Kohei Oikawa, Hisami Kobayashi, Masanori Tohno, Ryoh Nakakubo, Masaaki Matoba, Takahiro Nemoto, [Kosuke Minami](https://orcid.org/0000-0003-4145-1118), [Gaku Imamura](https://orcid.org/0000-0002-3130-7190), [Genki Yoshikawa](https://orcid.org/0000-0002-9136-8964)

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This is the peer reviewed version of the following article: Effects of fermentative quality of corn silage on lactation, blood metabolites, and rumen fermentation in dairy cows, which has been published in final form at https://doi.org/10.1111/asj.13880. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[Effects of fermentative quality of corn silage on lactation, blood metabolites, and rumen fermentation in dairy cows.](https://mdr.nims.go.jp/datasets/1c0c06e5-e352-4853-899c-4814083a9784)

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The DMI, milk calcium and bone turnover of primiparous cows during early lactation10Effects of fermentative quality of corn silage on lactation, blood metabolites, and rumen fermentation in dairy cowsResearch ArticleYuko KAMIYA1, Tomoyuki SUZUKI1, Kohei OIKAWA1, Hisami KOBAYASHI1, Masanori TOHNO1,2,3, Ryoh NAKAKUBO4, Masaaki MATOBA5, Takahiro NEMOTO5, Kosuke MINAMI5, Gaku IMAMURA5,6, Genki YOSHIKAWA5,71 Institute of Livestock and Grassland Science, NARO, Nasushiobara, Japan2 Japan Research Center of Genetic Resources, NARO, Tsukuba, Japan3 University of Tsukuba, Graduate School of Science and Technology, Tsukuba, Japan4 Institute of Livestock and Grassland Science, NARO, Tsukuba, Japan 5 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science (NIMS), Tsukuba, Japan6 Graduate School of Information Science and Technology, Osaka University, Suita, Japan 7 Materials Science and Engineering, Graduate School of Pure and Applied Science, University of Tsukuba, Tsukuba, JapanCorrespondence Yuko Kamiya, Institute of Livestock and Grassland Science, NARO, Nasushiobara, Tochigi 329-2793, Japan. Email: yukoiwm@affrc.go.jpABSTRACTWe investigated the effect of fermentation quality of corn silage on dry matter intake (DMI), milk yield, ruminal fermentation, methane (CH4) emissions and plasma metabolites in lactating cows. Six lactating Holstein cows were used in a 2 ( 2 crossover design with two dietary treatments containing high quality corn silage with lower pH (high group) or low quality corn silage with higher pH (low group). The cows were fed partial mixed ration (PMRs containing 50%DM of each corn silage) ad libitum plus 0.7 kg/day of concentrates at milking. The DMI of cows in the low group (24.8 kg/day) tended to be lower (P < 0.10) than that in the high group (26.8 kg/day). The dietary treatment did not affect milk yield or milk fat, protein, or lactose concentrations. The ruminal acetic acid proportion of the low group was significantly higher (P < 0.05) than that of the high group. The CH4 emission per DMI of the low group tended to be higher (P < 0.10) than that of the high group. The plasma concentration of the total cholesterol and the activity of aspartate aminotransferase and alanine aminotransferase of the low group were significantly higher than those of the high group. Key words: corn silage, dairy cow, fermentation quality, lactation INTRODUCTIONSilage quality affects the palatability, milk yield, and diseases occurrence of dairy cows (Borreani et al., 2008; Driehuis et al., 2018; Huhtanen et al., 2003). Low quality silage, for example, that including high ammonia and butyric acid, is well known to cause a reduction in dry matter intake (DMI) due to a decrease in palatability, as well as a decrease in nutritional value (Given et al., 1993; Grant & Ferraretto, 2018). Silage that had undergone aerobic deterioration is undesirable for feeding because of its lower nutritive value and the risk of negative effects on animal health (Gerlach et al., 2014). In addition, Kara (2016) reported that silage quality affected the nutritive value as well as methane (CH4) production. Thus, silage quality also is an important factor from the perspectives of mitigating global warming and preventing feed energy loss. For these reasons, silage quality should be evaluated frequently before feeding; however, analyzing silage fermentation requires a lot of time and effort. Several attempts have been made to estimate the fermentation quality of silage. Recently, we reported on a new sensor that can assess silage quality, which should enable the on-site measurement of silage samples with a compact device (Minami et al., 2023). For the practical use of the device, it is necessary to obtain many data of various silage, feeding trial, and clarify the relationship between silage quality as determined by the sensor and the performance of dairy cows, and to make the device more accuracy. Therefore, we used the two corn silages judged by a developed sensor to be of different qualities (Minami et al., 2023) and studied the feeding effects on dairy performance. Namely, we fed PMRs that contain these corn silages to lactating cows and examined the relationship between fermentation quality and feed intake, lactation performance, plasma metabolites, rumen fermentation, and CH4 emissions of dairy cows.MATERIALS AND METHODSPreparation of high and low quality corn silageThe preparation of corn silage has been shown in a previous report (Minami et al., 2023). Briefly, whole plant corn (Zea mays L.) was mown at the yellow ripe stage and ensiled in an underground silo for six months. Corn silage was taken out from the upper or lower side of the underground silo, made into round bale silage, and stored until the feeding trial. At the beginning of the experiment, corn silage from the upper side of the underground silo was put into the underground silo again and used with exposure to air (low quality). Corn silage from the lower side of the underground silo was opened each time the PMR was prepared (high quality).   Experimental designAll animal studies were approved by the Animal Care and Use Committee of the NARO, Japan (Approved No. 20C121CRARC) and conducted in accordance with the NARO Implementation Regulations on Animal Experiments.This experiment was carried out in a free stall barn equipped with an individual door feeder and two boxes of automatic milking systems (AMS) with one way traffic (MIone, GEA Farm Technologies GmbH, Steenstra, Germany) in the experimental barn of the Institute of Livestock and Grassland Science, NARO. Six lactating Holstein cows after peak lactation period (1.8 ± 0.9 [mean ± SD] parity (three primiparous cows and 3 multiparous cows), 303 ± 57 of days in milk, 699 ± 28 kg of initial body weight (BW)) were randomly assigned to a 2 ( 2 crossover experimental design with two dietary treatments a partial mixed ration (PMR) containing 50% (dry matter basis) high quality corn silage (high group) and PMR containing 50% (dry matter basis) low quality corn silage (low group). After an adaptation period for the door feeder, the experiment consisted of two 14-day experimental periods, each comprised of a 10 day treatment adaptation period, and a 4 day sample collecting period. The studies of Nishida et al. (1997) and Horiguchi et al. (2002) were referred for setting the duration of the experiment. The ingredients of the PMR are shown in Table 1. All experimental diets were formulated to meet the nutritional requirements of the Japanese Feeding Standard for Dairy Cattle (NARO, 2006). The experimental cows had ad libitum access to one of two PMRs offered two times daily (9:30 and 18:00 hr). Additionally, the cows were offered 0.35 kg (dry matter basis) of the same commercial concentrate (Nyuhai YAWARA, JA Higashi-Nihon kumiai siryo, Ota, Japan) in each milking. Daily milking at the AMS was permitted between 08:00 and 09:00 hr and between 17:00 and 18:00 hr, so that all cows were milked twice daily. During each sample collecting period, the amount of refused was weighed, and representative samples were collected daily before morning feeding. The samples of offered diets were dried at 60°C in an air forced oven for 48 hr, weighed, and then ground through a 1 mm mesh using a Wiley mill (1029-C; YOSHIDA SEISAKUSHO CO. LTD., Tokyo, Japan) for further analysis. Milk samples were automatically collected on the last 3 days of each experiment. The BW of each cow was measured before morning feeding on the last day of each experiment. During the sample collecting period, air around the feed bin in the AMS was collected and analyzed. The method of gas collection during milking in the AMS was described in a previous report (Suzuki et al., 2021). Briefly, air around the feed bin in the AMS was vacuumed with the pump (approximately 6.5 L/min), filtrated, and the dehumidified sample gas was sent to the infrared CH4 and CO2 analyzer (ZRF; Fuji Electric Co., Ltd., Tokyo, Japan). The background corrected CH4, and CO2 were averaged at each visit; consequently, the CH4/CO2 ratio was obtained for the CH4 emission prediction (Oikawa et al. 2022). Methane emissions of experimental cows were estimated using DMI, energy corrected milk (ECM), BW, and the CH4/CO2 ratio, according to the equation of Suzuki et al. (2021).Only the last day of each experimental period, the morning feeding was offered right after the ruminal fluid and blood samples were collected. Rumen fluid was collected orally using a catheter (FUJIHIRA INDUSTRY Co., Ltd., Tokyo, Japan) at 10:00 hr. The samples were filtered through 4 layers of cheese cloth, pHs were determined using a glass electrode pH meter (D-210P; HORIBA Advanced Techno, Co., Ltd., Kyoto, Japan), and they were stored at –30°C for further analysis. Blood samples were collected into heparinized tubes from the jugular vein of each cow at 10:00 hr. The samples were immediately centrifuged at 3,000 × rpm for 15 min at 4°C, and the plasma was stored at −30°C until the analysis. Sample analysisTo analyze the fermentative quality of silage, 100 mL of distilled water was added to 10 g (fresh weight) of silage material and the resulting solution was homogenized using a laboratory homogenizer (Pro･media SH-IIM, Elmex, Tokyo, Japan). After 5 min of extraction, the sample was filtered through 5A filter paper (Advantec, Tokyo, Japan). The resulting eluate was treated with Amberlite (Amberlite IR-120H+, Tokyo Chemical Industry, Tokyo, Japan) and centrifuged at 20,000 × g for 5 min. The supernatant was filtered through a membrane filter (pore size 0.45 μm; Advantec, Tokyo, Japan) and analyzed by a high performance liquid chromatograph (HPLC; JASCO Corporation, Tokyo, Japan) equipped with a Shodex RSpak KC-811 column (8 mm × 300 mm; Showa Denko, Tokyo, Japan) and a UV spectrometer (detection wavelength was 450 nm). The column was maintained at 60 °C. The flow rate of the mobile phase (3 mmol/L of HClO4 aq.) was 1.2 mL/min. BTB solution (0.2 mmol/L of bromothymol blue, 8 mmol/L of Na2HPO4, and 2 mmol/L of NaOH) was used as the reaction mixture.The DM was determined by drying the sample at 105°C. The ether extract (EE), Kjeldahl nitrogen (N), and crude ash (CA) values were determined by the AOAC method (AOAC International, 2000; methods 920.39, 990.03, and 942.05, respectively). The neutral detergent fiber (aNDFom) and acid detergent fiber (ADFom) (aNDFom was assayed with a heat stable amylase, and sodium sulfite and was expressed exclusive of residual ash; ADFom was expressed exclusive of residual ash) were analyzed according to the methods of Van Soest et al. (1991) and the AOAC method (AOAC International, 2000; method 973.18), respectively. Non fiber carbohydrates (NFC) were calculated as 100 – CP – EE – aNDFom – CA. The milk fat, protein, lactose, and milk urea nitrogen (MUN) were analyzed in Tochigiken Rakuno Kyoudo Kumiai (Tochigi, Japan) by using infrared spectroscopy methods. The samples of ruminal fluid were centrifuged at 3,000 × rpm for 10 min at 4°C. The supernatants were shaken with a cation exchange resin (Amberlite, IR-120B H AG; Organo Corp., Tokyo, Japan) and centrifuged at 12,000 × rpm for 10 min at 4°C. The supernatants were used for analyzing lactic acid and volatile fatty acids (VFAs) by high performance liquid chromatography (HPLC) (Hosoda et al., 2005). Ruminal ammonia was determined spectrophotometrically using commercially available kits for ammonia (Wako Pure Chemical Industries, Osaka, Japan). The plasma concentrations of total protein (TP), blood urea nitrogen (BUN), total cholesterol (TCHO), β-hydroxybutyrate (BHBA), glucose (GLU), non-esterified fatty acids (NEFA), calcium (Ca), and inorganic phosphorus (IP) and the activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactose dehydrogenase (LDH) were analyzed biochemical automatic analyzer (LABOSPECT 008, Hitachi, Tokyo, Japan) Statistical analysisThe data were analyzed statistically according to the crossover design by using R version 4.2.2. The model is given by Yijkl = μ + Ai + Bj + Ck(Bj) + Dl + Eijkl, where Yijkl is the observed dependent variable, μ is the overall mean, Ai is the effect of diet, Bj is the effect of the experimental group, Ck(Bi) is the effect of cow nested in the experimental group, Dl is the effect of the period, and Eijkl is the residual. The treatment is significantly different if the P value is < 0.05 and tends to be different if the P value is < 0.10. RESULTS The chemical composition of fermentation characteristics of experimental silage is shown in Table 2. The silage pH was 3.76 in high quality silage and 4.21 in low quality silage. The lactic acid concentration of high quality silage was 2.16% (on a fresh matter (FM) basis) and that of low quality silage was 0.94%FM. Acetic, propionic, and butyric acid concentrations of high quality silage were 1.02, 0.08, and 0.08%FM, respectively. Acetic acid, propionic acid, and butyric acid concentrations of low quality silage were 0.81, 0.14, and 0.10%FM, respectively.        Table 3 shows the chemical composition and fermentation characteristics of experimental PMR. The pH was 4.60 in high quality silage and 4.90 in low quality silage. The lactic acid concentration of high quality PMR was 1.54%FM, and that of low quality PMR was 0.89%FM. The acetic acid concentration of high quality PMR was 0.72%FM and that of low quality PMR was 0.57%FM.Table 4 shows the PMR intake and milk production, ruminal fermentation, and the CH4 emissions of experimental cows. The PMR intake of the cows in the low group (24.1 kg/day) tended to be lower (P < 0.10) than that of the cows in the high group (26.1 kg/day). There were no significant differences in milk yield or concentrations of milk fat, protein, and lactose. The MUN of the cows in the low group was significantly lower (P < 0.05) than that of the cows in the high group. The ruminal ammonia of the cows in the low group was significantly lower (P < 0.01) than that of the cows in the high group. The ruminal acetic acid concentration of cows in the low group was significantly higher (P < 0.01) than that of cows in the high group. The ratio of acetic acid to propionic acid (A:P ratio) in the low group tended to be higher (P < 0.10) than that in the high group. There were no significant differences in ruminal pH or the concentrations of total VFA, propionic acid, iso-butyric acid, butyric acid, iso-valeric acid, and valeric acid, between cows in the high and low groups. There were no significant differences in the CH4/CO2 ratio or estimated CH4 emissions. The CH4 emissions per total intake in the low group were significantly higher (P < 0.01) than those in the high group. Table 5 shows the plasma metabolites of the experimental cows. Plasma TCHO, AST, ALT, and ALP of cows in the low group cows was significantly higher than those in the high group. The plasma LDH of cows in the low group tended to be higher than those in high group. There were no significant differences in plasma concentrations of TP, BUN, BHBA, GLU, NEFA, Ca, or IP between cows in the high and low groups.DISCUSSIONWhen silage is exposed to air during feeding, aerobic deterioration may occur because of undesirable microbial activity (Wilkinson & Davies, 2013). Aerobic deterioration of silage is unavoidable after opening of the silo; therefore, silage quality needs to be checked frequently. For simplified monitoring the degree of aerobic deterioration, so far, changes in temperature or pH have been used as indicators (Shan et al., 2021). We recently demonstrated that one of the nanomechanical sensors MSS for detecting VFAs in silage and showed the sufficient discrimination of the silage samples (Minami et al., 2023). This sensor has clearly discriminated the quality of corn silage used in this study, and feeding trial was conducted for practical use of devise. It is known that the aerobic deterioration of silage causes a decrease in dry matter, an increase in pH, and an increase in temperature (Brüning et al., 2017; Chen & Weinberg, 2009; Gerlach et al., 2013). Also, losses of lactic acid, and acetic acid are observed in the aerobic exposure of silage (Brüning et al., 2017). Corn silage and the PMR of the low group had a higher pH and lower lactic acid and acetic acid concentrations, showing that aerobic deterioration progressed in silage containing PMR fed to the low group in this experiment.Feeding poor quality silage is well known to have various effects on dairy cows. The PMR intake of the low group tended to be lower than that of the high group. Brüning et al. (2017) showed that aerobic exposure post-opening of maize silage caused strong avoidance and low DMI of goats in a choice situation. Gerlach et al. (2013) demonstrated that strong changes concerning the fermentation products of maize silage and decreased DMI of goats. Wichert et al. (1998) reported that the silage exposed to air led to a decrease roughage of cows. These reports (Brüning et al., 2017; Gerlach et al., 2013; Wichert et al., 1998) showed that silage with aerobic exposure reduces the DMI in ruminants, as is shown in our data.Although the chemical composition of two PMRs are almost the same, the difference in silage fermentation quality affected ruminal parameters in this study. That is, a higher ruminal acetic acid concentration and A:P ratio were observed in cows in the low group. The silage that has deteriorated because of exposure to air often affects the nutritive value. Matsuoka & Fujita (1993) showed that the digestibility of NDF and ADF were significantly higher for the deteriorated silages than for the control silages in vitro trial. Furthermore, Tabacco et al. (2011) showed that DM and nutritional losses occurred during the exposure of corn silage to air. Amaral et al. (2014) reported that DM and organic matter (OM) digestibility was decreased in lactating cows fed deteriorated corn silage. Fujita et al. (1980) reported that the decrease in the nitrogen free extract (NFE) fraction digestibility in dry cows was most prominent in aerobic deteriorated grass silage and suggested the possibility of the disappearance of easily fermentable carbohydrates during aerobic deterioration. These changes in nutritional value in aerobic spoiled silage were thought to affect VFA concentrations in the rumen. Methane production in the rumen of ruminants is loses energy of the feed, and, in recent years, as a greenhouse gas, it must be reduced. It is well known that CH4 production is closely related to feed, including the level of intake, type, and quality of feeds (Shibata & Terada, 2010). Mitsumori et al (2008) mentioned that biological control of rumen fermentation is important for controlling CH4 production and the ruminal A/P ratio decreases, and CH4 production decreases because less hydrogen is available for CH4 production. Our results also showed that higher A/P ratio and higher CH4 production of cows in low groups. Hristov et al. (2013) suggested that improving forage quality is an effective way of decreasing CH4 emission. It is well known that silage that has deteriorated because of exposure to air often lowered the nutritive value. It is quite possible that the silage offered to experimental cows in low groups also had relatively low nutritional value. In addition, it is reported that the improved silage fermentation significantly decreased CH4 production, possibly because high lactic acid in silage increased propionic acid content in rumen (Kaewpila et al. 2021; Khota et al. 2017). In our study, it is possible that differences in nutrient value and lactic acid content of the offered silages affected rumen fermentation, resulting in differences in CH4 production.Plasma AST, ALT, ALP, and LDH —which were used as indicators of liver function—of cows in the low group were higher than those in the high group. Feeding poor quality silage is known to affect the health of cows. Some molds generated in aerobically deteriorated silage produce mycotoxins that are toxic to dairy cows and cause reduced liver function (Ogunade et al., 2018). Mold was not visually observed in the low quality silage; however, it is considered that the deterioration of silage may have put a strain on the liver. Plasma CHO level is used as an index for energy status (Kida, 2002). The plasma CHOs of cows in the low group were higher than those in the high group, although the DMI of the lower group tended to be lower with the same level of milk production. What causes the difference in CHO is not clear; the plasma CHO level in both groups is roughly within the standard value (Kida, 2002). Tabacco et al. (2011) indicated that substantial changes in nutritional quality occurred when silage was exposed to air and suggested that air exposure causes a reduction in the estimated milk yield. However, the difference in corn silage quality had no obvious effect on milk yield, milk fat, protein, or lactose concentration in this study. Kirklanda & Gordona (2001) reported that late lactation cows partitioned significantly less of the change in energy intake to milk yield than early lactation cows. Science the experimental cows was near dry off, therefore, it is supposed that the decrease of intake and poor silage quality did not lead to a reduction of milk yield.As the quality of silage changes day by day, the development of methods for quickly determining its quality is highly demanded. Silage that the sensor detects to have low quality has shown the possibility to reducing the intake of cows. Our results also indicated that poor quality silage might affect nutrient metabolism and the health of dairy cows. The monitoring of silage quality will lead to improved feeding management of dairy cows. AcknowledgementsWe thank the staff of the livestock management team at the NARO Institute of Livestock and Grassland Science for feed preparation and animal care. This study was financially supported by the Public/Private R&D Investment Strategic Expansion Program (PRISM), Cabinet Office, Japan; and Center for Functional Sensor & Actuator (CFSN), NIMS.CONFLICT OF INTERESTAll authors declare that they have no conflicts of interest.REFERENCES Amaral, R. C., Santos, M. C., Daniel, J. L. P., Sá Neto, A., Bispo, A. W., Cabezas-Garcia, E. H., … Nussio, L. G. (2014). The influence of covering methods on the nutritive value of corn silage for lactating dairy cows. 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