Effects of bupleurum extract on the haematological, mineral, and hormonal profiles of heat-stressed dairy cows

Abstract This study was conducted to investigate the effects of Bupleurum extract (BE) on the haematological profiles, the mineral and hormone levels of heat stressed dairy cows. Forty Holstein cows (75 ± 15 days in milk, 37.5 ± 1.8 kg of milk/d, and 1.7 ± 0.4 parity) with heat stress were randomly assigned to four treatments, which consisted of 0, 0.25, 0.5, or 1.0 g BE/kg DM (Dry Matter). Compared with the control group, cows that were fed 0.5 g/kg BE had a higher red blood cell count, haemoglobin, haematocrit, and white blood cell count. Compared with the control group, the supplementation of 0.25 and 0.5 g/kg BE decreased creatine kinase levels. Compared with the cows that were fed 0 or 1.0 g/kg BE, glutamic-oxaloacetic transaminase and alkaline phosphatase activities were lower in those given 0.5 g/kg BE, while serum glutamic-pyruvic transaminase was lower in those given 0.25 and 0.5 g/kg BE. Compared with the control group, the supplementation of 0.5 and 1.0 g/kg BE decreased sodium concentrations and increased potassium and calcium concentrations. In addition, the supplementation of 1.0 g/kg BE decreased phosphorus concentrations, while that of 0.5 g/kg BE increased chloride concentrations. As for hormone levels, triiodothyronine and prolactin levels increased in cows given 1.0 g/kg BE, while cortisol levels were lower in cows given 0.5 g/kg BE. Further, growth hormone levels were elevated in cows fed 0.25 g/kg BE. These findings suggest that supplemental BE at 0.5 g/kg could have positive effects on the blood metabolism of heat-stressed cows. HIGHLIGHTS Supplementation of 0.5 g/kg Bupleurum extract improved the haematological parameters of heat stressed dairy cows; Bupleurum extract can be used as a feed additive for cows.


Introduction
Heat stress is caused in dairy cows due to an imbalance between heat accumulation and heat dissipation in a hot environment (Bagath et al. 2019).The milk yield and dry matter intake (DMI) of dairy cows subjected to heat stress decreases significantly.In addition, heat stress can also destroy their immune system and lead to metabolic disorders (Tao et al. 2012;Sammad et al. 2020).Previous studies have shown that heat stress can cause changes in their haematological parameters and disorders related to hormone levels in their serum (Rhoads et al. 2009;Giri et al. 2017).Moreover, changes to the circulation of water and minerals caused by heat stress can destroy the osmotic balance and the ability to maintain blood pressure in animals (Bernabucci et al. 2010).The emergence of these problems further leads to a decline in the production and reproductive performance of dairy cows (Becker et al. 2020).Therefore, it is necessary to alleviate the heat stress of dairy cows during summer.
The genus Bupleurum belongs to the family Apiaceae and is widely distributed in the Northern Hemisphere.It has been widely used as a medicine in China for a long time (Li et al. 2019).In recent years, studies have shown that Bupleurum extract (BE) has immunomodulatory, anti-inflammatory, anti-tumour, antioxidant, antiviral, antibacterial, and other pharmacological effects (Ashour et al. 2011(Ashour et al. , 2018)).Its main active ingredient is saikosaponin, saikosaponin a (SSa) is believed to be most important active saikosaponin, which can have an anti-inflammatory effect by inhibiting the expression of certain inflammation-related factors and increases the expression of anti-inflammatory cytokine (Yuan et al. 2017).SSa dose-dependently inhibited the expression of ROS, TNF-a and IL-8 in human umbilical vein endothelial cells at treated of 0-200 lg/ml (Fu et al. 2015).Feed SSa to rats inhibited the production of IL-1b, IL-6, TNF-a, and increased the level of IL-10, TGF-b1 at the dose of 0.004%, respectively (Wu et al. 2010).In addition, saikosaponin can further inhibit the activation of T lymphocytes by inhibiting the activation of NF-jB and promote the apoptosis of tumour cells by activating caspase apoptosis pathways, which play an anti-tumour and immunomodulatory role (Li et al. 2018).In our previous study, BE was used as a feed additive to feed Holstein cows under heat stress, and it was found that the addition of BE could improve their production performance and alleviate their heat stress (Pan et al. 2014).Thus, we further studied the blood metabolites antioxidant status and immune function of heat-stressed dairy cows and found that the contents of antioxidant enzymes and the levels of IgA and IgG in the cows' serum increased, and BE improved their liver function because the levels of total protein and albumin in their serum increased as well (Cheng et al. 2018).However, the effects of directly ingested BE on the haematological profiles and the mineral and hormone levels of heat-stressed, lactating Holstein cows are still unknown.Therefore, the objective of this study was to evaluate the effects of BE supplementation on the haematological profiles, mineral metabolism, and hormone levels of early-lactation dairy cows during the summer months.

Animals and experimental design
This experiment was conducted in Shanghai Xinghuo Dairy Factory in summer from July to September, 2012.Forty lactating Holstein cows (75 ± 15 days in milk, 37.5 ± 1.8 kg of milk/d, and 1.7 ± 0.4 parity) were randomly divided into four groups, with 10 cows in each group.The cows were fed 1) a basal diet without BE addition (control group), 2) a basal diet with 0.25 g BE/kg DM, 3) a basal diet with 0.5 g BE/kg DM, or 4) a basal diet with 1.0 g BE/kg DM.The product used in this experiment is a commercial offering (Beijing Centre Biology Co. Ltd., China), in which the content of BE is 35%, while the rest is starch.The proportions of saikosaponins, essential oils, and polysaccharides in this BE were 6.6%, 2.5%, and 25.9%, respectively.
The basal diet fed to the cows during the trial (Table 1) was formulated to meet or exceed the relevant nutrient recommendations (NRC 2001).The ratio of forage to concentrate of the basal diet was 61:39.For the duration of the experiment, the cows were housed in tie stalls and had free access to fresh water.They were fed traditional separate-ingredients ration-roughage first and then concentrate feed three times daily at 05:30, 13:00, and 20:00 h-to ensure 5% refusals.Further, the BE supplements were top-dressed in equal portions to the concentrated feed used for the morning feeding, and the cows could eat the concentrated feed containing all of the BE supplements.All the cows were provided with forced-air ventilation using fans.Ambient temperature and humidity were recorded daily (0600, 1400 and 2200 h), and the mean Temperaturehumidity index (THI) in the barn in which the animals were housed was 78.2 (range: 71.9 to 80.8) at 06:00 h, 79.7 (range: 72.7 to 83.3) at 14:00 h, and 78.3 (range: 70.2 to 81.7) at 22:00 h.Rectal temperature (RT) and respiration rate (RR) were measured at 0700 and 1400 h twice a week.Rectal temperatures were measured using a glass mercury thermometer (Nasco, Ft.Atkinson, WI), and RR was determined by counting numbers of flank movements/min for 120 s (Pan et al. 2014).The experiment timeline consisted of one week for adaptation to the diet and nine weeks for sampling.

Sample collection
Blood samples were collected from all the cows before the morning feeding via the caudal vein on Day 0 (first week), 42 (sixth week), and 63 (9th week).A volume of 10 mL of blood from the samples was extracted into EDTA-treated vacuum tubes, and the haematological parameters were analysed.Seven millilitres of blood were collected in vacuum tubes without anticoagulant and centrifuged at 3,000 rpm for 15 min at 4 C.The serum obtained was stored at À40 C for the analysis of the constituent blood metabolites, mineral content, and hormone levels.

Data analysis
The data for the haematological profiles, enzyme activities, mineral content, and hormone levels were analysed by repeated measures using the mixed model procedure of SAS (2013).Week, treatment, and treatment Â week interaction were taken as the fixed effects, and the dairy cows were the random effects.Each variable was analysed with the corresponding data during the pre-treatment period as a covariate.The differences among the treatments were analysed using Tukey's multiple range tests.
The statistical model was as follows: Here, Y ij denotes the dependent variable, l denotes the overall mean, T i represents the treatment effect, W j represents the date effect, TW ij denotes the fixed interaction effect between the treatment and date, and E ij represents the error term.The value is expressed as the least square means with a standard error.Significant differences were identified at p 0.05.The differences at 0.05 < p 0.10 were considered as a trend towards significance.

Haematological profile
The effects of the BE treatment on the cows' haematological profiles are shown in Table 2. Cows given 0.5 g/kg BE had a higher (p < 0.05) RBC count and HGB and HCT levels; however, the ingestion of 0.25 or 1.0 g/kg BE had no effect (p > 0.05).Compared with the control group, the WBC count was higher (p < 0.05) in cows that were fed 0.5 g/kg BE and tended to increase (p < 0.1) in those given 0.25 g/kg BE; but no effect was found (p > 0.1) in cows given 1.0 g/kg BE.The supplementation with BE had no effect (p > 0.05) on the counts of MCV, MCH, MCHC, RDW, PLT, and MPV.The RBC and HGB levels were higher (p < 0.01) at six weeks than at the first week, and there was a significant (p < 0.01) Trt Â Time interaction in the RBC.

Serum enzyme
The effects of BE treatment on serum enzyme activities are shown in Table 3.Compared with the control group, the activities of GPT and CK were lower (p < 0.05) in cows given 0.25 and 0.5 g/kg BE.The activities of GOT and ALP were lower (p < 0.01) in cows given 0.5 g/kg BE and tended to be lower (p < 0.1) in cows that were fed 0.25 g/kg BE compared with the control cows.However, no effect was found (p > 0.05) in cows that were fed 1.0 g/kg BE.The activities of GPT, ALP, and CK were lower (p < 0.05) at 9 weeks than at the first week.

Serum mineral
The parameters of the serum mineral content are shown in Table 4. Compared with the control group, BE supplementation increased (p < 0.01) the concentration of K but had no effect (p > 0.05) on the Mg levels.The concentrations of Na were lower in cows given 0.5 g/kg BE.Ca was higher (p < 0.05) in those that were fed 0.5 and 1.0 g/kg BE, compared with control cows, and tended to be higher (p < 0.1) in those that were fed 0.25 g/kg BE.The concentrations of P were lower (p < 0.05) in cows given 1.0 g/kg BE and tended to be lower (p < 0.1) in those that were fed 0.25 and 0.5 g/kg BE.Further, the concentrations of Cl were higher (p < 0.05) in cows that were fed 0.5 g/kg BE and tended to be higher (p < 0.1) in those given 0.25 g/kg BE.Finally, the concentrations of K and Ca were higher (p < 0.05) at 9 weeks than the first week, and there was a significant (p < 0.01) Trt Â Time interaction for K and Cl.

Serum hormone
The serum hormone concentrations are shown in Table 5. BE supplementation decreased (p < 0.05) the concentration of COR and tended to reduce (p < 0.1) the level of GLU when compared with the control group, but it had no effect (p > 0.05) on the levels of T4, INS, NPY, IGF-I, LEP, and HSP70.Cows given 1.0 g/ kg BE had higher (p < 0.05) T3 and PRL concentrations compared with the control group, and the T3 level had a tendency to increase (p < 0.1) in cows that were  ,b Means with different superscripts within the same row differ significantly (p < 0.05). 1) WBC, white blood cell; RBC, red blood cell; HGB, haemoglobin; HCT, haematocrit; MCV, mean red blood cell volume; MCH, mean corpuscular haemoglobin; MCHC, mean erythrocyte haemoglobin concentration; RDW, red blood cell distribution width; PLT, platelets; MPV, mean platelets volume.
2) Cows were fed a basal diet (control) or basal diet supplemented with 0.25, 0.5, or 1.0 g BE kg-1 dry matter.
2) Cows were fed a basal diet (control) or basal diet supplemented with 0.25, 0.5, or 1.0 g BE kg-1 dry matter.
3) Trt, treatment; Int, treatment time Â interactions.fed 0.5 g/kg BE.Cows given 0.25 g/kg BE presented increased (p < 0.05) GH concentration, and this tended to be higher (p < 0.1) in cows that were fed 0.5 or 1.0 g/kg BE.The levels of T3 and COR were higher (p < 0.01) at 9 weeks than the first week, and there was a significant (p < 0.01) Trt Â Time interaction for PRL.

Discussion
The critical value for cow to suffer from THI is 72 (West et al. 2003).In the present study, minimum average THI was 78.2, exceeding the critical value.When the respiratory rate of a cow is more than 60 times per minute, and its rectal temperature reaches 39 C, the cow is said to be in a state of severe heat stress (Das et al. 2016).In this experiment, the average respiratory rate of the control group was 71.4 per minute, and the average rectal temperature was 39.3, indicating that the cows were suffering from severe heat stress.The detailed data for the same were reported in another study (Pan et al. 2014).
Heat stress can lead to an increase in reactive oxygen species (ROS), which leads to the attack of free radicals on the erythrocyte membrane and causes the lysis of RBC (Das et al. 2016).A lower concentration of saikosaponin can stabilise erythrocyte membranes; but at high concentration, it can induce erythrocyte lysis (Abe et al. 1981).This may explain why the RBC count increased for the dose of 0.5 g BE/kg DM and decreased for the dose of 1.0 g BE/kg DM.Thus, supplementation of 1.0 g BE/kg DM could induce erythrocyte lysis in a long term, but had no negative effect in a short term, it resulted RBC increase in the sixth week and then decrease in 9th week.During the experiment, the average THI in the first, sixth, and 9th week was 80, 78, and 71, respectively (Pan et al. 2014).This means that the animals suffered different degrees of heat stress during different times in the course of the experiment.Therefore, the Trt Â Time interaction of the RBC may have been caused by the different effects of BE in different degrees of heat stress.The RBC count was positively correlated with HGB and HCT; thus, HGB and HCT recovered with the recovery of the RBC count after BE supplementation (Mahmoud et al. 2013).In addition, long-term heat stress could increase serum osmotic pressure and decrease MCV and WBC (Gharibi et al. 2020).Furthermore, heat stress damaged lymph organs and reduced WBC by the reduction of feed intake in  -c Means with different superscripts within the same row differ significantly (p < 0.05). 1) Na, sodium; K, potassium; Mg, magnesium; Ca, calcium; P, phosphorus; Cl, chloride.
2) Cows were fed a basal diet (control) or basal diet supplemented with 0.25, 0.5, or 1.0 g BE kg-1 dry matter.
2) Cows were fed a basal diet (control) or basal diet supplemented with 0.25, 0.5, or 1.0 g BE kg-1 dry matter.
Japanese quail (Mahmoud et al. 2013).In another report of this experiment, the supplementation of BE increased the DMI of heat-stressed dairy cows, which could be one of the reasons for the increase in WBC (Pan et al. 2014).In the present study, concentration of HGB increased in sixth week, HCT and MCHC increased in 9th week, respectively.It is speculated that BE ameliorate hematological profiles of heat stressed dairy cows.
Heat stress caused the excessive accumulation of triglycerides and cholesterol in the liver of the dairy cows, affected the normal metabolism of the liver, and caused liver damage (Skibiel et al. 2018).The liver damage resulted in an increase in the serum GPT, GOT, ALP, and CK levels.The addition of BE significantly reduced the content of these enzymes in the serum, which proved that BE not only had a protective effect on the liver of dairy cows under heat stress but also might play a certain role in preventing the disorder of amino acid metabolism.This is because GOT and GPT are important intermediate enzymes in amino acid metabolism, and ALP is involved in vitamin B6 metabolism (Rader et al. 2017;Guo et al. 2020;Rawat et al. 2020).The decrease in CK levels after BE supplementation can be considered to indicate that BE promotes glycolysis in heat-stressed dairy cows, as, in the body, CK reduces the production of ROS mainly by coupling with ATP, and a large part of these available ATP comes from glycolysis (Wallimann et al. 2011).In addition, the up-regulation of ALP led to the calcification of medial vessels and stiffness of medial vessels; hence, BE may have a protective effect on blood vessels (Rader 2017).Compared with the first week, the concentration of GPT, ALP and CK decreased significantly at 9th week, revealed BE alleviate heat stress by improving serum enzymes.
Heat stress has been found to cause a decrease in serum Na and K and plasma Ca levels in dairy cows (Collier et al. 2017;Sugiharto et al. 2017).In this study, the higher K and lower serum Na concentrations of dairy cows that were fed BE may be due to the interaction between the saponins and the erythrocyte membranes, leading to the formation of pores in the membrane and making it permeable to ions, especially to the diffusion of Na from the plasma to the RBC and the diffusion of K from the RBC to the plasma (Hoffman 1962).In another study, the supplementation of BE increased the concentration of serum albumin, while the concentration of Ca in the blood was linked to the concentration of albumin; in addition, approximately 45-50% of the total plasma Ca was bound to the serum albumin, which may lead to an increase in Ca concentrations after BE supplementation (Srikandakumar and Johnson 2004).In addition, after the remission of the heat stress, the serum osmotic pressure decreased, which increased the concentration of Cl and led to the increase in H þ concentration to achieve electron neutralisation.This, in turn, led to a decrease in blood pH and an increase in serum Ca (Chan et al. 2006).The decrease in serum P levels may be a result of the anti-inflammatory effect of saikosaponin.The Trt Â Time interactions for K and Cl may be caused by the different effects of BE at different degrees of heat stress.
Triiodothyronine (T3) can be used as a reliable indicator of long-term heat stress.After heat stress, the levels of T3 and thyroxine (T4) decreased, and the difference in the concentrations of T3 was more obvious than those of T4.In this study, BE increased the levels of T3, but those of T4 did not change.It could be that BE stimulates the function of the peripheral tissues of dairy cows and converts a large amount of T4 into T3 in these tissues (Melesse et al. 2011;Morris and Galton 2019).Although the levels of GLU did not change significantly after BE supplementation, it tended to decrease, which is related to the increase in the GH and IGF-1 levels.The effect of GH on glucose metabolism will be complicated by IGF-1.When the level of GH is high, the level of IGF-1 will increase.IGF-1 has a biological function similar to insulin and can reduce blood sugar by stimulating glucose uptake and gluconeogenesis (Boucher et al. 2010;Kim and Park 2017).PRL is a poly-peptide hormone that has an immunomodulatory effect and is synthesised and secreted by specialised cells of the anterior pituitary.It affects breast development, milk synthesis, and the maintenance of milk secretion under heat stress.The hypothalamus-pituitary-gonadal (HPA) axis is activated, and PRL secretion decreases because of HPA axis disorders, resulting in a decline in lactation performance (Jacobi et al. 2001;Zhang et al. 2020).In another study, milk yield increased after BE supplementation, whereas, in this study, PRL increased after the supplementation of BE.This implies that BE has a certain inhibitory effect on the HPA axis (Lee et al. 2012;Pan et al. 2014).Similarly, there are Trt Â Time interactions among GLU, PRL, and IGF-1, which is speculated to be due to the different effects of BE on these hormones at different degrees of heat stress.The alleviating effect of BE on heat stress is reflected in the changes in the COR and HSP70 levels.The negative energy balancecaused by heat stress increases the concentration of COR.Because the amino acids and fats stored in the COR cells are used for the synthesis of energy, glucose, and other required compounds, the reduction in COR levels represents the relief of a negative energy balance.The changes in HSP are due to the molecular mechanism that maintains intracellular homeostasis at the cellular level, in which HSP70 has the highest temperature sensitivity and has a positive correlation with heat tolerance (Sahin et al. 2002;Sharma et al. 2013).However, although T3 increased in 9th week, concentration of COR increased in 10th week and the changes in HSP70 were not significant, these results revealed supplementation of BE improve some hormone levels in heat stressed cows, but cows still suffered from heat stress.

Conclusions
Bupleurum extract (BE) supplementation changed the hematological characteristics, serum mineral, and endocrine hormone levels of heat-stressed dairy cows.In the present study, supplementation of 0.5 g BE/kg DM was found to improve the hematological characteristics by increasing the concentration of WBC, RBC and HGB.It also decreased GPT, GOT, ALP, CK and increased K, Cl in serum, respectively.In addition, it inhibited COR level and promoted PRL level in serum hormone.Overall, these changes were the manifestation of the recovery of the pathological state of dairy cows that suffered from heat stress.

Table 1 .
Ingredients and chemical composition of the basal diet.

Table 2 .
Effects of different doses of Bupleurum extract (BE) on the hematological profile in heat-stressed dairy cows.

Table 3 .
Effects of different doses of Bupleurum extract (BE) on serum enzyme activities in heat-stressed dairy cows.

Table 4 .
Effects of different doses of Bupleurum extract (BE) on serum mineral metabolism in heat-stressed dairy cows.

Table 5 .
Effects of different doses of Bupleurum extract (BE) on serum hormone concentrations in heat-stressed dairy cows.