1. Introduction
Coccidiosis is an enteric disease affecting the performance, welfare, and health of chickens with an estimated economic loss of approximately GBP 10.36 billion in the global poultry industry [
1]. It is caused by several species of the genus
Eimeria that are known to invade specific sites of the gastrointestinal tract, inducing mild to severe gut lesions [
2]. Although anticoccidial medications have been used to control coccidiosis, concerns about the occurrence of resistant
Eimeria spp. and the residue on poultry meats have led the efforts to develop alternative nutritional strategies including probiotics, prebiotics, phytochemicals, and antimicrobial peptides [
3]. These potential alternative candidates may share working mechanisms such as reduction in the pathogenic (i.e., oocysts) load, mitigation of oxidative stress-induced gut damage, or enhancement of intestinal protective immunity in chickens [
2]. Recently, in-feed natural or synthetic antioxidants have been shown to lessen the severity of chicken coccidiosis, as
Eimeria infection is associated with lipid peroxidation in the intestinal mucosa [
4]. Among the antioxidants tested, plant extracts [
5], vitamin E [
6], and selenium [
7] are known to control avian coccidiosis.
Sulfur is an essential element for the growth of most animals including humans [
8]. Sulfur per se is not stored in the body, but animal diets need a supply of sulfur-containing macromolecules [
9]. In addition, sulfur has been used as an antimicrobial/anticoccidial agent for treating bacterial diseases and chicken coccidiosis due to its parasite killing, antioxidant, and immune-modulating activity [
10,
11,
12]. Chickens are generally resistant to the sulfur toxicity; sulfur tolerance is reported to be 14,000 ppm for broilers and 8100 ppm for laying hens [
13]. It is thus understood that sulfur toxicity in chickens is hardly seen at the commercial setting, and sulfur can be provided via consuming sulfur-containing water and ingredients or various forms of sulfate minerals.
Methyl sulfonyl methane (MSM) is an organic sulfur naturally found in all living organisms including insects, plants, animals, and humans [
14,
15]. It is an oxidized metabolite of dimethyl sulfoxide and contains 34% sulfur on a weight basis [
16]. MSM has been known to exhibit anti-inflammatory and antioxidant activities in vitro [
14] and in vivo [
17]. In addition, it is effective in treating parasitic infection by
Trichomonas vaginalis or
Giardia lamblia in animals including humans [
18]. The biological and parasitic activities of MSM make it a promising anticoccidial agent in chickens by inhibiting parasitic growth, augmenting preventive immunity, or mitigating parasite-induced oxidative stress. However, Abdul Rasheed et al. [
19] failed to see the anticoccidial effect of MSM in
Eimeria infected chickens. They noted an increase in oocyst counts and feed conversion ratios but a decrease in oxidative stress in the plasma samples of MSM-fed chickens compared with the
Eimeria-infected control group. Interestingly, MSM stimulated feed intake without affecting body weight gain compared with the infected control group. Thus, the lacking effect of dietary MSM or sulfur-containing molecules on avian coccidiosis remains to be concluded. When considering the antiparasitic effect of sulfur [
12,
18], we decided to re-test dietary MSM as a potential anticoccidial agent in broiler chickens. In addition to MSM as an organic sulfur, we included sodium sulfate (SS) as an inorganic sulfur to compare the effectiveness of different forms of sulfur in broiler chickens. It has been reported that both MSM and SS exhibit antimicrobial and antioxidant properties in laying hens [
20]. Nonetheless, Kim et al. [
20] concluded that SS vs. MSM might have different actions on feed or water intake, indicating different biological fates or functions between organic and inorganic sulfur sources in laying hens. Taken together, it would be of value to evaluate different forms of sulfur in a mild coccidiosis broiler chicken model.
4. Discussion
It is clear from this study that dietary organic or inorganic sulfur did not affect the growth performance of broiler chickens or those challenged with a coccidiosis vaccine. The lack of effect of MSM and SS on the naïve or coccidiosis vaccine-challenged broilers may not be due to the low addition level, the absence of a biological effect (e.g., antioxidant), or a failure in reproducing the coccidiosis disease model. On the contrary, dietary MSM exhibited a negligible tendency for a decrease in the performance of broilers in both the pre- and post-infection periods, indicating a low statistical power. In this study, we confirmed the antioxidative effect of MSM and SS as manifested by elevating the TAC concentration in liver samples of broiler chickens, which agrees with earlier studies in broiler chickens [
17,
19] and laying hens [
20]. The added levels of MSM and SS (i.e., 0.07% sulfur in diets) were effective in exhibiting the antioxidative effect in laying hens [
20]. In line with our findings, no effect of dietary MSM on productive performance was found in naïve ducks [
28], naïve broiler chickens [
17], or
Eimeria vaccine-challenged broilers [
19]. In contrast to our findings, increasing dietary MSM from 0.05 to 0.3% improved body weight gain and feed conversion ratio, but not feed intake, in broiler chickens [
29] and Pekin duck [
30]. It was found that those studies with MSM-induced improvement in growth performance were associated with an increase in immunity and antioxidant parameters coupled with a shift in gut microbiota [
29]. In this study, we used the coccidiosis vaccine challenge model to evaluate the protective effect of MSM and SS, if any, in broiler chickens. It is well reported that a high dose of coccidiosis vaccine challenge has been known to induce experimental coccidiosis with varying degree of gut lesions in broiler chickens [
19,
23]. We reproduced mild coccidiosis with an attenuated coccidiosis vaccine as manifested by
Eimeria-specific mild lesions on duodenum, jejunum, and cecum and the moderate reduction in growth performance. Thus, the anticoccidial efficacy of MSM or SS on chicken coccidiosis would have been detected, if present, in this challenge model. Abdul Rasheed et al. [
19] reported that dietary MSM did not affect performance, gut lesions, and fecal oocyst output in a mild coccidiosis disease chicken model. They speculated that mild
Eimeria infection might not be sufficient to disclose the anticoccidial effect, if any, of dietary MSM in broiler chickens. At this stage, a clear explanation on the lack of effect of MSM or SS on chicken coccidiosis is not readily available; that needs to be addressed. However, as the chicken exhibits resistance to sulfur toxicity [
13], higher inclusion doses in a clinical coccidiosis model using different doses or
Eimeria field strains might be needed in future studies. In addition, the clinical vs. sub-clinical disease model would be considered the better experimental model for testing potential anticoccidial agents as the former vs. the latter could increase the treatment effect size, and such practice could reduce the number of animals without lowering the statistical power.
The recent interest in natural or synthetic antioxidants in chickens has surged, as avian coccidiosis causes severe inflammation followed by lipid peroxidation of the intestinal mucosa [
4]. Indeed, Colnago et al. [
31] found that dietary selenium or vitamin E reduced an
Eimeria-mediated increase in mortality and growth depression in broiler chickens. The latter findings led us expect the anticoccidial activity of antioxidant sulfur (MSM and SS) in experimental avian coccidiosis. In contrast to our expectation, dietary MSM or SS did not affect growth performance nor lower gut lesions in coccidiosis vaccine-challenged broiler chickens, although both MSM and SS produced antioxidant activity at 21 days (i.e., before coccidiosis vaccine challenge). In line with our study, the antioxidant activity of MSM and SS in laying hens [
20] and MSM in broiler chickens [
17,
19] has been reported. In addition, it has been reported that antibiotic plant extracts [
5], vitamin E [
6], and selenium [
7] are known to lessen the gut severity of
Eimeria infection in broiler chickens. Tentatively, it is tempting to conclude that the antioxidant activity per se might not determine the anticoccidial activity of the potential candidates. Nonetheless, it was perplexing that both MSM and SS tended to increase duodenal lesions while lowering jejunal lesions compared with the challenged control group. In addition, dietary MSM tended to increase cecal lesions compared with the challenged control group. Due to the mild lesions produced by the attenuated coccidiosis vaccine, this marginal increase or decrease in the gut lesions of the sulfur-fed chickens over the challenged control group may not be considered an accurate indicator of anticoccidial activity. Commonly, it is well known that the field isolates of
Eimeria can cause more severe intestinal damage compared to overdose of attenuated coccidiosis vaccine strains [
22]. Whether dietary MSM or SS at different inclusion levels would be more effective in clinical coccidiosis with severe gut lesions waits to be addressed in future studies.
It is well reported that coccidiosis impairs nutrient digestion leading to a low productive performance in broiler chickens. For example, Amerah and Ravindran [
32] reported that a coccidia challenge with field isolates resulted in reducing the AID of dry matter, nitrogen, starch, fat, and energy of broiler chickens compared with the naïve control chickens. Of interest, the latter group [
32] found that the AID of crude ash was not affected by
Eimeria infection. Dunaway and Adedokun [
33] noted that a coccidiosis vaccine challenge lowered the digestibility of dry matter, nitrogen, and metabolizable energy in broiler chickens compared with the naïve control group. Similarly, we found that coccidiosis vaccine challenge lowered the AID of crude protein and crude ash, albeit that it did not affect that of dry matter. This finding coupled with lowered gut lesions suggests the negative effect of coccidiosis on the gut integrity of chickens. In this study, MSM and SS did not affect the
Eimeria-induced reduction in the AID of crude protein. However, MSM, but not SS, improved the
Eimeria-induced reduction in the AID of crude ash. The clear explanation for the improved effect of MSM over SS on ileal ash digestibility is not readily available, but it might be related to mineral interaction or dynamics in gut lumen. Summers et al. [
34] found that bone ash contents were linearly lowered with increasing dietary sulfur, indicating that calcium would be precipitated as an insoluble salt with a high sulfate concentration in the intestinal lumen. It would thus be likely that there is an interaction between sulfur and macro/micro minerals in the intestinal lumen. However, as both MSM and SS diets had equal sulfur contents, it might be less likely that the effect of sulfur per se on the mineral dynamics was the main explanation for the MSM vs. SS regarding favoring ash digestibility. Whether MSM vs. SS is more effective in mineral utilization/absorption at the gut level needs to be addressed. The latter statement can be addressed in analyzing macro/micro minerals in digestion trials to see which minerals are interacting with dietary MSM.
The SCFAs are known as the major end-products of fermentation by gut microflora on undigested carbohydrates including non-starch polysaccharides, are used as a nutrient source for colon epithelial cells, and have an inhibitory effect on intestinal pathogenic bacteria [
35,
36]. It is reported that sulfur is known to have antimicrobial activities in chickens as manifested by improved gut morphology and an increased
Lactobacillus but lowered
Escherichia coli [
37]. Similarly, Jiao et al. [
29] noted that increasing dietary MSM from 0.05 to 0.2% in diets linearly increased
Lactobacillus but lowered
E. coli in the excreta of broiler chickens. However, neither MSM nor SS affected cecal SCFAs in chickens in this study, although the former increased the relative percentage of BCFA by an average of 19.2% compared with the control group. The contradictory roles of BCFA have been reported, with both increases [
38] decreases in the inflammatory responses [
39]. It is however not clear whether the increased cecal BCFA concentration noted in this study could explain the slight increase in cecal lesions in the MSM-fed chickens, as the BCFA concentration remained low from 2.1 to 3.2% of total SCFA. Further studies are needed to delineate the interplay between gut microbiota and local/systemic immunity as highlighted elsewhere [
40].