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Effects of Radix Bupleuri extract supplementation on lactation performance

and rumen fermentation in heat-stressed lactating Holstein cows

Article  in  Animal Feed Science and Technology · January 2014

DOI: 10.1016/j.anifeedsci.2013.09.008

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 Animal Feed Science and Technology 187 (2014) 1–8

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Effects of Radix Bupleuri extract supplementation on lactation

performance and rumen fermentation in heat-stressed
lactating Holstein cows
L. Pan a , D.P. Bu a,∗ , J.Q. Wang a , J.B. Cheng a , X.Z. Sun a , L.Y. Zhou a , J.J. Qin b ,
X.K. Zhang c , Y.M. Yuan c
a
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan
West Road, Haidian District, Beijing 100193, PR China
b
Beijing Centre Biology Co., Ltd., Beijing 102206, PR China
c
Shanghai Bright Hostan Co., Ltd., Shanghai 200436, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Radix Bupleuri extract (RBE) has been shown to mitigate negative effects of high ambient
Received 8 April 2013 temperature. This experiment was conducted to investigate effects of RBE supplemen-
Received in revised form
tation on lactation performance and rumen fermentation in Holstein cows under heat
13 September 2013
stress. Forty Holstein cows (75 ± 15 d in milk, 37.5 ± 1.8 kg of milk/d, and 1.7 ± 0.4 par-
Accepted 15 September 2013
ity) were randomly assigned to one of four groups (n = 10). One of four treatment diets,
assigned randomly to one of four groups, consisted of RBE supplementation at 0, 0.25,
Keywords: 0.5 or 1.0 g/kg of the basal diet (concentrate and roughage) based on dry matter (DM).
Dairy cow Cows were housed in a tie-stall barn and were individually fed the treatment diets. The
Radix Bupleuri extract experiment lasted for 10 wk in hot summer. During the experiment, average ambient tem-
Heat stress peratures and temperature-humidity indexes (THI) were respectively 27.5 ± 1.5, 29.8 ± 1.9
Lactation performance and 28.1 ± 1.7 ◦ C, and 78.2 ± 2.7, 79.8 ± 3.3 and 78.3 ± 3.4 at 0600, 1400 and 2200 h. Aver-
Rumen fermentation age respiration rates (RR) with RBE at 0.25, 0.50 and 1.0 g/kg were 65.6, 60.3 and 67.4,
respectively, vs. 71.4 (breaths/min) for the control (P < 0.01). Average rectal temperatures
(RT) were 39.1, 39.0 and 39.1 vs. 39.3 ◦ C for the control (P < 0.01). Moreover, cows sup-
plemented with RBE increased dry matter intake (DMI, 22.8, 21.6 and 22.1 vs. 20.9 kg/d)
(P < 0.05) and milk production (34.2, 33.4 and 32.4 vs. 31.6 kg/d) (P < 0.01) compared with
control. Percentages of milk protein and fat were similar among groups, while milk protein
yield increased with increasing level of RBE (0.97, 0.95 and 0.92 vs. 0.89 kg/d for the control)
(P < 0.01). Milk fat yield also increased with RBE (1.13, 1.12 and 1.09 vs. 1.02 kg/d for the
control) (P < 0.05). There was no treatment effect on diet apparent digestibility or volatile
fatty acid (VFA) concentration among groups. Overall, supplemental RBE at 0.25 or 0.5 g/kg
could mitigate the negative effects of heat stress on production in lactating Holstein cows.
© 2013 Elsevier B.V. All rights reserved.

Abbreviations: RBE, Radix Bupleuri extract; DM, dry matter; DMI, dry matter intake; THI, temperature-humidity index; RR, respiration rate; RT, rectal
temperature; VFA, volatile fatty acid; AOAC, Association of Official Analytical Chemists; CP, crude protein; EE, crude fat ether extract; C, control; SCC,
somatic cell count; SCC, milk urea nitrogen; FCM, 4% fat-corrected milk; ECM, energy-corrected milk.
∗ Corresponding author. Tel.: +86 10 62813901; fax: +86 10 62897587.
E-mail addresses: burdenpan@gmail.com, panlong8809@163.com (D.P. Bu).

0377-8401/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.anifeedsci.2013.09.008
2 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8

1. Introduction

Environmental-induced hyperthermia compromises efficient animal production and jeopardizes animal welfare and
therefore is a significant financial burden in the dairy industry (Wrinkle et al., 2012). Dairy cows suffering from heat stress
during the summer markedly decreased feed intake and milk yield (Wheelock et al., 2010), compromised rumination and
nutrient absorption (Collier et al., 1982), and increased respiratory rate and sweating (West, 2003). Unabated heat stress
could decrease more than 50% of feed intake and more than 10% of milk production (Wheelock et al., 2010). Additional
maintenance requirements were thought to exceed 30% for homeothermia in heat-stressed cows (Fox and Tylutki, 1998).
Heat-stressed lactating cows presented a negative energy balance due to these collective changes. The decline in nutrient
intake has previously been identified as a major cause of reduced production (Collier et al., 1982). However, researches
(using a pair-feeding experimental design) have recently demonstrated that reduced nutrient intake, observed during heat
stress, accounted for only 50% of the heat stress-induced decrease in production (Baumgard et al., 2011), which indicates
that environmental-induced hyperthermia also affects production performance independently of reduced nutrient intake
(Wheelock et al., 2010). Mitigation strategies have been used to maximise milk yield during the summer months.
It is relatively common and cheap to remedy to the negative effects with shade and evaporative cooling with soakers and
fans in pens (Collier et al., 2006). Management strategies have been considered during heat stress including: increasing the
energy and nutrient density of the diet (Wang et al., 2010), supplementing with feed additives (Zimbelman et al., 2013) or
pharmaceutical additives (Liu et al., 2013). There is a recent interest in herbal feed additives for abating negative effects of
high ambient temperature and improving production in chickens (Wang et al., 2008), pigs (Dong et al., 2012) and rabbits
(Liu et al., 2011). These additives have nutritional and medicinal values and no toxic side effects; however they also have no
residue advantages compared with other feed additives (Wang et al., 2011). Few these herbal feed additives were effective
at improving lactation production. Consequently, it is essential to develop new secure and effective herbal feed additives to
mitigate environmental-induced hyperthermia.
Radix Bupleuri, a major component of oriental folk medicine, is widely known in Korea and Japan for the treatment of fever,
pain and inflammation associated with influenza and the common cold (Van Wyk and Wink, 2004). It is well-known that
Radix Bupleuri possesses a wide range of pharmacological functions, such as diaphoretic, antipyretic, and immunomodulatory
(Ashour and Wink, 2011) with abundant secondary metabolites including saikosaponins (Tan et al., 2008), essential oils
(Ashour et al., 2009) and polysaccharides (Xu et al., 2007), of which saikosaponin triterpenes generally constitute the main
class of secondary metabolites in the genus Bupleurum amounting to up to 7% of the total dry weight in roots (Ashour
and Wink, 2011). Saponins and essential oils have shown the possibility to modify rumen fermentation and enhance animal
production (Benchaar et al., 2008; Patra and Saxena, 2009; Bodas et al., 2012). However, the results have not been consistent.
Accordingly, this study was conducted to assess the effects of RBE (mixtures of essential oils and saikosaponins) on lactation
performance and rumen fermentation in heat-stressed lactating Holstein cows.

2. Materials and methods

2.1. Animals, diets and experimental design

Forty Holstein cows (75 ± 15 d in milk, 37.5 ± 1.8 kg of milk/d, and 1.7 ± 0.4 parity) were randomly assigned to one of four
groups (n = 10). The four treatment diets consisted of RBE supplementation at 0 (Control, C), 0.25 g/kg, 0.5 g/kg or 1.0 g/kg
of the basal ration (concentrate and roughage) consisting of 172 g/kg crude protein (CP), 399 g/kg neutral detergent fiber
assayed with a heat stable amylase (aNDF) and 41.2 g/kg ether extract (EE) on a dry matter (DM) basis, which was formulated
to meet or exceed nutrient recommendations (NRC, 2001). Ingredients and chemical composition of basal diet are presented
in detail in Table 1. Cows were individually fed the basal diet for 1 wk as the pre-treatment and the treatment diets for
the following 9 wk as formal period. Feed was offered at 0530, 1300 and 2000 h (roughage first followed by concentrate)
and water was available at all time. Cows were milked 3 times daily at 0600, 1330 and 2030 h, housed in a close-side barn
with tie stalls and cooled with a combination of sprinklers and fans during the experiment in hot summer from July 15 to
Septembers 23, 2012 in the municipality of Shanghai.

2.2. Sample collection and analysis

Ambient temperature and humidity were recorded daily (0600, 1400 and 2200 h) with a thermometer and hygrometer
in one instrument panel (Beijing Yaguang Equipment Co. Ltd., Beijing, China). Temperature-humidity index (THI) in the barn
was generated using the following formula: THI = 0.81 × T + (0.99 × T − 14.3) × R + 46.3, where T is the temperature and R is
the relative humidity. 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.
Feed consumption, allowing for approximately 50 g/kg feed refusal, was measured on two consecutive days of each week.
Daily dry matter intake (DMI) for individual cows was calculated by subtracting the orts from feed offered. Weekly dried feed
samples were ground through a Cyclotec 1903 mill (Tecator 1093, Tecator AB, Hoganas, Sweden) using a 1-mm screen, and
weekly representative feed and ort samples were analyzed for DM content (AOAC, 2000; method 930.15), CP (AOAC, 2000;
 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8 3

Table 1
Ingredients and chemical composition of the basal diets (dry matter basis).

Composition Content

Ingredient (g/kg dry matter)

Alfalfa hay 127
Chinese wildrye 94
Corn silage 126
Oat grass 37
Cotton seed meal 89
Beet pulp 71
Corn 230
Dry distillers grains 37
Barley 94
Soybean 74
Sodium bicarbonate 6.4
Dicalcium phosphate 5.4
Salt 4.9
Vitamin-mineral premix1 4.3
Chemical composition (g/kg DM)
Net energy for lactating cow2 (Mcal/kg of DM) 1.6
Crude protein 172
aNeutral detergent fiber3 399
Acid detergent fiber 251
Ether extract 41.2
Calcium 10.0
Phosphorous 4.8
1
Contained (per kg of DM): a minimum of 2000,000 IU of vitamin A; 450,000 IU of vitamin D; 10,000 IU of vitamin E; 3000 mg of niacin; 4560 mg of Cu;
4590 mg of Mn; 12,100 mg of Zn; 200 mg of Se; 270 mg of I; 60 mg of Co.
2
Calculated according to NRC (2001) based on actual DMI.
3
Neutral detergent fiber assayed with a heat stable amylase inclusive of residual ash.

method 976.05), ash (AOAC, 2000; method 942.05), EE (AOAC, 2000; method 920.39), calcium and phosphorous (AOAC,
2000; method 935.13). Acid detergent fiber and aNDF analysis were determined by the basic procedure of Van Soest et al.
(1991) using heat-stable amylase for aNDF determination, and expressed inclusive of residual ash for all samples of diets
and feces.
Milk yield was recorded twice weekly from individual cows. Milk samples (approximately 50 ml) from individual cows
were collected every ten days from three consecutive milkings and combined at a ratio of 4:3:3 by volume (this ratio reflecting
the milk yield of morning, afternoon and night). Milk samples were preserved with potassium dichromate and stored at
4 ◦ C until being analyzed for fat, protein, lactose, total solids, somatic cell count (SCC) and milk urea nitrogen (MUN) using
mid-infrared spectrophotometry (BactoScan FC CombiFoss 6000; Foss Electric, Hillerød, Denmark). The following equations
were used for the calculation of energy-corrected milk (ECM): 0.327 × milk (kg/d) + 12.95 × fat (kg/d) + 7.65 × protein (kg/d)
and 4% fat-corrected milk (FCM): 0.4 × milk (kg/d) + 15 × fat (kg/d) (NRC, 2001).
On week 6 and 10 of the trial, rectal fecal grab samples were collected from each cow every 6 h each day for 3 d, but offset
by 2 h daily. Twelve fecal samples were mixed fully for each cow and 200 g of composite samples, accounting for diurnal
variation, were mixed with 20 ml of hydrochloric acid (6 mol/l). Another 200 g sample was dried, ground to pass a 1-mm
screen to perform the same analyses as the one on feed. The fecal and ration samples were also analyzed for acid insoluble
ash (Van Keulen and Young, 1977) to calculate nutrient apparent digestibility.
Rumen fluid samples were collected at 2 h post-feeding using stomach-tube on week 6 and 10 of the trial. The first 200 ml
sample of rumen contents per animal was discarded to minimize contamination with saliva, and the following 200 ml rumen
fluid samples were collected. Rumen samples were strained through 4 layers of cheesecloth and immediately analyzed for
pH with a portable pH meter (370 model pH meter; Jenway, London, UK) equipped with a combination electrode. A portion
of this strained ruminal fluid (5.4 ml) was then acidified with 25 g/ml metaphosphoric acid (1.8 ml) and frozen at −20 ◦ C
until being analyzed for volatile fatty acid (VFA) and ammonia–N. Ammonia–N was determined by the phenol–hypochlorite
method using spectral-photometric determination, and VFA were determined with a gas chromatograph (Agilent 6890N,
Salt Lake, UT, USA) using a DB-FFAP and FID.

2.3. Statistical analysis

Milk production and composition, DMI, RR and RT data were analyzed as a repeated measurement using the Proc Mixed
procedure of SAS (version 9.2, 2008), and week, treatment and treatment·week interaction were the fixed effects with cow
as random effects. Each variable was analyzed using corresponding data in pre-treatment period as covariate. The VFA,
pH, ammonia N and apparent digestibility data were analyzed by GLM followed by Duncan’s multiple range tests. Values
were presented as least squares means with standard error of the mean. Significant differences were declared at P ≤ 0.05.
Differences at 0.05 < P≤0.10 were considered as a trend toward significance.
4 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8

Fig. 1. Weekly THI pattern and ambient temperatures at 600, 1400, 2200 h and mean during the 10 wk experimental period in the barn.

Table 2
Effects of Radix Bupleuri extract supplementation on respiratory rate (RR) and rectal temperature (RT) in heat-stressed dairy cows.

Item Treatment Week SEM P-value

0 g/kg 0.25 g/kg 0.5 g/kg 1.0 g/kg 1 wk 6 wk 10 wk Treatment Week Interaction

RR (breath/min)
0700h 71.2a 64.2b 58.3c 67.03b 68.9b 75.2a 57.7c 2.01 <0.01 <0.01 0.78
1400h 71.6a 66.0ab 60.9b 67.6ab 74.1a 76.3a 63.2b 2.16 0.03 <0.01 0.80
Average 71.4a 65.6a 60.3b 67.4a 71.0a 75.9a 60.4b 2.86 <0.01 <0.01 0.77
RT (◦ C)
0700h 39.1x 39.0xy 38.9y 38.9y 39.1b 39.4a 38.6c 0.05 0.08 <0.01 0.87
1400h 39.5a 39.3b 39.2b 39.2b 39.4b 39.7a 38.9c 0.06 <0.01 <0.01 0.93
Average 39.3a 39.1b 39.0b 39.1b 39.2b 39.5a 38.8c 0.05 0.01 <0.01 0.92

Mean values within a row with different letters (a , b and c ) differ at P ≤ 0.05. Mean values within a row with different letters (x and y ) differ at 0.05 < P ≤ 0.10.

3. Results

3.1. Ambient temperature and THI, RR and RT

During the 10 wk of the experiment, mean weekly ambient temperatures in the barn was 27.5 ◦ C (range 24.6 to 29.2 ◦ C) at
0600 h, 29.8 ◦ C (range 26.3 to 31.7 ◦ C) at 1400 h, and 28.1 ◦ C (range 25.3 to 30.0 ◦ C) at 2200 h. The mean THI was 78.2 (range
71.9 to 80.8) at 0600 h, 79.7 (range 72.7 to 83.3) at 1400 h, and 78.3 (range 70.2 to 81.7) at 2200 h (Fig. 1).
Compared with C, all doses of RBE reduced RR in the morning, whereas in the afternoon RR was only lower with the
0.5 g/kg dose (P < 0.05). All doses of RBE decreased RT at 1400 (P < 0.01), while there was a tendency (P = 0.08) for decreased
RT in the morning compared with C for RBE doses of 0.5 and 1.0 g/kg (Table 2).

3.2. Milk production and composition and DMI

Dry matter intake was increased (P < 0.05) with all doses of RBE (9.1%, 3.4% and 5.7% vs. C, with 0.25, 0.50 and 1.0 g/kg,
respectively) (Table 3). Milk yield was increased (P < 0.01) by 8.2 and 5.7% vs. C, with 0.25 and 0.50 g/kg RBE, respectively).
Energy-corrected milk and 4% FCM were increased (P < 0.01) by 9.2% and 7.6%, and 9.0 and 7.9% respectively with both
0.25 and 0.50 g/kg RBE. Moreover, the feed efficiency (milk yield/DMI, 4% FCM/DMI and ECM/DMI) increased, especially
at 0.25 and 0.5 g/kg levels. There was no treatment effect on milk protein or fat concentration, whereas milk protein
yield (P < 0.01) and milk fat yield (P < 0.05) were increased compared with C, with both 0.25 and 0.50 g/kg RBE. Milk
lactose, total solids and MUN concentrations were not different, while milk SCC tended to decrease (P = 0.06) compared
with C.
 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8 5

Table 3
Effects of Radix Bupleuri extract supplementation on dry matter intake, milk production and composition, and feed efficiency in heat-stressed dairy cows.

Item Treatment Week SEM P-value

0 g/kg 0.25 g/kg 0.5 g/kg 1.0 g/kg 1 wk 6 wk 10 wk Treatment Week Interaction

Dry matter intake, kg/d 20.9c 22.8a 21.6b 22.1b 22.2ab 21.6b 23.6a 0.16 0.02 0.01 0.09
Milk yield, kg/d 31.6c 34.2a 33.4b 32.4bc 33.6a 32.2b 33.6a 0.41 <0.01 <0.01 0.54
4% FCM1 , kg/d 27.9b 30.4a 30.1a 29.3ab 29.4ab 28.9b 29.9a 0.53 <0.01 0.05 0.94
ECM2 , kg/d 30.4b 33.2a 32.7a 31.8ab 31.3b 31.6b 32.8a 0.48 <0.01 <0.01 0.81
Efficiency
Milk yield/DMI 1.48c 1.64a 1.58b 1.54b 1.51a 1.53a 1.42b 0.02 <0.01 <0.01 0.17
4%FCM/DMI 1.32c 1.49a 1.43b 1.40ab 1.38x 1.40x 1.31y 0.02 <0.01 0.07 0.67
ECM/DMI 1.43c 1.58a 1.54b 1.51ab 1.46a 1.50a 1.38b 0.02 <0.01 <0.01 0.49
Components
Fat, g/kg 32.4 32.6 34.0 33.2 32.4 34.1 33.4 0.7 0.41 0.37 0.99
Fat yield, kg/d 1.02b 1.13a 1.12a 1.09ab 1.09 1.08 1.12 0.03 0.03 0.25 0.99
Protein, g/kg 27.9 27.9 28.9 28.1 27.6b 28.5a 28.8a 0.4 0.19 <0.01 0.88
Protein yield, kg/d 0.89b 0.97a 0.95a 0.92ab 0.88b 0.91ab 0.97a 0.02 <0.01 <0.01 0.46
Lactose, g/kg 49.6 49.6 49.9 49.9 49.9 49.7 49.4 0.3 0.87 0.23 0.99
Total solids, g/kg 123 123 125 124 125ab 126a 123b 1.2 0.37 0.02 1.00
a ab
Solids-not fat, g/kg 89.7 90.3 91.9 90.8 92.9 91.6 89.9b 0.9 0.36 <0.01 0.99
MUN3 , mg/d/l 13.9 13.7 14.2 14.4 12.48b 13.27b 14.74a 0.28 0.38 <0.01 0.96
SCC4 , ×104 /ml 46.6x 18.5y 22.6y 19.1y 36.7 37.0 33.4 8.65 0.06 0.97 0.86

Mean values within a row with different letters (a , b and c ) differ at P ≤ 0.05.
Mean values within a row with different letters (x and y ) differ at 0.05 < P≤ 0.10.
1
4% FCM (kg/d) = 0.4 × milk (kg/d) + 15 × fat (kg/d), (NRC, 2001).
2
ECM (kg/d) = 0.327 × milk (kg/d) + 12.95 × fat (kg/d) + 7.65 × protein (kg/d).
3
MUN = milk urea nitrogen.
4
SCC = somatic cell count.

Table 4
Effects of Radix Bupleuri extract supplementation on apparent digestibility coefficients in heat-stressed dairy cows.

Item Treatment Week SEM P-value

0 g/kg 0.25 g/kg 0.5 g/kg 1.0 g/kg 6 wk 10 wk Treatment Week Interaction

Dry matter 0.66 0.66 0.65 0.65 0.68 0.63 0.08 0.37 0.01 0.91
Crude protein 0.67 0.68 0.68 0.68 0.70 0.65 0.09 0.91 0.01 0.89
aNeutral detergent fibera 0.56 0.56 0.54 0.55 0.62 0.50 0.12 0.61 0.01 0.58
Acid detergent fiber 0.42 0.43 0.40 0.40 0.47 0.31 0.16 0.19 0.01 0.69
Organic matter 0.64 0.64 0.61 0.64 0.66 0.60 0.09 0.11 0.01 0.89
a
Neutral detergent fiber assayed with a heat stable amylase inclusive of residual ash.

3.3. Apparent digestibility and rumen fermentation parameters

Apparent digestibility of DM, CP, NDF, ADF and OM were similar among groups (Table 4). Rumen pH tended to increase
(P = 0.09) with RBE at the dose of 0.25 and 1.0 g/kg, while there was no treatment effect on VFA. Similarly, ammonia-N content
was same among groups (Table 5).

Table 5
Effects of Radix Bupleuri extract supplementation on rumen fermentation parameters in heat-stressed dairy cows.

Item Treatment Week SEM P-value

0 g/kg 0.25 g/kg 0.5 g/kg 1.0 g/kg 6 wk 10 wk Treatment Week Interaction

Rumen pH 6.24y 6.58x 6.32xy 6.40x 6.30 6.57 0.11 0.09 0.03 0.38
Ammonia-N mg/d/l 12.0 8.02 10.3 8.99 8.68 7.20 1.36 0.18 0.81 0.70
Rumen VFA, mmol/l
Acetate 68.2 65.2 70.4 65.2 67.0 67.2 2.70 0.43 0.95 0.81
Propionate 23.4 20.9 22.7 24.0 22.1 23.2 1.47 0.42 0.51 0.59
Isobutyrate 0.82 0.76 0.86 0.83 0.79 0.83 0.04 0.31 0.21 0.17
Butyrate 11.8 10.5 11.8 10.7 11.4 10.9 0.68 0.37 0.36 0.72
Isovalerate 1.15 1.07 1.28 1.23 1.17 1.18 0.10 0.36 0.92 0.76
Valerate 1.27 1.16 1.26 1.16 1.17 1.25 0.06 0.16 0.09 0.38

Total 107 100 108 104 104 105 4.16 0.41 0.87 0.64
Acetate/propionate 2.96 3.17 3.14 2.74 3.07 2.96 0.17 0.28 0.56 0.86

Mean values within a row with different letters (x and y ) differ at 0.05 < P ≤ 0.10.
6 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8

4. Discussion

Heat stress in dairy cows is a non-specific response to hot weather and high humidity (West, 2003). Due to its body and
physiological characteristics, heat stress begins to occur in dairy cows when THI exceeds 72 (Armstrong, 1994; Bohmanova
et al., 2007). When ambient temperature is above 25 ◦ C, the cow reaches a point where she can no longer cool herself
adequately and enters heat stress (Berman et al., 1985), and lactating dairy cows begin to seriously experience the neg-
ative effects of heat stress when RT exceeds 39.2 ◦ C and when RR exceeds 60 breath/min (Staples and Thatcher, 2011).
Furthermore, average RR exceeded 60 breath/min, and RT exceeded 38.9 ◦ C (Table 2). Both are characteristics of heat-
stressed dairy cattle (Kadzere et al., 2002). Therefore, dairy cows were subjected to heat-stress condition during the
experiment.
Rectal temperature, an indicator of thermal balance and a representative measurement of animal core temperature
(Nielsen, 1997), may be used to assess the adversity of the thermal environment (Johnson, 1980). Even small upward shifts
in core temperature have profound effects on tissue and endocrine function that, in turn, can reduce fertility, growth and
lactation (McDowell et al., 1976). In the experiment, cows fed RBE supplementation, especially at 0.5 g/kg, had decreased
RR and RT in the morning and afternoon (Table 2). It meant that RBE could reduce core temperature to mitigate heat-
stressed effect to some degree. As is well-known that cows in heat stress environment are more dependent upon peripheral
vasodilatation and water evaporation to enhance heat loss compared with cows in optimal temperature conditions (Berman
et al., 1985). About 85% of the endogenous metabolic heat must be transferred to the skin, where it is dissipated to maintain
the homeothermy (McDowell et al., 1976). Accordingly, no matter where the heat load, external or internal, comes from,
the peripheral vasodilatation and water evaporation are responsible for heat loss. The significantly decreased body core
temperature in heat-stressed cows fed RBE may be due to increasing vasodilation and thus transferring heat to the skin
where it is dissipated by water evaporation.
The reduced body temperature increased the comfort for cows, and accordingly the DMI were increased (Table 3). Sub-
sequently, milk production was improved partly owing to increased nutrient intake. Additionally, recent researches have
demonstrated that reduced nutrient intake accounted for a little less than 50% of the heat stress-induced decrease in milk
synthesis (Rhoads et al., 2009; Baumgard et al., 2011). This indicates that heat directly affects production performance by
impairing metabolism in dairy cattle, independently of feed intake (Wheelock et al., 2010). Reversely, the improved milk
production in the experiment was probably not only due to the increased nutrient intake, but to the direct effect of heat-
stress relief providing more energy for production rather than for homeothermy (Armstrong, 1994). This would explain the
increased feed efficiency in the present study. Accordingly, milk production was improved following both increased nutrient
intake and increased energy availability, accounting for increased milk fat and protein yields without difference in milk fat
or protein concentration. The reason for decreased lactation performance observed with the highest dose of RBE (1.0 g/kg)
compared with 0.25 or 0.50 g/kg could come from hepatotoxicity of RBE at this level of incorporation. Indeed, previous
studies with multiple doses of RBE given to rats have shown that serious liver injury was dependent on the dose of RBE and
the content of saikosaponins (Huang et al., 2010; Sun and Huang, 2010).
Somatic cell count in milk was increased during heat stress (Hammami et al., 2013), while RBE tended to decrease SCC
in the present study (Table 3). This indicated that the increase in cow comfort owing to decreased RT and RR may improve
the health of heat-stressed cows in some extent. There was no significant difference in apparent digestibility or rumen
fermentation for cows fed RBE in heat stress, which meant that RBE did not affect negatively diet digestibility or rumen
fermentation (Tables 4 and 5). In general, saponins and essential oils have been demonstrated to favourably improve rumen
function, such as increasing total VFA concentrations and feed digestibility, and decreasing ammonia concentrations in the
rumen (Wina et al., 2005; Patra and Saxena, 2009). Such discrepancies might have resulted from differences in type, dose and
content of sapogenins, which is the active component of saponins, of saponin products used in the rumen (Patra et al., 2012).
In another way, it has been suggested that microbial populations would adapt to essential oils or saponins in the rumen
over time after long exposure to these extracts, which would explain the absence of effects of the plant extracts on rumen
fermentation as well (Hart et al., 2008; Benchaar and Greathead, 2011). Additionally, effects of saponins and essential oils
on rumen VFA production seem to be pH dependent (Spanghero et al., 2008; Hart et al., 2008). However, rumen pH tended
to increase with RBE at the doses of 0.25 and 1.0 g/kg in this study. Because rumen pH was only measured at one time point
post-feeding and because it is known to be very variable in time, it is difficult to speculate on the effect of RBE over rumen
pH in the present study.

5. Conclusion

Supplemental RBE at 0.25 or 0.5 g/kg could decrease RT and thus mitigate negative effects of high ambient temper-
ature. Accordingly, the increased comfort in heat-stressed cows accounted for the increased DMI. Subsequently, milk
production was probably improved with both more nutrient intake and more energy available, while there was no dif-
ference in milk components, apparent digestibility or rumen fermentation. Overall, supplemental RBE at 0.25–0.5 g/kg could
palliate the negative effects of environmental-induced hyperthermia and improve lactation performance in heat-stressed
cows.
 L. Pan et al. / Animal Feed Science and Technology 187 (2014) 1–8 7

Acknowledgements

This investigation was financially supported by the Research Program of the State Key Laboratory of Animal Nutrition
(2004DA125184F1102) and National Key Technologies R & D Program (2012BAD12B02; 2012BAD12B08-5). The authors
express their appreciation to Shanghai Bright Dairy Co. (Shanghai, China) for use of animals and assistance with animal care
and feeding throughout the study and the staff of the State Key Laboratory of Animal Nutrition (Beijing, China) for sample
analyses.

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