Impact of dietary supplementation of chromium, sodium nitrate or mineral mixture on growth performance and rumen microbes of Brahman crossbred cattle

Ruminants utilize a wide variety of dietary substrates via microbial fermentation primarily in the rumen, which is crucial for the growth and production of ruminants. The composition of the microbial community in the rumen and the composition of end products of fermentation depend on the diet fed to the animals. Therefore, finding changes in the structure, function and diversity of the rumen microbial populations in response to diet changes is important (Kittelmann et al., 2013) as ruminant feed is a necessary substrate of rumen fermentation (Anantasook and Wanapat, 2012). Recent studies have focused on exploiting compounds such as feed additives to improve ruminal fermentation to enhance protein metabolism, reduce methane production (Kamra et al., 2008; Sarkar et al., 2016) and improve animal health and productivity (Decandia et al., 2000).
       
In animals, chromium plays an important role in using glucose, reducing muscle fat and increasing lean percentage (Prasad and Gowda, 2005). It is thus recognized as an important chemical element in mammalian diets. Some studies have shown that dietary supplementation of chromium increases protein accumulation (up to 5.4%), decreases lipid accumulation (fat loss to 8.2%) (Mooney and Cromwell, 1995) and increases the mean daily weight, which reduces cholesterol levels in 6-month old calves (Mondal et al., 2007). In addition, sodium nitrate improves the efficiency of fermentation and stimulates digestion. Furthermore, nitrate supplementation under certain conditions can replace nonprotein nitrogen sources such as urea to assist in protein synthesis in rumen (Li et al., 2012). Minerals are essential for health, survival and production of animals due to their involvement in the physiological, structural, catalytic, and regulating functions (Underwood and Suttle, 2001) and it was shown by Satapathy et al., (2019) that a mixture of mineral supplementation (Ca, P, Mn, Cu, Zn) at 50g/day/animal has improved daily gain of crossbred cattle. In order to achieve the optimum results in growing cattle, the simultaneous provision of conditions for microbial growth and the balanced provision of nutrients are essential. This study was conducted with the objectives to compare the effect of chromium, sodium nitrate and mineral complex supplementation on growth performance, feed intake, blood parameters and rumen fermentation characteristics in Brahman crossbred cattle.

Animals and design
 
The experiment was conducted in An Giang province (10°23 ‘N, 105°26 ‘E) on 20 Brahman crossbred bulls in the growing stage with an average of 217 ± 22.6 kg body weight and arranged in a completely randomized blocks with 5 replicates in 4 treatments (Control: basic diet; Treatment 1: basic diet + 2.8 mg chromium/head/day; Treatment 2: basic diet + 28 g sodium nitrate/head/day; Treatment 3: basic diet + 15 g mineral mixture/head/day). The basic diet was natural grass, rice straw sprayed with urea (60 g urea/head/day) and concentrate (0.5% body weight) provided twice a day based on the intake during the experimental diet. Mineral mixtures included biotin, K⁺, Na⁺, Mn⁺⁺, Zn⁺⁺, Cu⁺⁺, Fe⁺⁺, Iodine and organic selenium. The amount of dry matter was calculated based on the daily feed intake. During the experimental period, cattle were fed twice a day as per requirements with ad libitum straw. Experimental period lasted 105 days, including 15 days for adaptation and 90 days for data collection.
 
Measurements and sampling
 
Cattle were weighed in the morning before feeding on two consecutive days at the beginning of the experiment, after 30 days, 60 days and at the end of 90 days. At the end of the trial, rumen fluid was collected through a stomach tube before feeding. About 200 ml of rumen fluid was used for immediate pH measurement using a Delta-320 pH meter (Mettler Toledo, USA). The rest of rumen fluid samples were screened through 4 layers of cheesecloth and stored at -80°C for DNA extraction.
 
Quantification of rumen bacteria by real-time PCR
 
Rumen samples from the same cattle were pooled for DNA extraction. Microbe genomic DNA extraction was done following the CTAB-based protocol described by Minas et al., (2011). The primers used in PCR and real-time PCR were taken from Denman and McSweeney (2006) for total bacteria (TB) and from Koike and Kobayashi (2001) for Ruminococcus albus (RA), Fibrobacter succinogenes (FS), and Ruminococcus flavefaciens (RF) quantification. For methanogens, the primer pair was picked up from Denman et al., (2007). The standard curves were generated following the instruction of the manufacturers. In brief, purified PCR products were cloned in TOPO® TA Cloning^® Kits (Invitrogen). The recombinant plasmids were then extracted by the PureLink^® Quick Plasmid Miniprep Kits (Invitrogen). Real-time PCR assays were performed on ABI Prism® 7000 SDS machine (Applied Biosystems), based on the fluorescent component changes with the increase of the product.
 
Blood sampling and analysis
 
At the end of the feeding trials, about 10 ml of blood samples was collected into tubes containing 10% Na-heparin. Samples were then centrifuged at 1000 rpm for 20 min at 4°C and plasma was separated for laboratory analysis. The plasma concentrations of metabolites and protein were analyzed by an automated biochemical analyzer (Humalyzer 3000, USA).
 
Statistical analysis
 
The data were subjected to analysis of variance using the General Linear Model procedure of Minitab software version 16.2.1. Tukey’s pairwise comparisons (P<0.05) were applied to determine the differences between dietary treatments.

Total bateria and cellulolytic bacteria species
 
The results from Fig 1 showed that the number of total bacteria at the end of the experiment did not differ significantly in basal treatment and treatments supplemented with chromium and sodium nitrate. However, the supplementation of mineral mixture significantly increased the number of total bacteria from 9.91x10⁹/mL at the beginning of the experiment up to 10.81x10¹⁰/mL at the end of the experiment. On the other hand, the variation of F. succinogenes, R. flavefaciens, and R. albus in the basic treatment and treatments supplemented with chromium was non-significant. In the treatment with sodium nitrate supplementation, the number of F. succinogenes and R. flavefaciens community did not show any significant changes; however, the number of R. albus increased remarkably from 2.78x10²/mL up to 4.45x10⁴/mL. In the treatment with mineral mixture supplementation, number of R. flavefaciens also increased noticeably while the growth of R. albus was negligible at the end of the experiment. By contrast, F. succinogenes population slightly decreased from 7.2x10⁷/mL to 6.54x10⁶/mL. From the presented data, it is suggested that the addition of mineral mixture to the diet of animals increased the number of total ruminal bacteria, especially cellulose-degrading bacteria that contributed to better cellulose digestion (Koul et al., 1998). Similar results were observed by Nurhaita et al., (2014), who suggested that bacteria needed not only nitrogen but also other nutrition such as energy and mineral for optimum growth that could lead to the increase of rumen microbial activity, thereby affecting digestibility. In addition, current results implied that the addition of sodium nitrate increased the number of total bacteria, R. flavefaciens and R. albus. This was consistent with the report of Zhao et al., (2015), who found that the addition of nitrate in vivo (1-2% dry matter, nitrate type unknown) was associated with a rise in various fibroblasts, including R. flavefaciens, R. albus and F. succinogenes. However, the number of F. succinogenes, R. flavefaciens, and R. albus dropped when supplemented with 12 mM sodium nitrate and did not affect the total bacterial population as reported by Zhou et al., (2012). Even though the addition of chromium to the diet almost did not affect the change of the number of total ruminal bacteria in the present study, chromium supplementation seemed to slightly improve the rumen fermentation in the study of Soltan et al., (2012).
 

 
 Methanogenic bacteria
 
Changes of methanogenic bacteria in the rumen of cattle consuming different diets are presented in Fig 2. Statistical analysis of the obtained data reveals that treatment with chromium and mineral mixture supplementation showed no significant increase in the number of methanogenic bacteria. In addition, in the treatment with sodium nitrate supplementation, the number of bacteria remained unchanged although the in vitro study by Zhou et al., (2011) showed that nitrite, a toxin for methanogen, could contribute to lowering methane production. Zijderveld et al., (2010) also found a significant reduction in the number of methane-producing bacteria when nitrate was added to the diet. The difference was probably because of low level of sodium nitrate supplementation (28 g/d). A previous report by Hulshof et al., (2012) also indicated that the addition of sodium nitrate to cow diets at high level (85-125 g/d) would reduce methane-producing bacteria.
 

 
Change of bacteria in the rumen of cattle under different diets
 
Fig 3 showed that the the number of total bacteria in the mineral mixture supplementation treatment (10.81x10¹⁰/mL) was higher than that of the control treatment (10.05x10¹⁰/mL). There was a difference in the number of F. succinogenes with sodium nitrate supplementation (7.22x10⁷/mL) compared to the treatment supplemented with chromium (6.31x10⁶/mL). In general, it was shown that supplementing mineral mixture in the diet increased in total bacteria as well as cellulolytic bacteria and these results were in agreement with those obtained by Sayed et al., (2014).
 

 
 Growth performance
 
The average initial live weight, final life weight, weight gain and daily weight gain of animals from 0-90 days of the trial period are presented in Table 1. The results showed that the final live weight increased on supplementation of mineral mixture and was higher than that in the control (62.0 kg). Simimar trend was also indicated for average daily gain (688 g/day) and DM intake (98.2 g/kg BW^0.75/d). In line with this study, Satapathy et al., (2016) also concluded that cattle supplemented with mineral mixture at 50 g/head/day had better daily gain (200 g/d) than those without mineral mixture addition (144 g/d). Similarly, Pangestu et al., (2015) also showed that goats fed minerals supplementation in diet had higher average daily gain than those having treatments without supplementation. According to Church (1988), the supply of minerals to the diet increased the source of nutrients for the ruminal bacteria and supported tissue metabolism. Thus, the increase in nutrients and metabolic activity has accelerated tissue synthesis, resulting in a rise in average daily gain. In addition, live weight went up significantly in animals fed nitrate supplemented diets and it was observed that nitrate supplementation resulted in better weight gain for cattle (Sangkhom et al., 2012).
 

 
Blood biochemical profiles
 
It is shown in Table 2 that both metabolites and protein content in blood of cattle consuming chromium, mineral mixture and sodium nitrate supplemented diets were similar. When animals are stressed, more corticosteroids would inhibit the immune system function, leading to increased blood glucose levels. The addition of chromium will help to reduce the concentration of corticosteroids and thus reduce the amount of glucose in the blood, alleviating stress in animals (Pechova et al., 2002). This was consistent with the present study that the amount of glucose in animal blood was slightly lower when supplemented with chromium.
 

Supplementation of chromium, sodium nitrate and mineral mixture to cattle diets affected the number of total and cellulolytic bacterial species. However, the addition of these supplements to the diets did not affect the number of methanogenic bacteria. In addition, the supplementation of the mineral mixture increased the number of total bacteria with the best weight gain and DM intake. All blood metabolites and proteins are not influenced by either chromium, sodium nitrate or mineral mixture supplementation.

This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-NN.05-2013.04.