Effects of Lonicera japonica extract on performance, blood biomarkers of inflammation and oxidative stress during perinatal period in dairy cows

Objective An experiment was conducted to evaluate the effects of Lonicera japonica extract (LJE) on milk production, rumen fermentation and blood biomarkers of energy metabolism, inflammation and oxidative stress during the perinatal period of Holstein dairy cows. Methods Eighteen Holstein dairy cows were used in a complete randomized design experiment with 3 dietary treatments and 6 cows per treatment. All cows received the same basal total mixed ration (TMR) including a prepartal diet (1.35 Mcal of net energy for lactation [NEL]/kg of dry matter [DM], 13.23% crude protein [CP]) from −60 d to calving and a postpartal diet (1.61 Mcal of NEL/kg of DM, 17.39% CP) from calving to 30 days in milk (DIM). The 3 dietary treatments were TMR supplemented with LJE at 0 (control), 1 and 2 g/kg DM, respectively. LJE was offered from 21 d before calving to 30 DIM. Dry matter intake (DMI) and milk production were measured daily after calving. Milk and rumen fluid samples were collected on 29 and 30 d after calving. On −10, 4, 14, and 30 d relative to calving, blood samples were collected to analyze the biomarkers of energy metabolism, inflammation and oxidative stress. Results Compared with control diet, LJE supplementation at 1 and 2 g/kg DM increased DMI, milk yield and reduced milk somatic cell count. LJE supplementation also decreased the concentrations of blood biomarkers of pro-inflammation (interleukin-1β [IL-1β], IL-6, and haptoglobin), energy metabolism (nonesterified fatty acid and β-hydroxybutyric acid) and oxidative stress (reactive oxygen metabolites), meanwhile increased the total antioxidant capacity and superoxide dismutase concentrations in blood. No differences were observed in rumen pH, volatile fatty acid, and ammonia-N (NH3-N) concentrations between LJE supplemented diets and the control diet. Conclusion Supplementation with 1 and 2 g LJE/kg DM could increase DMI, improve lactation performance, and enhance anti-inflammatory and antioxidant capacities of dairy cows during perinatal period.


INTRODUCTION
Dairy cows experience substantial metabolic and physiological changes during the perinatal period due to their negative energy balance [1], inflammation [2], oxidative stress [3], and immune dysfunction [4]. For example, failure to meet the energy requirements for fetal growth and lactation during this period may lead to metabolic disorders such as ketosis and fatty liver and compromised antioxidant mechanisms [5,6]. Polymorphonuclear neutrophilic leukocyte function was compromised during the transition period and may lead to peri partum mastitis and metritis [7]. Oxidative stress is induced by the increased production of free radicals and reactive oxygen species and consequently results in a decrease in anti oxidant defense. Oxidative stress also often leads to damage of biologic macromolecules and dysregulation of metabolism and physiology [8], which further results in the imbalance of immune function and inflammatory responses during the transition period [9].
Lonicera japonica, also known as Japanese honeysuckle or goldenandsilver honeysuckle, is a traditional folk medicine and usually used to treat common colds, fevers, enteritis, pain, and swellings in East Asian countries including China, Japan and Korea. More than 140 compounds have been isolated and identified from Lonicera japonica, including organic acids, flavonoids, iridoid glycosides and saponins etc. [10]. There has been more and more interest in Lonicera japonica extract (LJE) because of its unique antiinflammatory [11,12], anti oxidative [13] and hepatoprotective activities [14]. However, the effects of LJE on performance and health of dairy cows during transition period has rarely been reported. Therefore, the objectives of this study were to evaluate the effects of LJE on milk production, rumen fermentation and blood bio markers of energy metabolism, inflammation and oxidative stress during the peripartal periods of Holstein dairy cows.

MATERIALS AND METHODS
All experimental procedures for this study were approved by the Animal Care and Use Committee of the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences (IAS20181).

Animal management
Animals were fed twice daily at 0700 h and 1800 h during the experimental period ad libitum. Dry cows were housed in a ventilated free stall barn until 5 d before expected parturition date, and then they were moved to individual maternity barn with straw bedding until calving. The cows stayed in the mater nity barn for 5 to 9 d. After parturition, they were transferred to a lactation barn and milked at 0700 h, 1300 h, and 1900 h daily.

Experimental design and dietary treatments
Eighteen Holstein dairy cows were used in a complete ran domized design experiment with 3 dietary treatments (n = 6). The cows in each group were balanced according to their parity (3.3±1.2), body condition score (3.48±0.07), initial body weight (759±13 kg) and expected calving date. All cows re ceived the same basal total mixed ration (TMR) including a prepartal diet (1. [15] to meet or exceed the nutrition requirements. Diet ingredients and chemical composition are shown in Table 1. Lonicera japonica extract (LJE) was added from 21 d before expected calving date until 30 DIM. The 3 dietary treatments were basal TMR supple mented with LJE powder at 0 (control), 1 and 2 g/kg DM, respectively. The ethanol extract of lonicera japonica was pur chased from a commercial supplier (Zhongxing Biotechnology Co. Ltd., Xian, China). Chlorogenic acid has been reported as the major organic acid and used as the chemical marker for the quality evaluation of LJE [16]. The content of chlo rogenic acid was 10% in the LJE used in the current study analyzed by liquid chromatography (LC20A, Shimadzu, Kyoto, Japan).

Diets sampling and analysis
Automatic feeding equipment (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China, and NanShang Husbandry Science and Technology Co. Ltd., Henan, China) was used to record DMI after calving. Samples of each diet were collected twice per week, stored at -20°C and composited weekly for analysis of DM, CP, ether extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), ash, calcium (Ca), and phosphorus (P). The DM content was determined by oven drying at 105°C until the weight is constant (method 930.15, AOAC) [17]. The CP content was determined using Kjeldahl nitrogen analysis (method 945.16, AOAC) [17]. The EE content was deter mined using a Soxhlet extractor (method 945.16, AOAC) [17]. The NDF and ADF were analyzed using heatstable amylase (Sigma no. A3306, Sigma Chemical Co., St. Louis, MO, USA) and sodium sulfite according to the procedure of Van Soest et al [18]. Ash was measured by combustion using a muffle furnace (method 942.05, AOAC) [17]. The colorimetric method was used for analysis of phosphorus (Spectrophotometer UV752N, Yoke Instrument Co. Ltd., Shanghai, China) and calcium was measured using atomic absorption spectrometry (PerkinElmer AAS800, Waltham, MA, USA).

Blood sampling and analysis
Blood samples were collected from the coccygeal vessel before the morning feeding on -10 d relative to expected calving and on 4, 14, and 30 d after calving. Samples were collected into 10mL vacutainer tubes (Kindly Enterprise Development Group Co., Ltd., Shanghai, China) containing either clot ac tivator or sodium heparin for serum and plasma, respectively. Serum and plasma were harvested by centrifugation at 3,000 ×g for 15 min at 4°C and stored at -80°C for further analysis. Serum and plasma were analyzed using a clinical autoanalyzer (GFD200, Shandong Caihong Analytical Instruments Co. Ltd., Shandong, China) for glucose, urea, βhydroxybutyric acid (BHBA), nonesterified fatty acid (NEFA), aspartate ami notransferase (AST), gammaglutamyl transferase (GGT), cholesterol, total antioxidant capacity (TAOC), superoxide dismutase (SOD), and glutathione peroxidase (GSHPx). The concentrations of ceruloplasmin, haptoglobin, reactive oxy gen metabolites (ROM), interleukin1β (IL1β) and IL6 were determined according to the procedures described by Jacometo et al [19] and absorbance was read with a microplate reader (Multiskan MK3, Thermo Labsystems, Philadelphia, PA, USA).  [15].

Ruminal fluid sampling and analysis
Rumen contents were collected at 2 h postfeeding using a stomachtube (gastric lavage flexible pipe hose) on 29 and 30 d after calving. To reduce the contamination of saliva, the first 250 mL rumen fluid was discarded. Rumen contents were strained through 4 layers of cheesecloth with a mesh size of 250 μm and immediately determined pH with a portable pH meter (370 model pH meter, Jenway, London, UK). Each 10 mL of filtered rumen fluid of samples was mixed with 2 mL of 250 g/L metaphosphoric acid and stored at -20°C for vol atile fatty acid (VFA) and NH 3 N determination. Individual and total VFA (TVFA) were analyzed by gas chromatograph (GC2010, Shimadzu, Japan) and NH 3 N content was deter mined by phenolhypochlorite method using a microplate reader (Multiskan MK3, Thermo Labsystems, USA).

Statistical analysis
Data were analyzed as a completely randomized design using analysis of variance for evaluation of the effects of LJE on animal performance, rumen fermentation parameters and blood biomarkers of energy metabolism, inflammation and oxidative stress. The statistical program used in the present study was SAS 9.4 (SAS Institute Inc., Cary, NC, USA) with probability levels of p≤0.05 for significance and 0.05<p≤0.10 for tendency in treatments.

Milk production and composition
As shown in Table 2, LJE supplementation at 1 and 2 g/kg DM both had greater DMI (p<0.05), milk yield (p<0.05), milk fat yield (p<0.01), milk protein yield (p<0.01), 4% FCM (p< 0.05) and ECM (p<0.05) and less milk SCC (p<0.05) than the control diet. Moreover, LJE supplementation at 1 g/kg DM had better DMI and milk yield than 2 g/kg DM group (p<0.05). However, there was no significant effect of LJE supplementation on milk protein, fat or lactose content.

Blood biomarkers
The effects of LJE supplementation on blood biomarkers are www.ajas.info

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Zhao et al (2020) Asian-Australas J Anim Sci 33:1096-1102 shown in Table 3. The NEFA, BHBA, IL1β, IL6, ROM and haptoglobin concentrations were lower (p<0.05), meanwhile the TAOC and SOD concentrations were higher (p<0.05) in LJE supplementation groups than those in the control group. Moreover, the 1 g/kg DM LJE supplementation group had greater TAOC and less ROM and haptoglobin concentra tions in the blood than those in the 2 g/kg DM LJE group. There was no significant effect of LJE supplementation on the concentrations of glucose, cholesterol, urea, GGT, AST, GSHPx, albumin and ceruloplasmin in blood.

Rumen fermentation parameters
The effects of LJE supplementation on rumen pH, NH 3 N and VFA concentrations are shown in Table 4. No differences between LJE supplementation diets and the control diet were observed in any variables of pH, NH 3 N, acetate, propionate, butyrate, isovalerate, valerate, and TVFA concentrations, and the ratio of acetate/propionate. However, TVFA concentration  was tended (p = 0.07) to increase in the LJE supplementation groups compared with the control group.

Milk production and composition and dry matter intake
During the perinatal period, negative energy balance occurs in dairy cows due to reduced DMI and increased nutrient requirements, which has been reported associated with in creased disease susceptibility. In the present study, we found that LJE supplementation at doses of 1 and 2 g/kg DM in creased DMI of the dairy cows. Doepel et al [20] reported that inflammatory response reduced DMI during the peripartum period of dairy cows. In the current study, LJE supplementa tion significantly decreased blood inflammatory cytokine concentrations (e.g. IL6 and IL1β, Table 3), indicating that inflammation was possibly eased. Hence, LJE supplementation increased DMI and consequently increased the milk yield. However, the milk fat and protein concentrations were not different between the LJE supplementation and the control. Therefore, the milk fat and protein yields, 4% FCM and ECM were not affected by LJE supplementation either. Somatic cell count is the indicator of infection within the udder, and SCC >200,000 cells/mL is considered as being infected by bacteria [21]. In the current study, SCC values of all groups were below 200,000 cells/mL, and the milk SCC in the cows offered LJE supplementation diets were even lower than those offered the control diet. This indicated that adding LJE in diets could effectively reduce the risk of udder infection during the prepartum period.

Biomarkers of energy metabolism
To balance the negative energy during the transition period, dairy cows mobilize adipose tissue to meet energy require ments [2]. However, excessive lipid mobilization leads to increased blood and liver NEFA concentrations and conse quently results in proinflammatory periparturient diseases and fatty liver [5]. Furthermore, as a major ketone body, BHBA can be produced by NEFA oxidation in the liver, and the extreme accumulation of BHBA may lead to ketosis [6].
In the present study, supplementation of LJE at doses of 1 and 2 g/kg DM reduced both NEFA and BHBA concentrations in blood. Previous study has demonstrated that supplemen tation with chlorogenic acid regulated energy metabolism and decreased serum BHBA concentration in rats [22]. More over, LJE supplementation increased the intake of the cows in the current study, therefore reduced the concentrations of NEFA and BHBA by alleviating the mobilization of adipose tissue. These results indicated that LJE supplementation at 1 and 2 g/kg DM could possibly improve the negative energy balance of dairy cows during the transition period.

Biomarkers of oxidative stress
The imbalance between the production of ROM and anti oxidants leads to oxidative stress [23]. The ROM is one of the most abundant free radicals in animals, and has been widely used as a biomarker for oxidative stress [24]. On the other hand, TAOC is an important indicator in assessing concen trations of antioxidants which reflects the ability of reducing free radicals in the animal body [25]. Furthermore, SOD is recognized as a critical antioxidant enzyme in eliminating free radicals [26]. LJE has been reported possessing antioxi dant activity in which chlorogenic acid in LJE is confirmed as the main effective ingredient [13]. Palíková et al [14] also found that the phenolic components isolated from Lonicera japonica alleviated oxidative damage of rat liver microsome and human umbilical vein endothelial cells. In the present study, LJE supplementation at doses of 1 and 2 g/kg DM de creased ROM and increased TAOC and SOD concentrations. This may be attributed to the chlorogenic acid in LJE which has been approved with appreciable antioxidant potential in eliminating hydroxyl radicals and superoxide anion radical [16]. Therefore, LJE supplementation could improve the anti oxidant capacity of the dairy cows during the perinatal period.

Biomarkers of inflammation
Proinflammatory cytokines including TNFα, IL1β, IL6, and IL8 are closely linked to the severity of coliform mastitis during the periparturient period [26], which could effectively stimulate neutrophils to produce prostaglandins and leu kotrienes, and sequentially increase the local inflammatory reactions [27]. Zhou et al [24] demonstrated that proinflam matory cytokines were increased during the transition period due to dramatic metabolic changes. In the current study, IL1β and IL6 concentrations were lower in the cows offered LJE supplementation diets than those fed the control diet. Park et al [11] found that the polyphenol components iso lated from Lonicera japonica had antiinflammatory effect on mouse peritoneal macrophage cells. Kang et al [12] also reported that luteolin from the Lonicera japonica inhibited inflammatory cytokines release by regulating nuclear factor kappa B and mitogenactivated protein kinases pathways in human mast cell. As a major acute phase protein, hapto globin has been used as a diagnostic biomarker due to its quick increase during the acutephase response. Acute phase proteins are mainly produced by hepatocytes activated by the proinflammatory cytokines (e.g., IL1β and IL6) [28].
In the current study, LJE supplementation decreased the blood haptoglobin concentration, which, on the other hand, agreed with the decreased concentrations of IL1β and IL6 as previously described. These results indicated that LJE could contribute to reducing the risk of inflammation during the periparturient period.

Rumen fermentation parameters
Supplementation of LJE tended to increase TVFA concen tration in the current study. This could be explained by that LJE supplementation increased DMI, therefore providing more fermentation substrates for the rumen microorganisms. However, LJE had no significant effect on pH, NH 3 N and individual VFA concentrations. This may be due to the micro organism ecosystem gradually adapted to the LJE addition in the rumen owing to longterm (from 21 d before calving to 30 DIM) feeding [29]. It has been well documented that rumen individual VFA concentrations are closely corelated with the milk composition [30]. Similarly, the unaffected VFA composition could possibly explain the corresponding stable milk ingredients across the three dietary treatments in the current study.

CONCLUSION
Overall, supplementation with 1 and 2 g LJE/kg DM during the periparturient period could increase DMI and milk production and tend to increase TVFA concentration. By analyzing the blood biomarkers of lipid mobilization, oxi dative stress and inflammation, LJE supplementation could also contribute to improving the capabilities of antiinflam mation and antioxidant of dairy cows. Therefore, LJE could be a potential natural plant substitute for the antibiotics to prevent metabolic diseases during perinatal period and im prove animal performance. However, its antiinflammation and antioxidant mechanisms still need to be further inves tigated.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.