4.1. Effect of Arsenic-Containing Rice Bran on Growth Performance of Pigs
Previous research suggested that As is required by the pig as an ultra-trace mineral [
23,
24]. However, the level of As dietary requirement by the pig is uncertain [
13], although As feed additives have been used in some swine farms, which is driven not only by some commercial benefits but also by certain traditional practices [
1,
18,
25]. To humans, as aforementioned, inorganic As is a class-I non-threshold carcinogen and it can lead to numerous human diseases [
6]. To the pig, evidence has indicated that upwards of 450 ppm arsanilic acid in the ration fed daily for one week could produce symptoms of acute poisoning [
18]. In this study, however, all the pigs were healthy looking, performed normally, and displayed no poisoning symptoms throughout the trial.
Sun et al. (2008) reported that the total As content of rice bran products ranged from 0.71 to 1.98 mg/kg (or 710 to 1980 ppb) with the inorganic As ranging from 60 to 95% [
11]. The total As content in the rice bran product used in this study was approximately 830 ppb, a mild level in general, and the total As contents in Diets II and III were approximately 450 and 630 ppb, respectively, which are far below the level of As in 450 ppm arsanilic acid as stated in Gary (1966) [
18]. This is likely the reason why the pigs in this study did not show any clinical poisoning symptoms.
The growth performance data of this study clearly indicate that the ADFI and ADG of the pigs were negatively affected by the high level (approximately 70%) of dietary rice bran inclusion, but not significantly affected by the moderate level (approximately 35%) of the inclusion. Although the effect of dietary As content on the growth performance of pigs cannot be defined in this study, a conclusion regarding the effect of dietary rice bran as a whole on the ADFI and thus the ADG of pigs can be drawn. As shown in
Table 2, both the crude fiber and contents in Diet III were higher than in Diet II, and the same was true for Diet II vs. Diet I. Same trends can also be found for the contents of crude fat, crude protein, gross energy, and ash (
Table 2). Thus, the reduction of ADFI by dietary rice bran inclusion (at the level of 73% in this study) may be attributed to the contents of dietary fiber, crude fat, crude protein, gross energy, and ash. It is commonly recognized that fiber can stay in the digestive tract for a longer period and, therefore, reduces feed intake and weight gain. The negative effect of dietary fiber intake on energy and nutrient digestibilities is well documented in the literature [
26,
27,
28,
29,
30], although Kaufmann et al. (2005) reported that the protein and amino acid digestibilities of rice bran have no relationship with the content of neutral detergent fiber [
31].
Slightly different from the result of this study, de Campos et al. (2006) reported that the inclusion of 30% rice bran in a corn and soybean meal-based diet reduced ADFI by approximately 0.27 kg/day in growing-finishing pigs [
32]. Casas et al. (2018) also reported that a dietary inclusion of full fat rice bran at 10 to 30% linearly reduced ADFI in finishing pigs. With weanling pigs [
29], Warren and Farrell (1990) even reported that the ADFI was slightly increased when a defatted Australian rice bran was included from 10 to 30% [
33]. The discrepancies between this present study and these afore-mentioned reports may be attributed to the differences in the chemical compositions of the neutral detergent fiber and crude fat in different rice bran products, as well as the different pigs, used in the studies.
As shown in
Table 3, the G:F ratio was not significantly affected by the dietary inclusion of rice bran, which suggests that the reduced final BW of Diet III pigs was mainly due to the high level of dietary fiber inclusion and the reduced ADFI. These results are partially in agreement with Casas et al. (2018), who reported that the G:F ratio and final BW of pigs were not affected by the inclusion of rice bran [
29]. Several factors, such as the dietary nutrient ratio balance and the health status of pigs, can affect the G:F ratio. The contents of dietary net energy, essential amino acids, as well as minerals and vitamins, are similar among the three experimental diets (
Table 2). In addition, no pig showed any unhealthy activities during the entire feeding period. Thus, the feed efficiency was not significantly affected by the dietary inclusion of rice bran at a level up to 73%.
4.2. Transportation and Distribution of Arsenic in Pig Body
To date, the studies of As nutrition in pigs are not available or are very limited, so data regarding the As absorption, transportation, distribution, retention, and excretion, in and by the pig are scarce. Rice bran, a widely used animal feed, usually contains a high amount of As [
11]. Thus, this present study was designed to investigate the tissue distribution of As (released from the rice bran consumed) within the pig body, and the fecal and urinary excretion of As out of the pig body.
It has been known that the As component from dietary source is readily absorbed in the intestine and rapidly transported by the blood stream throughout the body [
34]. In pigs approximately 25% of dietary As are usually absorbed, whereas in humans the absorption rate is much higher, approximately 80% [
35,
36]. After absorption, most of the As is cleared rapidly from the blood stream of pigs with some being retained in various organs and others being eliminated through feces and urine [
25]. In this study, the concentrations of As in the blood of the pigs were below the detection level, which, however, is discrepant from the results of Ledet et al. (1973), who reported that the average As concentration in the blood of pigs was 1.80 ppm during the 27-day feeding period [
25]. The discrepancy between Ledet et al. (1973) and ours might be due to the low dietary As concentrations in our study, which were 448 and 633 ppb (analyzed values) for Diets II and III, respectively, whereas the pigs of Ledet et al. (1973) were fed the diet contained 1000 ppm arsanilic acid [
25]. Thus, it can be predicted that the concentration of As in pig’s blood will be very low and, in most cases, undetectable when a mild amount of As (roughly 400 to 600 ppb) is fed.
To study the retentional distribution of As in various tissues, this study measured the total As concentrations in the liver, kidney, muscle, and hair samples of the pigs fed with or without the As-containing rice bran. The results showed that the As retention is less than or around 20 to 30 ppb in the liver and kidney of the pigs fed the moderate to high level of As-containing rice bran (
Table 4). The As concentration in the muscle was under detection limit (<10 ppb) of the updated detection method current available for analytical laboratories. Similar As distribution pattern was observed by Ledet et al. (1973), who reported that the concentrations of As in the liver and kidney were much higher than in the muscle of the pigs consumed high As diets [
25,
37]. Ledet et al. (1973) reported that the As concentrations in liver and kidney were close (9.67 and 8.33 ppm, respectively) in the pigs fed with 1000 ppm As-containing diet. Moreover, López-Alonso et al. (2007) investigated the concentrations of As and some other essential metal elements in muscle, liver, and kidney of pigs in Galicia, Spain [
38]. The authors reported that appreciable As contents were found in the liver and kidney, whereas As was not detected in most (98%) muscle samples.
The major biochemical mechanism of As retention in body tissues is that the arsenicals, which include trivalent, monomethyl, and dimethyl As that have high affinity for sulfhydryl groups, can bind to the reduced cysteine residues in peptides or proteins [
39]. By this mechanism, As can be distributed ubiquitously to all major tissues; however, the actual capacity of As retention may vary in different tissues. For example, Ducoff and Neal (1948) used [
76As] sodium arsenite to study As tissue distribution patterns and excretion rates in rats and rabbits [
38]. Rats retained the most of As in their red blood cells, with smaller concentrations in the spleen, heart, lungs, kidneys, and liver. On the other hand, the As was lowest in the blood and highest in liver, kidneys, and lungs in rabbits [
40,
41]. The As tissue distribution pattern observed in this present study is principally supported by those previous studies in pigs, rats, and rabbits.
Arsenic retention was the highest in the hair when compared to the other tissues tested in this study, whereas no hair As concentration data for pigs were found in the literature. Olguín et al. (1983) studied a human population exposed with As and found that the second highest As deposition was in the hair (1240 ± 610 ppb) followed by the nails (4550 ± 3250 ppb) [
42]. In addition, Katz (2019) summarized the hair As concentration data from multiple studies and claimed that the hair As concentration is positively correlated with and can be considered as a biomarker for evaluation of As toxicity in humans [
43]. The scientific basis of this claim is that the keratin in hair is rich in disulfides that can easily incorporate As into the growing portion of hair root [
43]. These references [
42,
43] support our findings in this study that the pigs’ hair retained the highest level of residual As after chronic exposure to dietary As.
4.3. Excretion of Arsenic from Pig Body
Along with the tissue distribution pattern, the concentrations of As in the feces and urine of the pigs were also measured in this study to explore the pattern of As excretion from pig body. The results of this study concerning the fecal As content provide some novel data on the pattern of As excretion. As illustrated in
Figure 2, the fecal As concentrations were similar among the three groups of pigs before the feeding trial. However, after the 6-week feeding trial, the fecal As concentrations were increased drastically in pigs fed Diets II and III, which suggest that the degree of gastrointestinal As absorption was not high. This finding agrees with Mandal (2017) who stated that the As compounds are poorly absorbed in the intestine of pigs (only about 30% of dietary intake) when compared to rodents and humans (approximately 90% of the dietary intake) [
44]. As a result, most of the unabsorbed As was excreted through the feces of the pigs. That being said, please also keep in mind that since As can circulate in the enterohepatic and can be reabsorbed by the intestine, an increased fecal As excretion may not necessarily be consistent with a poor As absorption.
The fact that As was excreted in the urine is an evidence of As absorption. In terms of As excretion through urine, studies with laboratory animal models have shown that the inorganic As is metabolized by the reduction of arsenate (As
V) to arsenite (As
III), followed by the sequential methylation to monomethylarsonic acid (MMA) and dimethyl As acid (DMA). These methylation reactions have traditionally been regarded as a detoxification mechanism since the methylated metabolites exert less reactivity and acute toxicity with tissue constituents than the inorganic As [
34,
45,
46,
47]. The methylated forms of As are water soluble and mostly can be eliminated from the body via glomerular filtration of the kidney or through urinary excretion [
48,
49,
50]. Most mammalian species can efficiently eliminate As from the body after withdrawal of the As-containing diet [
37].
In this study, the As concentration was increased by more than 200% in the urine of pigs fed Diets II and III relative to Diet I. There is a lack of reference value in the literature to support this finding of ours regarding urinary As excretion pattern in pigs. Previous studies in humans and experimental animals suggested that urination is one of the primary routes for the arsenical compounds to be eliminated from the body, and the intake of As from dietary or other sources affects the urinary As concentration [
49,
51,
52]. Bae et al. (2013) studied the relationship between daily dietary As intake and the urinary As concentration in a Korean population and found a significantly positive (r = 0.096,
p < 0.05) relation between the two parameters, which suggested that the dietary As intake affects the total urinary As concentration [
53]. A study by Choi et al. (2012) also found high As concentrations in the urine of seafood-consuming population in Korea [
54]. Therefore, the high As concentration in the urine of pigs fed high As diets suggests that pigs are capable of efficiently eliminating most of the absorbed dietary As through urine.