Effects of dietary fumonisins on nutrients digestibility in weanling pigs

The aim of the study was to ascertain the potential effects of fumonisins on nutrients’ digestibility in the gastrointestinal tract. Eighteen (n= 6 x 3) weanling pigs from the age of 35 days were administered 0, 15 or 30 mg/kg dietary fumonisins (FB1, FB2 and FB3) for a period of 21days, after an acclimatization period of 14 days. Titanium dioxide (0.5%) was added as indigestible marker to the feed and representative fecal samples were taken during a 5day collection period in order to determine the digestibility of crude protein, crude fat, crude fiber, starch, ash, calcium, phosphorus and energy. The final body weights, cumulative feed intake and relative organ weights of all groups were were not influenced by treatment (p>0.05). Digestibility of energy, crude fiber, ash, calcium and phosphorus were significantly higher (p<0.05) in the control group relative to either 15 or 30 mg/kg treated pigs. These findings suggest that fumonisins in a dose of 15 or 30 mg/kg potentially distorts total tract nutrient digestibility in weanling pigs and thus, compromise the nutritive value of the mixed feed.


Introduction
The Fusarium verticillioides (Sacc.) is the major producer of fumonisins (FUM); a group of water-soluble secondary fungal metabolites of moulds commonly found in maize and maize-based products intended for both human and animal consumption. At least 28 FUM analogues have been characterized (Rheeder et al. 2002) namely the A-, B-, C-and P-series. The B-series FUM which includes FB 1 , FB 2 , FB 3 , and FB 4 are the predominant analogues produced by wild-type isolates of F. verticillioides (Nelson et al. 1993). Amongst these, FB 1 is extensively reviewed due to its diverse toxicological characteristics which includes the notorious porcine pulmonary edema (PPE) and equine leukoencephalomalacia (ELEM) (Harrison et al., 1990), both of which are fatal to these animal species. Humans have also had their share on this fungal metabolite through the induction of oesophageal cancer and some neural tube defects (Gelineauvan Waes et al., 2009;Islami et al., 2009). Thus, falling in the group 2B (possibly carcinogenic) category of the International Agency for Research in Cancer (IARC) classifications (IARC, 2002). Structural analogy of FB 1 to the sphingoid bases sphingosine and sphinganine is the key mode for its toxicity. Through this, FB 1 is able to distort the enzyme ceramide synthase (a key enzyme in the de novo biosynthesis of sphingolipids) activities which subsequently leads to cellular disturbances in cell growth and differentiation, cell survival, and apoptosis (Merrill et al., 2001).
Just like other major existing mycotoxins, FUM cause several clinical disorders in most livestock species. However, the effect of FUM exposure on potential symptoms in gastrointestinal tract (GIT) till now, has not been thoroughly investigated. The pigs' gut nurtures several microbial species, produces several hormones, digests and absorbs a huge majority of nutrients, and is responsible for approximately 20% of body energy expenditure (Ewing, 2008;Choct, 2009). Thus, anything that hampers the normal functioning of the GIT is of utmost significance. The growing interest of intestinal health in the study of mycotoxins in recent years is due to how these ubiquitous substances have found the GIT as a newest functional target (Grenier & Applegate, 2013). Regarding FUM, the poor intestinal absorption of this mycotoxin in monogastric animal species (1 to 6 percentage; Bouhet & Oswald, 2007) suggests that, the gut epithelium is constantly exposed to significantly higher concentrations of FB 1 relative to other tissues (Grenier & Applegate, 2013).
Unfortunately, the amount of knowledge garnered on the biochemical/toxicological actions of FUM barely accentuates the causal mechanisms of FUM toxicity on nutrients digestion and absorption and gut morphology especially in in vivo trials. Younger farm animals like weaned piglets are mostly at risk due to the delicate nature of their gut when transitioning from liquid to solid feed i.e. the inescapable stress of weaning. Furthermore, reduced/compromised nutrient absorption at an early age may increase the incidence of intestinal disorders post-weaning and thus an overall reduction in animal performance.
In this study, we sought to evaluate the effects of FUM on total tract nutrients digestibility in weanling pigs exposed to either a diet containing no fungal culture of FUM (FB 1 , FB 2 and FB 3 ), one containing 15 or another containing 30 mg FUM/kg feed for a period of 21 days.

Material and Methods
The research protocol was reviewed and authorized by the Animal Use and Care Administrative Advisory Committee and approved by the Agricultural Administrative Authority (Protocol SOI/31/00308-10/2017).

Animals, Experimental Diets and Methods
Eighteen male Danbred weanling pigs of 5 weeks of age, after an acclimatization period of 2 weeks (at exactly 7 weeks old) with body weights of 13.5 ± 1.3 kg were randomly assigned, in a completely randomized design to each of 3 diets (n=6). A commercial piglet feed was used as a basal diet complemented with FUM (FB 1 , FB 2 and FB 3 in a fungal culture), either 15 or 30 mg/kg whiles the control diet was FUM free. Feed was offered as an amount that covers 2.5 times the maintenance energy requirement and was provided twice a day, in two equal portions. Refused feed was measured back every day. In Table 1 the result of the proximate analysis of the feed is given. Drinking water was available ad libitum. Piglets were kept in individual metabolic cages (80x80 cm) and weighed individually with g precision at the beginning of the trial and at weekly intervals. Piglets were monitored every day. The room temperature was adjusted according to the needs of the piglets and diarrhoea score was monitored throughout the trial.
A Fusarium verticillioides fungal culture of high FB 1 concentration; a culture named RL 596, according to Bartók et al. (2010) was mixed into the ration of the experimental animals, so as to provide a daily FUM (FB 1 +FB 2 +FB 3 ) feed concentration of 15 and 30 mg/kg. The mycotoxin concentration of the control and the experimental feed was determined with LC-MS (Shimadzu, Kyoto, Japan). The limit of detection (LOD) for FB 1 and FB 2 were 0.031 and 0.051 mg/kg respectively; LOD for FB 3 was not measured. The diet fed to the control group did not contain detectable amounts of FUM (complete absence of deoxynivalenol, zearalenone and T-2 toxin was as well controlled and confirmed). Fumonisin B contents were determined with a LC-MS method as described by Bartók et al. (2010).

Experimental Methods and Feed Analysis
The digestibility trial consisted of the inclusion of titanium dioxide (Ti 2 O) at a level of 0.5% to the feed. During a 5-day collection period, representative fecal samples were taken 4 times a day (approximately 200g sample per day). The urine was collected separately in closed plastic containers. Fecal samples were weighed and frozen at -18 °C immediately after collection. At the end of the trial, piglets were euthanized by exsanguination after sedation (Euthanyl-Pentobarbital Sodium, 400 mg/mL, Dechra Veterinary Products, Shrewsbury, UK) in order to evaluate the effect of FUM exposure on visceral organs.
The chemical compositions of the diets and fecal samples collected were used to calculate the apparent total tract digestibility (ATTD) of crude protein, crude fat, crude fibre, starch, ash, calcium and phosphorus and determined according to the methods of AOAC (2000). The gross energy content of the feed and the fecal samples were determined by analysing duplicate samples using IKA-Calorimeter C4000 adiabatic bomb calorimeter with benzoic acid used as a standard.

Data Calculation and Statistical Analysis
Apparent total tract digestibility of crude protein, crude fat, crude fiber, starch, calcium, phosphorus and ash, as well as dietary energy was calculated using the Ti 2 O concentration in feces and feed by using the equation: where Marker_feed and Marker_feces are % of Ti 2 O in the feed and in the feces, and Nutr_feed and Nutr_feces are nutrients (g/kg) and energy (kJ/g) in the feed and in the feces, respectively on dry matter basis.
Statistical analyses were performed using the SPSS 20.0 software (IBM Corp., Armonk, NY, USA). Results were subjected to a one-way analysis of variance (ANOVA). In case of a significant treatment effect, the intergroup differences were checked by a Tukey post-hoc test. A p value <0.05 was described significant.

Clinical Signs and Growth Rate
No mortality or diseases of any sort was observed during the 21-day long exposure of weanling pigs to either a control, 15 or 30 mg/kg FUM contaminated diet. There was diarrhea during the 14-day adaptation period but the piglets recovered before the start of the trial.
There were no significant differences (p>0.05) in the final body weights of pigs but numeric increments were observed in the 15 and 30 mg/kg dosed groups (data not shown). Except for one, all animals ate the daily ration, so generally, there was no feed refusal. A pig in treatment 30 mg/kg left some orts and therefore, its feed consumption was 95.5% of the others. In addition, there were no significant differences (p>0.05) in the relative kidney, liver and pancreas weights at the end of the trial (data not shown).

Nutrients Digestibility
The apparent total tract nutrient digestibility (ATTD) values in weanling pigs were higher for the control group as compared to those on the 15 and 30 mg/kg diets for each variable except for starch which remained the same (0.99) in all treatments (Table 2). Crude fiber, phosphorus, calcium and ash digestibility were significantly higher (p<0.05) in the control group relative to either the 15 or 30 mg/kg groups. The crude fat digestibility albeit did not differ significantly (p>0.05) in different treatments, but appeared numerically higher in the control group as compared to the two FUM treated groups. As a result of the difference in fiber and the shift in fat digestibility, the total tract digestibility of dietary energy was also compromised by the FUM exposure.

Discussion
The present study was designed to evaluate the effects of FUM on the fecal digestibility of nutrients in weanling pigs. According to the scientific literature, mycotoxins challenge typically and frequently impairs growth and productive performance in most examined animal species. Yet, our results revealed no such physiological disorders on the weanling pigs fed either a 15 or 30 mg FUM/kg feed. Corroborating to our results is that of Tóth and co-workers (2000) who reported no remarkable effect of an even much higher dosage of dietary fumonisin B1 (FB 1 ; 40 ppm) on feed intake and body weight gain of pigs, although pigs developed strong porcine pulmonary edema.
Regardless, it seems that the FUM managed to interfere with the nutrients digestion of the pigs. In line with this, the establishment that mycotoxins, including FUM are capable of inducing non-specific GIT hypo-functionality in animals is attested (Gbore et al., 2010). Although the rate of digestibility of crude protein, crude fat and starch was unperturbed by FUM intoxication, the decline in digestibility values of calcium, phosphorus and ash suggests a potentially distorted absorptive functions along the gut of the pigs. Concurring to this outturn, a sub-chronic study by Gbore et al. (2010) revealed a remarkable reduction in digestibility values of wistar rats exposed to increasing levels of FB 1 (0.2, 10 or 20 mg/kg feed) albeit along with this outcome, the authors observed a negative impact on growth performance. Moreover, the decline in crude fiber digestibility signifies a possible shift in the composition of the residential microbes that harbor the gut of the pigs since dietary fiber is a key player in the state of existence of intestinal microbes.
Furthermore, even though digestibility of crude fat did not differ statistically, the observed numeric decline in its digestibility values in both treatment groups suggests a possible interference in the lipid metabolic pathways of the pigs; the major toxicity pathway of FUM widely established. In slight attestation, Gbore & Egbunike (2007) observed, in a dose-response fashion, a decrease in crude fat and crude protein digestibility when an increasing dose of dietary FB 1 (0.2, 5.0, 10 and 15 mg/kg) was fed to growing pigs in a 6-month feeding trial. In this regard, an appropriate suggestion could be that our 21-day long dietary FUM exposure period was not enough to provide reliable difference in fat digestibility even in a dosage of 30mg/kg feed.

Conclusion
This study has proven a detrimental effect of dietary FUM on the nutritive value of the feed for weanling pigs. In reference to the above findings, it is proper to conclude that, increasing levels of dietary FUM up to 30 mg/kg for a period of 21 days potentially distorts nutrients, particularly fiber and minerals as well as energy digestion and absorption in the GIT of weanling pigs without necessarily depressing growth performance.