Introduction
Phytoestrogens are widely found in soybeans, alfalfa and other plant feedstuffs. Daidzein is the main component of soybean isoflavones (Franke et al., Reference Franke, Custer, Cerna and Narala1994; Liggins et al., Reference Liggins, Mulligan, Runswick and Bingham2002). Due to its chemical structure, similar to mammalian oestrogens, daidzein displays weak oestrogenic activity in animals and has been studied extensively for possible biological activities such as antioxidant, anti-stress, modulating glucolipid metabolism and promoting animal growth (Wong et al., Reference Wong, Emery, Preedy and Wiseman2008; Liu et al., Reference Liu, Suzuki, Laxmi, Okamoto and Shibutani2012). Researchers have shown that daidzein improves the feed efficiency of chicken, castrated piglets and weaned piglets (Wang et al., Reference Wang, Hang and Chen1994; Guo et al., Reference Guo, Han and Wang2002; Li et al., Reference Li, Zhu, Zhu, Gao, Meng, Guo and Zhang2015), increases the daily weight gain of chicken, castrated piglets and Northeast fine wool sheep (Guo et al., Reference Guo, Han and Wang2002; Ren et al., Reference Ren, Geng, Liu and Guo2009; Li et al., Reference Li, Zhu, Zhu, Gao, Meng, Guo and Zhang2015) and elevates milk production, milk protein content, milk fat content and serum antioxidant capacity of dairy cows (Liu et al., Reference Liu, He, Jin, Liu, Tang, Li and Zhong2013). In beef cattle, research has focused mainly on the effect of daidzein on rumen fermentation characteristics (Zhao et al., Reference Zhao, Zhou, Bao, Song, Ouyang, Xu, Pan, Liu and Qu2018). In a previous study, supplementation with daidzein changed the bacterial community composition and enhanced the rumen fermentation function of beef cattle (Liang et al., Reference Liang, Xu, Zhao, Bai, Chen, Zhou, Song, Ouyang, Pan, Liu and Qu2018).
As a result of rapid economic development, the demand for high-quality beef has grown in popularity in China. Improving the production and quality of beef has become an urgent issue for the Chinese beef industry. Daidzein has been reported to be effective at affecting the meat quality of animals, but a few animal studies published have given conflicting results. Zhao et al. (Reference Zhao, Yang, Bao, Wang, Zhou, Gong, Fu, Xu, Liu and Qu2015) found that daidzein supplementation improved the tenderness of beef by increasing the intramuscular fat (IMF) content and marbling score. On the other hand, Rehfeldt et al. (Reference Rehfeldt, Adamovic and Kuhn2007) reported that supplemental daidzein changed the muscle fibre type in piglets. By measuring the IMF content and tenderness of beef, the current research was conducted to clarify the controversial results.
Xianan cattle (Bos taurus) is the first cattle breed developed especially for beef in China (Ru et al., Reference Ru, Lou, Qi, Li, Wang and Yang2006). Xianan cattle are crossbreeds of Nanyang cattle (female parent) and Charolais cattle (male parent) which have the characteristics of fast growth, high-meat quality, coarse feedstuff tolerance and strong adaptability (Wang et al., Reference Wang, Garrity, Tiedje and Cole2007, Reference Wang, Han and Wang2008). However, few investigations have focused on the meat quality of Xianan cattle. Moreover, there are no reports about the effect of daidzein on growth performance, blood biochemical parameters and meat quality of finishing Xianan cattle. Besides, the supplemental dosages of daidzein in previous studies were below 500 mg/kg. A higher dosage (1000 mg/kg) of daidzein has not been tested. Therefore, the purpose of the current study was to evaluate the effects of different dosages of daidzein on growth performance, blood biochemical parameters and meat quality of finishing Xianan cattle, and explore the mechanisms of daidzein on improving meat quality.
Materials and methods
Animals, diets and experimental design
Thirty 2-year-old finishing Xianan steers (body weight (BW) = 686 ± 50.9 kg) were used in an 80-day feeding study during the finishing stage. These steers were segregated by live body weight (LBW) and then divided into three experimental groups using a randomization procedure to ensure that groups were equilibrated by LBW (similar number of animals from each weight group in experimental groups). Each group randomly received one of three dietary treatments (concentrate ratio = 80%): (1) control; (2) 500 mg/kg daidzein (500 mg daidzein/kg concentrate) and (3) 1000 mg/kg daidzein (1000 mg daidzein/kg concentrate). The cattle were untethered in individual stalls whose floors were paved with brick and fed with an 800 g/kg concentrate diet in quantities sufficient to provide ad libitum consumption (Table 1). For daidzein treatments, daidzein was mixed into the mineral premix and added to the concentrate. Diets were offered to the cattle twice daily at 07.00 and 14.00 h. Fresh water was available for ad libitum consumption throughout the study.
Table 1. Basic diet composition (daidzein was included according to treatment)
a Mineral-vitamin pre-mix (per kg): vitamin A, 250 000 IU; vitamin D3, 30 000 IU; vitamin E, 800 IU; Cu, 1 g; Fe, 5 g; Mn, 4 g; Zn, 3 g; Se, 10 mg; I, 50 mg; Co, 10 mg.
Growth performance
Amount of feed offered and refused were recorded daily for each steer. The feed intake was calculated daily for each steer as the dry matter (DM) offered minus DM refused. Steers were weighed for two consecutive days at the start and end of the experiment. The average daily gain (ADG) was determined for the overall experiment by subtracting initial BW from BW at the end of the experiment and dividing by the number of days. Feed conversion rate (FCR) was determined as the ratio of dry matter intake (DMI) to ADG.
Carcase characteristics
Steers were slaughtered at a commercial abattoir following the standard procedures. The carcases were then put into a chiller at 0 °C and aged for 14 days. The Longissimus dorsi muscle (LM) area and 12th-rib fat thickness were obtained for each carcase after ageing. The ribeye area was obtained by tracing the transverse section of each LM on sulphate paper, and then calculated by the volume-curve method using Leica QWin software. Fat thickness over the LM was determined by means of vernier callipers (triplicate in each carcase).
Physicochemical analysis
The meat quality was measured from an LM sample removed from the 12th and 13th ribs on the right side of the carcase, with no connective tissue or subcutaneous fat. The pH value was determined by using a Delta 320pH Meter (Mettler Toledo, Greifensee, Switzerland) with the probe inserted into the middle of the LM. Meat colour values of L*, a* and b* were measured by using a colorimeter (WSC-S, Shanghai, China) with an illuminant D65, 10° observer and CIELAB system.
To determine the cooking loss and shear force values of LM, steaks (3 × 4 × 5 cm) were dissected from the LM sample, placed in package bags and subsequently cooked in a 75 °C water bath with automatic temperature control until the internal temperature reached 70 °C. Then, the cooked beef was cooled naturally to room temperature. Cooking losses were calculated as the difference between initial and cooked weight as a percentage of initial weight. Six beef pieces of 3 × 1 × 1 cm (length × width × height) were removed parallel to the muscle fibre direction and cut with a Warner-Bratzler shear device (C-LM4, Haerbin, China) by running it perpendicularly in the muscle fibre direction. The shear force was expressed as the mean of six shear force values (kg f). To determine the drip losses of LM, the samples were covered with valve bags to avoid evaporation and placed in a container at 4 °C for 48 h. The samples were kept away from contacting the container and with the muscle fibre direction parallel to gravity, and drip losses were calculated as the difference between initial and refrigerated weight as a percentage of initial weight.
Determination of chemical composition of the basal diet and LM was carried out following the procedures of AOAC methods (AOAC, 2000). Briefly, the daidzein content of the basal diet was determined by high performance liquid chromatography (HPLC; LC-10A; Shimadzu, Tokyo, Japan) as described by Vyn et al. (Reference Vyn, Yin, Bruulsema, Jackson, Rajcan and Brouder2002). DM content was determined by drying feed and meat samples at 105 °C to constant weight. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) contents were determined by using an automatic fibre analyser (Ankom 2000i full; Ankom Technology Corporation, Macedon, NY, USA) (Van Soest et al., Reference Van Soest, Robertson and Lewis1991). Ether extract content was determined by Soxhlet extraction with diethyl ether, preceded by an acid hydrolysis treatment when analysing meat samples. Ash content was determined by combustion at 550 °C for 5 h. Crude protein content of meat was measured by the Kjeldahl nitrogen method.
Determination of blood biochemical parameters
At the end of the experiment, the feed was removed from the stalls and blood samples were collected from each steer at 07.00 h, 24 h before slaughter, via jugular venepuncture using 5 ml capacity evacuated blood-collecting tubes (YL003, Nanjing, China). After coagulation at room temperature for 30 min, the blood samples were centrifuged at 3000 g for 10 min at 4 °C. The serum samples were divided into tubes and stored at −20 °C for analysis.
Blood biochemical parameters including total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), glucose, total protein (TP), albumin, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), urea, glutamic-pyruvic transaminase (ALT), glutamic-oxaloacetic transaminase (AST), total superoxide dismutase (T-SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), immunoglobulin (Ig)A, IgM, IgG, triiodothyronine (T3), tetraiodothyronine (T4), growth hormone (GH), insulin, leptin and adiponectin were determined by assay kits (Table 2). All the assay kits were purchased from the same biotech company (Jiancheng, Nanjing, China). All measurements were operated in an AU5421 Automatic Biochemistry Analyzer (Backman-Kelt, USA) at the First Affiliated Hospital of Nanchang University.
Table 2. The assay kits for blood biochemical parameters
a All assay kits were purchased from the same biotech company (Jiancheng, Nanjing, China).
Statistical analysis
Differences between means were tested for statistical significance with one-way analysis of variance. The least significant difference test was conducted to determine the significance level of the particular comparison between treatment means. All statistical procedures were performed with SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Significance was declared at P⩽0.05. The model including random and fixed effects was as follows:
where Y ij is the dependent variable, μ is the overall mean, T j is the fixed effect of daidzein (j = 1–3) and E ij is the error term.
Results
Intake and growth performance
Steers fed with the 1000 mg/kg daidzein diet had greater DMI than those fed with the control diets (P = 0.025; Table 3). However, the final BW, ADG and FCR did not differ among three groups.
Table 3. Effects of supplemental daidzein on growth performance in finishing steers
BW, body weight; ADG, average daily gain; DMI, dry matter intake; FCR, feed conversion rate.
a s.e.m. = standard error of the mean (n = 10 steers per treatment).
Carcase characteristics and meat quality
Compared with the control steers, fat thickness was greater for steers fed with 1000 mg/kg daidzein diet (P = 0.042; Table 4). The shear force (P = 0.038) and L* (P = 0.026) of LM in steers fed with 1000 mg/kg daidzein were significantly lower than those fed with control diets. No difference in LM area, pH, drip loss, cooking loss, a* or b* of LM were observed among the three groups.
Table 4. Effects of supplemental daidzein on meat quality of the Longissimus dorsi muscle
L*, lightness; a*, redness; b*, yellowness.
a s.e.m. = standard error of the mean (n = 10 steers per treatment).
Supplementation with daidzein did not affect the moisture, ash, crude protein or IMF of LM (Table 4).
Blood biochemical parameters
Daidzein supplementation at 1000 mg/kg significantly increased the concentrations of FFA (P = 0.002) and ALT (P = 0.024), by 164.8 µmol/l and 11.5 U/l, respectively, compared with the control group (Table 5). Supplemental daidzein did not affect the concentrations of TC, TG, glucose, TP, albumin, HDL-C, LDL-C, urea or AST.
Table 5. Effects of supplemental daidzein on blood biochemical parameters of finishing steers
TC, total cholesterol; TG, triglyceride; FFA, free fatty acid; TP, total protein; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; ALT, glutamic-pyruvic transaminase; AST, glutamic-oxaloacetic transaminase.
a s.e.m. = standard error of the mean (n = 10 steers per treatment).
Blood antioxidant ability and immune parameters
Daidzein supplementation did not affect the blood antioxidant ability including T-SOD, MDA, GSH-PX and T-AOC (Table 6), or blood immune parameters including IgA, IgM and IgG.
Table 6. Effects of supplemental daidzein on blood antioxidant ability and immune parameters of finishing steers
T-SOD, total superoxide dismutase; MDA, malondialdehyde; GSH-PX, glutathione peroxidase; T-AOC, total antioxidant capacity.
a s.e.m. = standard error of the mean (n = 10 steers per treatment).
Blood hormone parameters
The concentration of T4 was 36% higher with 500 mg/kg daidzein than the control (P = 0.016; Table 7). The concentration of insulin was higher for steers fed with 1000 mg/kg daidzein in comparison with the steers in the control group (P = 0.043). Concentrations of T3, GH, leptin and adiponectin did not differ among the three groups.
Table 7. Effects of supplemental daidzein on blood hormone parameters of finishing steers
T3, triiodothyronine; T4, tetraiodothyronine; GH, growth hormone.
a s.e.m. = Standard error of the mean (n = 10 steers per treatment).
Discussion
To date, there are very few studies on the effects of daidzein supplementation in beef cattle diet and, in particular, limited information is available regarding the effects of daidzein on meat quality of beef cattle. Furthermore, in a few animal studies which have been published, the results of daidzein affecting meat quality have been contradictory. Since there are very few studies on the relationship between beef cattle and daidzein, this paper only compared the current results with those obtained from other animal species.
During high-grade beef production, feeding a high-concentrate diet continuously would undermine the health of finishing cattle, which might cause a reduction in feed intake and low efficiency in fat deposition (Brown et al., Reference Brown, Krehbiel, Galyean, Remmenga, Peters, Hibbard, Robinson and Moseley2000; Khafipour et al., Reference Khafipour, Krause and Plaizier2009). The results from the current study showed that daidzein supplementation at 1000 mg/kg increased the feed intake of steers, which was significant for high-grade beef production. The increase of feed intake did not affect ADG of steers but fat thickness increased significantly. These results could be related to changes in rumen fermentation parameters. In earlier studies, adding 1000 mg/kg daidzein significantly increased acetate content in the rumen (Liang et al., Reference Liang, Xu, Zhao, Bai, Chen, Zhou, Song, Ouyang, Pan, Liu and Qu2018). As a precursor of adipose, acetate is conducive to lipogenesis in ruminants (Kristensen and Harmon, Reference Kristensen and Harmon2004).
Contrary to previous results, in which supplemental daidzein improved the capacities of antioxidant (Foti et al., Reference Foti, Erba, Riso, Spadafranca, Criscuoli and Testolin2005; Mishra et al., Reference Mishra, Kar and Kale2009; Liu et al., Reference Liu, He, Jin, Liu, Tang, Li and Zhong2013) and immunity (Morimoto et al., Reference Morimoto, Watanabe, Yamori, Takebe and Wakatsuki2009; Mohammad-Shahi et al., Reference Mohammad-Shahi, Haidari, Rashidi, Saei, Mahboob and Rashidi2011) in serum, these were not affected for finishing Xianan cattle in the current study. It should be noted that the regulative effect of daidzein in animals was influenced by several factors, such as supplementing amount of daidzein, animal breed, age, sex and methods of feed management (Lozupone et al., Reference Lozupone, Stombaugh, Gordon, Jansson and Knight2012). In the current study, soybean meal in the basal diet could supply daidzein in significant amounts (Kašparovská et al., Reference Kašparovská, Dadáková, Lochman, Hadrová, Křížová and Kašparovský2017). Besides, soybean oil in the basal diet could supply di-tert-butylhydroxytoluene which is an antioxidant (Blanco et al., Reference Blanco, Giráldez, Morán, Mateo, Villalobos-Delgado, Andrés and Bodas2017). These reasons might lead to a high antioxidant activity for the basal diet.
There are no consistent effects of daidzein on lipid metabolism in animals. Some researchers have shown that daidzein has a positive effect on fat deposition. Zhao et al. (Reference Zhao, Yang, Bao, Wang, Zhou, Gong, Fu, Xu, Liu and Qu2015) found that daidzein supplementation significantly increased the IMF content of crossbred steers. Li et al. (Reference Li, Li, Yang, Yuan, Zhang, Geng, Xiao and Yin2011) and Rehfeldt et al. (Reference Rehfeldt, Adamovic and Kuhn2007) reported that adding a high dose of daidzein significantly increased the fat proportion in carcases of Guangxi minipig and piglets, respectively. But there was also a viewpoint that supplemental daidzein inhibited the synthesis of lipids in animals. Szkudelska et al. (Reference Szkudelska, Szkudelski and Nogowski2002) and Choi et al. (Reference Choi, Jung, Kim, Kim, Park, Jang and Lee2005) found that feeding daidzein reduced glucose uptake and inhibited the synthesis of TG in mouse cells. Liu et al. (Reference Liu, Zhang and Zhang2003) reported that supplementation with daidzein decreased the fat thickness and abdominal fat percentage of broilers. The results of the current study showed that daidzein supplementation significantly increased fat thickness and enhanced the synthesis of lipids in finishing Xianan cattle. This result may be caused by the weak oestrogenic activity of daidzein. Oestrogen could inhibit the β oxidation of fatty acids in the liver and lead to elevated serum concentrations of FFAs, which are precursors for the synthesis of lipids (Bauman et al., Reference Bauman, Harvatine and Lock2011). Kadegowda et al. (Reference Kadegowda, Piperova, Delmonte and Erdman2008) reported that adipose tissue increased the intake of FFAs and decreased the intake of β-hydroxybutyrate and acetic acid for synthesis of lipids with the increase of FFA concentrations in blood, which was consistent with the current study. Besides, there was an interaction between plasma FFA and insulin. It is known that plasma FFA could inhibit insulin signalling in different physiological and metabolic processes, inducing insulin resistance. The increase of FFA content inhibited insulin's anti-lipolytic action and promoted the synthesis of lipids (Ovadia et al., Reference Ovadia, Haim, Nov, Almog, Kovsan, Bashan, Benhar and Rudich2011).
As an important indicator to measure the tenderness of beef, shear force was affected by the IMF content and muscle fibre type of beef meat (Belew et al., Reference Belew, Brooks, McKenna and Savell2003; Koohmaraie and Geesink, Reference Koohmaraie and Geesink2006). Daidzein supplementation significantly decreased the shear force of LM in Xianan cattle. Considering that supplemental daidzein did not affect the IMF content of LM, the current study hypothesized that daidzein may change the muscle fibre type of LM to reduce the shear force. Due to the characteristic of weak oestrogenic activity, daidzein could regulate expression of the oestrogen receptor gene, which could affect the differentiation of marrow stromal cells (Zhou et al., Reference Zhou, Zilberman, Wassermann, Bain, Sadovsky and Gazit2001) and change the muscle fibre type.
Meat colour plays an important role in purchasing decision and price (Liu et al., Reference Liu, Lanari and Schaefer1995). Different meat colours are largely related to the relative proportions of metmyoglobin (grey-brown), myoglobin (dark red) and oxymyoglobin (bright red, the ferrous oxygenated form of myoglobin) (Kim et al., Reference Kim, Keeton, Smith, Berghman and Savell2009). There was a strong correlation between the content of myoglobin and L* value in meat, and the L* value decreased with the increase of myoglobin content (O'Keefe and Hood, Reference O'Keefe and Hood1982). In the current results, daidzein supplementation significantly decreased the L* value of LM. The lowest values of L* could be related to a lower proportion of white glycolytic fibres, which are known to be harder than oxidative ones (Chen et al., Reference Chen, Niu, Huang and Zhou2012).
With respect to the dosage effect of daidzein, supplementing with high doses (1000 mg/kg) of daidzein in diet had a better effect than low dose (500 mg/kg). This result was consistent with studies by Li et al. (Reference Li, Li, Yang, Yuan, Zhang, Geng, Xiao and Yin2011), Šošic-Jurjević et al. (Reference Šošic-Jurjević, Filipović, Ajdzanović, Brkić, Ristić, Stojanoski, Nestorović, Trifunović and Sekulić2007) and Dang and Löwik (Reference Dang and Löwik2004), in which daidzein supplementation at high doses had a better effect on enhancing the synthesis of lipids in swine, rat and mouse, respectively. However, the dosage of daidzein (1000 mg/kg) used in the current study was much higher than the supplemental dosage in Li et al. (Reference Li, Li, Yang, Yuan, Zhang, Geng, Xiao and Yin2011) (500 mg/kg), Šošic-Jurjević et al. (Reference Šošic-Jurjević, Filipović, Ajdzanović, Brkić, Ristić, Stojanoski, Nestorović, Trifunović and Sekulić2007) (3 mg/kg) and Dang and Löwik (Reference Dang and Löwik2004) (30 mg/kg). The reason for those results might be that daidzein can be partly metabolized to equol by ruminal microorganisms (Lundh, Reference Lundh1995).
In conclusion, daidzein supplementation above 500 mg/day modifies feed intake and metabolic and hormonal profile, with positive and negative effects on meat quality. Further studies should be conducted to optimize daidzein dose and to identify the modulating factors influencing the potential antioxidant activity of isoflavones.
Author ORCIDs
M. R. Qu, 0000-0002-8918-4904
Financial support
This work was supported by National Beef Cattle Industry Technology & System (CARS-37).
Conflict of interest
No potential conflict of interest was reported by the authors.
Ethical standards
All animal procedures such as ethical and animal welfare issues were approved by the ethics committee of Jiangxi Agricultural University. Daidzein was purchased from Ci Yuan Biotechnology Co., Ltd. (purity >98%, Shanxi, China).
