The Effect of Fermented Aflatoxins Contaminated Feed on Digestibility and Performance of Broiler Chickens

Broiler chicken farming is a major sector of the poultry industry. Poultry is susceptible to mycotoxicoses caused by aflatoxins. The experiment was carried out, where 144 DOCs were allocated to six diets. The diets were diet1 (no aflatoxin and not fermented), diet2 (no aflatoxin and fermented without yeast), diet3 (no aflatoxin and fermented with yeast), diet4 (contained aflatoxin and not fermented), diet5 (contained aflatoxin and fermented without yeast) and diet6 (contained aflatoxin fermented with yeast). The aflatoxin level was 30.08 ppb. Each diet was assigned to 6 chicks, replicated 4 times for 21 days. Leftovers and mortalities were recorded daily and chicks were weighed on a weekly basis. Feed consumption and body weight gain were not different across treatments. However, gain to feed ratio was significantly (p=0.048) better in broilers fed diets fermented naturally. The mortality rate was 75.0% in chicks fed on a non-fermented aflatoxin diet. Therefore, natural fermentation is the best method of improving the quality of aflatoxin contaminated feed for broilers.


Introduction
The consumption of poultry meat keeps growing across the globe in both first and third world countries (Kralik et al., 2018). Broiler chicken farming is a major sector of the poultry industry. Additionally, chicken meat is a high biological value protein (Da Silva et al., 2017) and a nutritious diet since it contains moderate energy (Marangoni et al., 2015). Having a lower fat content and higher PUFA than other meats, poultry meat is the recommended item in a balanced diet. (Riovanto et al., 2012). The quality of feed may be compromized when it is exposed to any contamination (bacterial, fungal, and mold) during storage; consequently, the feed is spoilt and the poultry is more likely to get infected (Sugiharto and Ranjitkar, 2018). For example, broilers carrying aflatoxin B1 could make the economy and public health suffer tremendously (Yunus et al., 2011). The negative effect of aflatoxin to birds is most significant in production aspects, such as weight gain, feed consumption, feed conversion ratio (FCR) and harvest (Hussain et al., 2010). Aflatoxins were discovered in 1960 and are widely associated with maize, groundnuts, tree nuts, figs, dates and oil seeds, such as cotton seeds (Kanyi, 2018;Negash, 2018). Aflatoxin B1, B2, G1 and G2 are the most prevalent toxins to interfere with metabolism of carbohydrates, fats and nucleic acids in livestock (Negash, 2018). In maize, the most vigorous aflatoxin (Aflatoxin B1) is produced from an abundant amount of A. flavus and parasiticus (Zaki et al., 2012).
According to many regulatory bodies on aflatoxins, the tolerable levels of total aflatoxins in foodstuffs and animal feeds are different. The World Health Organization (WHO) set aflatoxins limits of 5ppb for animals (Kajuna et al., 2013), while the United States Food and Drug Administration and European Commission set 20ppb as the maximum tolerable level of total aflatoxins for poultry feed (Syahidah et al., 2017). The concentrations of AFB1 in poultry meat and the edible parts may be elevated irrespective of whether the aflatoxin levels in the diet is low (22ppb) or high (2500ppb) (Fouad et al., 2019). Fermenting the feed could lower the non-nutritive substance, increase the overall nutritive value of the feed (Aljuobori et al., 2014;Çabuk et al., 2018) and reduce total aflatoxin levels (Mukandungutse et al., 2019). Additionally, digestive tract (crop and gizzard) may contain less Campylobacter and Salmonella due to consuming fermented feed. (Jawad et al., 2016). Sugiharto et al. (2015) reported that grain fraction (not complete feed), which is fermented prior to mixing with compound feed may help avoid losing some essential nutrients in fermented feeds. The use of fermented liquid feed appears to be a cost-effective alternative to the use of antibiotics growth promoters (Missotten et al., 2013). This study evaluated the effect of fermented contaminated feed with aflatoxins on the digestibility and performance of broiler chickens.

Source of aflatoxin
Clean (tested, aflatoxin-free) maize kernels were inoculated with Aspergillus flavus obtained from contaminated samples of maize. The incubation of maize (31°C, 60 days) was conducted while retaining the moisture intermittently to allow fungi to grow well and aflatoxin to produce. ELSA technique was performed to obtain AF level following manufacturer's instructions.

Fermentation
After inoculation, the contaminated maize and clean maize was milled using a sieve of 0.8mm into flour from which the water was added to the maize flour in the ratio of 1:1.5w/v (weight of maize flour/volume of water). The fermentation was done either without or by adding Saccharomyces cerevisiae (NCYC 125) at room temperature (25°C) for 72hours, then sun dried. After drying, fermented maize flour was used to compound six experimental diets.

Experimental diets, animals, design and treatments
A total of 144 DOC (broilers) were purchased from a commercial breeding place (Kenchic). All vaccination procedures against Gumboro and New Castle diseases were carried out by the hatchery before supply. The chicks were then put in a room which was well ventilated and fitted with fluorescent lighting. It was cleaned with liquid soap and disinfected before occupation by the chicks. The chicks were weighed individually before feeding. During brooding, the room was warmed to 30-34 o C using infrared bulbs and there was continuous lighting. In the poultry house, 24 partitions, with 1.2 x 1.2m each, were made. Each diet was assigned to 6 broiler chickens of similar body weights and replicated four times. Chickens were offered the six diets for 21 days. The six experimental diets were offered in clean disinfected feeders daily at 09:00hrs. The leftover feed was collected, weighed, recorded and fresh feed provided before the next feeding. The broilers were given clean, ad libitum drinking water. The diet composition was based on NRC standard (1994) for starter broilers (23% crude protein and about 3200Kcal/kg metabolizable energy). The experimental design was a "three by two factorial" arrangement where factor one was the type of fermentation (not fermented, fermented without yeast and fermented with yeast) and factor two was aflatoxin (0 and 30.08ppb). The six experimental diets are shown on Table 1. Every morning at 09:00hrs, the leftover feed was collected, weighed, and recorded before providing fresh feed. Daily feed consumption was calculated as total feed offered minus total leftover. The chicks were weighed once every week before feeding. Body weight gain (BWG) and average daily gain (ADG) of each chicken were calculated. BWG is the score after subtracting final weight by the initial weight, while ADG is BWG divided by seven days. Gain to Feed ratio (the total feed needed to gain one unit of weight) was obtained by dividing (ADG) by (ADFI).

Results and Discussion
The feed intake of the non-contaminated and contaminated diets was not improved by fermentation. Fermentation without yeast and fermentation with yeast had similar effects on feed intake whether diet is contaminated or not. The fermentation types did not significantly affect the feed consumption and body weight gain. The gain to feed ratio of chicken had been affected by fermentation types with p-value=0.048. The total aflatoxin levels had no significant effect on the chickens' performance.  The feed intake, body weight gain and gain to feed ratio were not significantly (p>0.05) different within the six diets (Table2). The types of fermentation did not improve feed consumption and body weight gain during 21days of study, but improved the gain to feed ratio (Table2). The study done by Naji et al. (2016) indicated that fermented feed with probiotics was economically beneficial since it improved broiler feed conversion ratio. Broiler chickens fed on fermented moist feed showed a detrimental effect on early bird growth but affected beneficially feed efficiency (Missotten et al., 2013). Pre-arranged probiotics for wet fermented feed may cause a significant improvement in chicken feed conversion ratio (Jawad et al., 2016). The total aflatoxin level of 30.08ppb did not significantly affect intake, body weight gain and feed conversion ratio (Table2). Many studies reported decreased feed consumption and body weight gain as well as an increased feed conversion ratio when higher levels of aflatoxin were used in the feed (Table  4).
The mortality rate was 75.0% (Table 3) in the non-fermented feed with 30.08ppb total aflatoxin levels which was greater than the United States Food and Drug Administration (USFDA) and European Union Commission (EUC) maximum tolerable limit of 20ppb (Morrison et al., 2017). Sobrane et al. (2016) observed the 20.13%±9.45 mortality rate of broilers fed contaminated feed with 2000ppb aflatoxin B1. During this study, some of the chicks fed on aflatoxins-contaminated feed were not able to stand on their feet. Lowered body weight, depressed feed intake, increased FCR (Marchioro et al., 2013) 1500ppb AFB1 Impaired growth (Chen et al., 2016) 1000ppb AFB1 Lowered growth rate (Ali Rajput et al., 2017) 200ppb and 400ppb AFs Reduced body weight, daily weight gain, feed intake and increased FCR (Valchev et al., 2017)

Conclusions
Fermentation with or without yeast did not affect feed intake and body weight gain but improved gain to feed ratio of broiler chickens. The mortality rate was high (75.0%) in nonfermented feed containing 30.08ppb aflatoxin. Ameliorative effects of grape seed proanthocyanidin extract on growth performance, immune function, antioxidant capacity, biochemical constituents, liver histopathology and aflatoxin residues in broilers exposed to aflatoxin B1. Toxins. 9(11): 371.