Dietary rambutan peel powder as a rumen modifier in beef cattle

Objective The experiment was conducted to study the effect of rambutan (Nephelium lappaceum) fruit peel powder (RP) on feed consumption, digestibility of nutrients, ruminal fermentation dynamics and microbial population in Thai breed cattle. Methods Four, 2-year old (250±15 kg) beef bull crossbreds (75% Brahman×25% local breed) were allotted to experimental treatments using a 4×4 Latin square design. Four dietary supplementation treatments were imposed; non-supplementation (control, T1); supplementation of RP fed at 2% of dry matter intake (DMI) (low, T2); supplementation of RP fed at 4% of DMI (medium, T3) and supplementation of RP fed at 6% of DMI (high, T4). All cattle were given a concentrate supplement at 1% of body weight while Napier grass was provided as a free choice. Results The findings revealed that RP supplementation did not negatively affect (p>0.05) DMI of Napier grass, while RP intake and total DMI were the greatest in the RP supplementation at 4% and 6% DMI. Nevertheless, the nutrients (dry matter, organic matter, crude protein, neutral detergent fiber, and acid detergent fiber) digestibilities were not changed in the RP supplementation groups. Rumen fermentation parameters especially those of total volatile fatty acids, acetate and butyrate were not significantly changed. However, the propionate concentration was remarkably increased (p<0.05) in the RP supplementation. Notably, the ratio of acetate to propionate, the number of protozoa, as well as the methane estimation were significantly reduced in the RP supplemented groups (4% and 6% of DMI), while the counts of bacteria was not altered. Conclusion Supplementation of RP (4% of DMI) improved rumen propionate production, reduced protozoal population and methane estimation (p<0.05) without a negative effect on feed consumption and nutrients total tract digestibilities in beef cattle. Using dietary rambutan fruit peel powder has potential promise as a rumen regulator.


INTRODUCTION
and Rychlick [1] stated the importance of rumen ecology on rumen fermentation efficiency and the subsequent production of livestock. The use of feed additives such as antibiotics can improve rumen fermentation efficiency. While, research and development regarding methane (CH 4 ) production in ruminants have been receiving considerable atten tion in which mitigation of the rumen CH 4 has been the main issue [2]. The ruminal methane production is associated with global warming and the loss of digestible energy intake (8% to 12% of total energy intake) [3]. Currently, plants rich in plant metabolites (condensed tannins [CT], saponins [SP]) and essential oils have been receiving more interest regarding their selective inhibition of some rumen microbes and their fermentation [4]. Rambutan (Nephelium lappaceum) is a tropical fruit, grown in Southeast Asia e.g. Thailand, Malaysia, Indonesia, Vietnam and eaten fresh and can produce the products such as jams, juice and canned products. Rambutan peel s contains a high level of phenolic compounds such as tannins, SP, flavanoi, garaiin etc [5]. Locally available feed resources containing plant metabolites (polyphenols) espe cially those of anthrocyanidins and/or SP have been shown to increase the rumen propionate (C 3 ), decreased rumen degradation of protein, reducing CH 4 production and in creasing conjugated linoleic acid in ruminant products [6]. Therefore, rambutan peel powder (RP) is a potential sup plement to manipulate the rumen process. Nevertheless, modification of rumen fermentation in ruminants by using RP containing CT and SP has been relatively limited. Plant secondary metabolites namely SP and tannins which can impact on rumen microorganisms and fermentation, in creased total volatile fatty acid (VFA), supporting the ruminant hosts, whilst mitigating rumen methane production as stated by Poungchompu et al [7]. Hence, this experiment aimed at investigating the influence of RP levels on feed consumption, total tract nutrients digestibilities, rumen fermentation dy namics and methane production in Thai native cattle.

Feed preparation, experimental design of animals
In brief, the RP was prepared as follows; the rambutan peels were collected from fresh fruit peel at Malee Group Public Company Limited in Nakhon Pathom province, Thailand. The peels were sundried for about 5 days to attain about 90% DM and ground into powder form. Feed ingredients and their nutritive values are presented in Table 1. Four, 2year old (250±15 kg) crossbred beef cattle (75% Brahman×25% Thai native), were assigned to receive ex perimental diets in a 4×4 Latin square design. The dietary treatments were as follows; nonsupplementation (T1); sup plementation of RP fed at 2% of dry matter intake (DMI) (T2); supplementation of RP fed at 4% of DMI (T3) and sup plementation of RP fed at 6% of DMI (T4), respectively. The experimental cattle were offered concentrate mixture at 1.0% of body weight (BW), fed two times a day, in the morning and in the afternoon, while Napier grass (Pak Chong I) was fed freely. Mineral blocks and water were provided as a free choice during which the animals were kept in individual pens. This experiment was conducted comprising of the preliminary and the actual feeding regimes for four periods, and each period lasted for 21 days. After the first 14 days, all beef cattle were well adjusted to dietary treatments and samples of the supplement and Napier grass including the leftover were collected, every day during the entire feeding periods. Dur ing the last 7 days, feeds and fecal were collected from each animal in the morning before feeding time (07:00 am) samples of feces w in each period. All samples were then composited for each animal period. They were stored in freezer until preparation for chemical analysis.

Data collection, sample collection, and chemical analyses
Feeds and fecal samples were analyzed for their chemical com position by the method of AOAC [8], neutral detergent fiber (NDF), acid detergent fiber (ADF) according to Van Soest et al [9] and acidinsoluble ash (AIA). The AIA was used as an internal indicator to predict the digestibility of nutrients as described by Van Keulen and Young [10]. Plant secondary metabolites especially proantrocyanidins or CTs were chemi cally analyzed by the VanillinHCL method [11] and SP using methanol extraction as described by Wanapat and Ngamsaeng [12].
Respective rumen fluid from each bull was sampled via stomach tube using vacuum pump to withdraw rumen fluid at 0, 4 h post feeding. The rumen pH and temperature were immediately measured using the measurement meter (HANNA instrument HI 8424 microcomputer, Singapore). Respective samples of rumen fluid from treatments were then thoroughly filtered. The second portion of rumen fluid was then fixed with 10% formalin solution (1:9 v/v, rumen fluid:10% for malin) for total direct counts of microorganisms (bacteria, protozoa, and fungal zoospores) by haemacytometer [13]. Samples were analyzed for rumen VFAs produced from ru men fermentation, for NH 3 N, where 5 mL of H 2 SO 4 solution (1 M) was added to 45 mL of rumen fluid. The mixture was centrifuged at 1,600×g for 15 min, and the supernatant was later stored at -20°C prior to VFA analyses using highper formance liquid chromatography [14]. The blood sample from jugular vein, about 10 mL, was collected into tubes with ethylenediaminetetraacetic acid, separated by centrifu gation at 500×g for 10 min at 4°C then the plasma was stored at −20°C until later analysis of blood urea N according to the method of Crocker [15]. Details of the experimental protocols used under this experiment were fully reported in Wanapat et al [16].
The VFAs (C 2 , C 3 , C 4 ) were used in the equation as de scribed by Moss et al [17] in order to predict the methane estimation.

Statistical analyses
All the data were statistically analyzed using procedure gen eral linear model (Statistical Analysis System [SAS], 2013) [18] according a 4×4 Latin square design. Treatment trends were statistically compared using orthogonal polynomials. The results were presented as mean values with the standard error of the means. Difference among means with p<0.05 was accepted as statistical differences.

Experimental ingredients used in diets and their chemical analyzes
The composition of the concentrate, rambutan peel powder and Napier grass, are shown in Table 1. The nutritive values of fibrous components (NDF, ADF), and crude protein (CP) were 184 and 107 and 141 g/kg DM in the concentrate, re spectively. Napier grass contained 102, 697, and 435 g/kg DM of CP, NDF, and ADF, respectively. The rambutan peel powder contained 120 and 105 g/kg DM of CTs and SP, respectively.

Feed consumption and nutrients digestibilities
The DMI measured by kg/d, % BW/d, and g/kg BW 0.75 /d data are presented in Table 2. DM intake of Napier grass and con centrate were similar by RP supplementation. As shown, the DM of Napier grass ranged from 5.7 to 5.8 kg DM/d, while the concentrate intakes were provided as 1% of BW. However, total DMI and DM intake of RP were linearly increased, while CT and SP intake were increased when RP was added at 2%, 4%, and 6% DMI. Furthermore, nutrient digestibilities were not affected by the RP supplementation except at the high level of RP (4% and 6% of DMI).

Ruminal fermentation, blood, methane estimation, and microbial population
Ruminal fermentation characteristics by supplementation of RP are shown in Table 3. The results of rumen pH (6.6 to 6.7), temperature (39.0°C to 39.8°C) remained unchanged (p> 0.05). Rumen NH 3 N (17.5 to 18.1 mg/dL) and blood urea nitrogen (BUN) (9.7 to 10.4 mg/dL) were not altered (p>0.05). Furthermore, ruminal fermentation parameters (VFAs, C 2 , C 4 ) were not significantly impacted by dietary treatments imposed. However, C 3 was significantly increased in the RP supplementation at both 4% and 6% of DMI (T3, T4). More over, the C 2 :C 3 ratio and methane estimation were significantly deceased in the supplementation at 4%, 6% of DMI (T3, T4). Nevertheless, the counts of total bacteria were not affected by RP the supplementation whilst, the number of protozoa was significantly reduced with an increased level of RP sup plementation, meanwhile the number of fungal zoospores were similar in the RP supplementation groups.

Feed consumption and digestibilities of nutrients
Supplementation of RP did not change DMI of Napier grass, whilst the total DMI, RP intake, CT intake, and SP intake were linearly increased when RP was added at 2%, 4%, and 6% DMI. However, the lack of effect on apparent digestibility under this experiment could be explained by the low con centration of CT in the supplementation levels used (0%, 2%, 4%, 6% DMI), which RP (6% DMI) contained 0.7% CT and 0.6% SP of DMI, while as reported by Beauchemin et al [19] higher concentrations of CT (>50 g/kg DMI reduces DMI and digestibility. of Poungchompu et al [7], who used a higher level of CT (>3% CT of total DMI), also reported a decrease of DMI and digestibility. This could be attributed to its coating of feed particles and an effect on the cellular membrane of the rumen bacteria.

Ruminal fermentation, blood, methane estimation, and microbial population
Under this study, the rumen pH, temperature and ruminal NH 3 N were not affected in the RP supplemented groups. The rumen pH of 6.5 to 6.8 was suitable for the bacterial ac tivity especially cellulolytic bacteria, whilst BUN concentrations did not differ among treatments [20]. Wanapat and Pimpa [21] who stated that rumen degradable protein in the form of rumen NH 3 N was essentially to be used for rumen micro bial protein production efficiency. The optimal concentration of rumen NH 3 N (15 to 30 mg/dL) required for digestion by microorganisms has been reported when the ruminants were fed on rice straw [20,21].  In this experiment, the ruminal fermentation parameters (total VFA, C 2 , and C 4 ) remained similar by the RP supple mentation, while the C 3 production was increased in the RP supplemented groups. It could be due to the relationships with feed intake improvement and the rumen microorganism activity. The ratio of C 2 :C 3 was reduced as a result of the RP supplementation groups, which agreed with the data of Foi klang et al [22] who revealed that using grape pomace powder (GPP) supplement remarkably increased the total VFA and C 3 production, while rumen methane estimation was dra matically decreased in the supplemented treatments. Similarly, Gunun et al [23] also reported that C 3 was increased, while C 2 and C 4 were similar when supplementing with plants con taining of CT and SP less than 3% of total DMI. Norrapoke et al [24] who found that dietary of mangosteen peel powder (MSP) enhanced total VFA concentration, increasing C 3 , reducing C 2 :C 3 and methane production in the MSP sup plementation in swamp buffaloes.
Under this experiment, the total bacterial and fungal zoo spores were similar among treatments, while the number of protozoa was reduced in the RP supplementation (4% to 6% DMI) groups. Norrapoke et al [24] who used MSP pellet at 300 g/hd/d in dairy cows, found no influence on rumen bacterial and fungal zoospores. Rumen protozoa and metha nogens have a symbiotic relationship, in using CO 2 to produce methane, and the methanogens adhering to the protozoa were reported to be responsible for 9% to 37% of the methane emissions in cattle. Plant metabolic compounds, essentially SP and CTs, have been reported to suppress rumen CH 4 pro duction [2]. The influence of tannins on methane emission depends on fermentation of microbes and the enzymes se creted [25]. Plant secondary metabolites (CT and SP) have been shown to exert impact on activity of rumen metha nogens and protozoa activities that would then limit the H 2 availability for methanogenesis in the rumen [26]. Further more, plant secondary compounds were found to react on the sterols membrane of the protozoa [27]. The possible ac tions of CT in reducing methane production include the indirect effect on reducing H 2 formation and the activity of protozoa and methanogens [28]. In the present experiment, the t methane production and protozoal population were sub sequently decreased while fungal zoospores were not affected by the RP supplementation. This agrees with Shokryzadan et al [29] who found that supplementation of MSP reduced the rumen microbial population, especially protozoa and methanogens, as well mitigating methane production in ru minants. Paengkoum et al [30] also found that using CTs extracted from mangosteen peel could reduce gas production and concentration of ruminal methane in an in vitro gas experiment. Methanogens and CH 4 production were sig nificantly decreased when the MSP supplementation level was increased, the least value was obtained at 300 g/d. During the rumen fermentation process, enteric CH 4 is produced when the metabolic hydrogen produced by rumen cellulo lytic bacteria reacted with CO 2 [31]. Bhatta et al [3] explained that tannins considerably suppressed the rumen methane production by decreasing the protozoal population found in an in vitro study. While, Foiklang et al [22] discovered that supplementation of GPP could suppress protozoal popula tions and mitigate methane production in cattle. Similarly, Poungchompu et al [7] revealed that using 4% of MSP as a supplement could decrease rumen protozoa and, hence re duced methane production, accordingly. Currently, Aditya [32] and Gunun et al [23] also reported that using rambutan peel powder at 16 to 20 mg, remarkably decreased methane concentration in in vitro gas kinetic experiment. These results reiterate the impact of CTs and SP on rumen fermentation and the endproducts.