INTRODUCTION
The application of ferrous sulfate (FS) to enhance the nutritional value of lignocellulosic biomass during roughage preservation for ruminant feed is well established [
1]. Incorporating FS during the ensiling process has the potential to accelerate the breakdown of biomass, leading to the formation of more functional microbes and compounds that lower nutrients loss and formation of toxic substances, consequently increasing the productivity in ruminants [
1,
2]. Several direct-fed FS studies have shown to increase the health and productivity of small ruminants [
2,
3]. Direct-fed FS method either administered FS orally in the form of a capsule or incorporating it into the diet [
4]. More recently, study showed that addition of FS to molasses (MS) as a mixed additive (FS-MS) enhanced the effectiveness of the ensiling process and goat performance [
5].
Heat-induced oxidative stress is a well-known limiting factor affecting animal health and performance in the tropics [
6,
7], and feeding anthocyanin-rich black cane (
Saccharum sinensis Robx.) to tropical ruminants, including slow-growing indigenous goats, can be a practical approach to overcome the above production constraint [
8]. In Thailand, anthocyanin-rich black cane (hybrid of
Saccharum spontaneum and
Saccharum officinarum) was recently recognized as a potential alternative method for sugar production, while the leftover biomass (stalks and leaves), including in the form of silage, could be used as ruminant feed [
1,
5]. It is possible that, in the tropics, feeding diet containing anthocyanin to goats is beneficial because anthocyanin has the potential to enhance the antioxidant capacity of the rumen [
8] which in turn reduces oxidative stress in the animals by limiting the availability of free radicals and the generation of additional oxidations [
6,
8]. However, the highly lignified components of anthocyanin-rich black cane limit anthocyanin bio-accessibility during feeding. Thus, incorporating FS-MS mixture to anthocyanin-rich black cane prior to ensiling can be useful. Earlier studies revealed that treating anthocyanin-rich black cane with FS-MS mixture (0.15% to 0.03% FS and 4% to 8% MS on fresh weight silage basis) decreased lignin contents, improved anthocyanin stability, and
in vitro ruminal fermentation of black cane silage [
1,
5]. Recent
in vivo studies reported that the microbial population in rumen fluid of goats fed purple corn [
9] and black cane [
8] containing anthocyanin enhanced the acetate to propionate ratio. The anthocyanin-induced acetic acid production may result in more substrate available for
de-novo fat synthesis [
10], however, because the molecular structure of anthocyanins varied depending on the plant species or during processing, it is difficult to accurately generalize the earlier findings [
8,
9].
As far as we know, evaluation of FS-MS treated anthocy anin-rich black cane silage as ruminant feed has been limited only to laboratory scale silos and
in vitro procedures [
1,
5]. Therefore, the present study investigated the effects of feeding a standard total mixed ration (TMR) containing anthocyanin-rich black cane silage treated with FS-MS mixture on animal performance, rumen fermentation, microbial community, blood biochemical indices, and carcass characteristics in meat goats.
DISCUSSION
To the best of our knowledge, the current study provides the first
in vivo results on the feeding of FS-MS treated anthocyanin-rich black cane silage-based diet in ruminants. The results showed that treating anthocyanin-rich black cane silage with 0.03% FS and 4% MS (TBS) resulted in a relatively higher degradation of lignin and possibly its silica content. Most likely, the function of FS during biomass fermentation was to enhance the breakdown of lignin, hemicellulose, and cellulose during ensiling [
1,
5]. This was often a highly dehydrating process. In addition, MS appeared to enhance the FS catalytic process by increasing the moisture and soluble carbohydrate (WSC) content during the silage process, which promotes the growth of anaerobic and lactic acid bacteria [
5].
Results of the current study showed that feeding a TMR diet containing 50% TBS to Thai-native×Anglo-Nubian male goats increased their ADG and feed conversion efficiency. The improvement in feed conversion efficiency observed in goats fed TBS is consistent with the fact that TBS diet significantly increased (p = 0.04) total rumen VFA concentration as compared with those fed untreated BS diet which is in agreement with earlier
in vitro studies that FS-MS-induced VFA production [
1,
5].
The use of FS-MS to treat anthocyanin-rich black cane (TBS) could be a viable strategy to reduce anthocyanin loss during fermentation (
Table 2). We postulate that the higher dietary anthocyanins because of feeding TBS improved total VFA concentration because anthocyanins play a regulatory role in growth of bacterial populations in the rumen as demonstrated by the increased relative abundance of
R. albus in this study. It was indicated earlier that FS could hasten the breakdown of the lignin part in the TBS, which would result in an increase in the availability of sugar and, therefore, an increased production of fermentation acids for
R. albus [
1]. Although the nutrient digestibility of the diets was not determined, the higher relative abundance of
R. albus in goats fed TBS diet appeared to support this notion. Others [
20] reported that incorporating
Andrographis paniculata leaves rich in plant active compounds (such as lactones, anthocyanin, flavonoids, and sterols) into goat diets enhanced ruminal
R. albus population without altering the total bacteria which eventually led to an improvement in nutrient digestibility.
Our results reaffirmed a previous
in vitro study [
5] that black cane treated with 0.03% FS and 4% MS increased relative abundance of
R. albus but no other bacteria, including
R. flavefaciens,
F. succinogenes,
B. fibrisolvens,
M. elsdenii, and
S. bovis. The above findings also concord with those reported using black cane silage [
8] and purple maize anthocyanin [
9].
The lower ruminal ammonia-N concentrations in goats fed TBS diet deserves some mentioning. The above could be because TBS provided more fermentable carbohydrates which play an important role in increasing rumen microbes by converting rumen degradable CP to microbial protein [
21,
22]. However, it cannot exclude the possibility that greater anthocyanin consumption decreased rumen solubility of dietary proteins, which in turn decreased rumen ammonia-N concentrations [
9]. Also, the above result could be affected by the 24 h fasting before slaughtering and collection of rumen fluid [
16] used in this study. After 24 h of fasting, rumen fermentation may be quite mild and not reflecting its true potential [
16]. It is unfortunate that the present study did not provide data to explain for anthocyanin induced protein solubility reduction, thus more research is needed on this interesting subject. In addition, feeding TBS diet resulted in a decrease in the relative quantity of methanogens in the rumen fluid sample. In fact, both BS or TBS diet contained anthocyanin components. The result of anthocyanin reducing methanogens in presence of higher
R. albus population observed in this study is rather difficult to explain, but similar observations of reduced relative abundance of methanogens in rumen fluids in sheep and goats after feeding mulberry leaf [
23] and black cane [
8] had been reported. Perhaps, the reduced relative abundance of methanogens in TBS goats could be explained by the higher anthocyanin components and FS residue in the rumen fluid, which might inhibit methanogenesis by making electron exchange more challenging for FS-reducing bacteria and methanogens harboring Fe oxides [
24]. This hypothesis, however, contradicts the increased rumen acetate to propionate ratio in TBS fed goats in this study. The current finding might be read as feeding of TBS diet reduced methane production via reductive acetogenesis [
25], because synthesis of acetate, as opposed to the production of propionate, is commonly acknowledged as a source of hydrogen [
25,
26]. It is difficult to explain the apparent difference between present acetic acid results and the relative abundance of methanogens, but it cannot be ruled out that acetogenic bacteria used hydrogen to produce acetate. Acetogenic bacteria can, in fact, transform carbon dioxide and hydrogen straight into acetate [
27]. Further research is required to shed additional light on this subject.
Feeding of TBS had no effect on any of the carcass param eters investigated (e.g., dressing percentage, LMA, and pH value) in this study, which is in line with other reports in small ruminants [
8,
19]. However, feeding of TBS to cattle resulted in favorable effect, including external fat color [
28]. The discrepancy in the results among studies could be attributed to a number of factors, including the composition of the anthocyanin-based diets, the animals and the bioavailability of flavonoids or anthocyanins. Also, neither the adipose nor the lean tissue color (L*, a*, b*, or c*) of the goats was influenced by diets. Anthocyanins containing malvidin, malvidin-3-O-glucoside, and peonidin-3-O-glucoside were found to be capable of stabilizing muscle membranes, resulting in improved meat color [
8,
19]. In this study, both the BS and TBS diets contained peonidin-3-O-glucoside, malvidin-3-O-glucoside, and malvidin, which may have reduced lipid peroxidation and preserve meat coloration. Our results showed that TBS diet promotes higher intramuscular fat content. The increased intramuscular fat accumulation in the TBS-fed goat muscles could most likely due to the increased rumen acetate synthesis as increasing acetate production opens up additional substrate for
de novo fat synthesis [
10]. Previous research [
29] reported a link between intramuscular fat content and WBSF tenderness values. Therefore, it is postulated that the lower WBSF values obtained in current study are, at least in part, attributable to greater intramuscular fat concentrations.
Blood biochemical indices can be useful indicators for animal’s health status relating to dietary absorption and metabolism [
7]. We hypothesized that the anthocyanins and other bioactive compounds in the TBS diet can help to reduce oxidative stress in animals (goats in this case) under stress in the tropics. Results of the blood biochemical analysis in this study, specifically, the lower TBARS values and higher TAC levels supported the above. Similar findings were also reported involving anthocyanin in purple maize [
9], mangosteen peel [
7], and piper meal [
6]. It is believed that the higher level of anthocyanins (such as peonidin-3-O-glucoside, malvidin-3-O-glucoside, and malvidin) provided better protect against free radicals and decrease inflammation [
8] in the TBS fed goats. Simultaneously, feeding TBS resulted in an increase in the activity of antioxidant enzymes such as SOD, CAT, and GSH. In this aspect, our results concord with previous study from this laboratory [
6] that lactating goats fed a mixed diet that included piper meal rich in flavonoids (possibly anthocyanin as well), essential oils, and phenolic acids altered oxidative markers (SOD, CAT, and GSH) activities in the rumen, blood, milk, and mammary tissue. Increasing antioxidant enzyme activity in ruminal fluid or blood may result in a decrease in TBARS levels by optimizing the rumen and its rumen bilayers for dietary bioactive chemicals produced from piper meal by increasing membrane fluidity [
30]. Based on the current results, we postulated that the higher level of anthocyanin in the TBS diet acts as an electron donor, reducing the accumulation of reactive oxygen species (ROS), as SOD is the first line of defense against ROS scavenging in intracellular enzymes. In contrast, activation of CAT and GSH-Px or GSH-Rx implies that the superoxide anion radical in the dismutation exceeded the limit, resulting in the ROS being scavenged enzymatically by CAT or GSH before being reduced to water [
8]. It appeared that SOD, CAT, and GSH were all acting together to reduce ROS scavenging. Therefore, the lower TBARS values and the higher TAC levels are not only induced by the antioxidative activity of anthocyanin, but also by the greater expressions of SOD, CAT, and either GSH-Px or GSH-Rx.