Research progress on the harm of feed mold to pigs and its prevention and control technology
Mycotoxins are toxic metabolites produced by molds during their growth and reproduction on substrates. Common mycotoxins in feed include aflatoxin B1 (AFB1), ochratoxin (OTA), deoxynivalenol (DON), T-2 toxin (T-2), zearalenone (ZEN), and fumonisins (FUM) [1]. Mycotoxins are commonly found in starch-containing feed or feed raw materials. Many molds may metabolize and produce large amounts of mycotoxins during the growth, harvesting, storage, feed processing, transportation, and sales of grains. Molds can be identified by judging whether food or feed is moldy, and can be removed by high temperature or sunlight. Mycotoxins are colorless, odorless, invisible, and odorless, and it is difficult to determine their content in feed and whether they exceed the standard with the naked eye. Mycotoxins are chemically very stable and resistant to high temperatures (e.g., aflatoxin is decomposed at 237-306°C; the melting point of ochratoxin A is 169°C; fumonisin B1 is also relatively stable (100-120°C) [2]. In addition, although mycotoxins are sensitive to high-intensity ultraviolet rays, the irradiation effect is difficult to achieve the expected effect [3].
- Harm of mycotoxins to pigs
Mycotoxins are highly toxic. For example, aflatoxin is defined by the WTO as a class 1 carcinogen, which is 68 times more toxic than arsenic. Once mycotoxins in feed exceed the standard, they are very harmful to pigs. In mild cases, toxins remain in organs, growth is stunted, and feed utilization is reduced. In severe cases, pig organs are damaged and immunity is blocked. In more serious cases, anorexia, illness, and even death occur. Mycotoxins have special targeted hazards to animal organs and body tissues. AFB1 poisoning is prone to liver lesions[10]. OTA poisoning is prone to kidney lesions[11-13]. T-2 causes vomiting, anorexia, and necrotizing hemorrhagic gastroenteritis in pigs[14-16]. ZEN poisoning affects the reproductive performance of sows and causes redness and swelling of the vulva[17-21].
Fu Jianchao[22] studied the harm of AFB1 to piglets and found that AFB1-contaminated diet (372.8 μg/kg) had no significant effect on pig production performance, but it remained in the liver and kidneys, reducing the content of red blood cells, white blood cells, monocytes and neutrophils in the blood. Chang Siying[23] added 0.5-2.0 mg/kg of ZEN to the diet and found that as the ZEN level in the diet increased, the area of the piglet’s vulva increased, became slightly red and swollen, the estrogen level increased, and the levels of estradiol, follicle-stimulating hormone, luteinizing hormone and progesterone in the blood increased significantly. At the same time, the activities of alanine aminotransferase and aspartate aminotransferase in the piglet’s blood increased significantly, and the ZEN residue in the liver increased. Zhang Yuanyuan[17] fed pregnant sows a diet containing 2.77 mg/kg ZEN and found that ZEN could cause apoptosis of uterine and placental cells and ovarian hyperplasia. The number of embryos in a litter at 70 days of gestation was reduced by 2.17, and the weight of each embryo was reduced by 0.04 kg. The number of live piglets in a litter was reduced by 3.33, and the weight of each piglet was reduced by 0.11 kg. The number of weaned piglets at 21 days of age was reduced by 3.67, and the weight of each piglet was reduced by 1.72 kg. The daily weight gain of growing and fattening pigs was reduced by 70 g when they were marketed.
- Feed mold prevention and control technology
In order to solve the complex global problem of ZEN contamination, many scientists have conducted research on mold inhibition, mold removal and mold decomposition at three levels: in vitro, in the digestive tract and in the liver. This can also be understood as building three lines of defense to solve or alleviate the harm of mold in feed. ① Elimination in vitro. Create good storage conditions before feeding, control temperature and humidity, and prevent the growth and reproduction of mold in feed raw materials or compound feed. Antifungal agents (such as propionic acid, etc.) can be used to prevent feed mold. Seeds with better mold resistance can also be selected and genetically modified[24]. ② Elimination in the digestive tract, which is mainly achieved in the digestive tract through decomposition methods such as adsorbents, microorganisms, and enzymatic hydrolysis. ③ Elimination in the liver, which is achieved by using detoxifiers to increase the activity of metabolic enzymes in the body and decompose mycotoxins.
Feed mildew inhibitors refer to feed additives that can reduce the number of microorganisms in feed, control the metabolism and growth of microorganisms, inhibit the production of mycotoxins, prevent the loss of nutrients in feed during storage, prevent feed mildew and prolong storage time[25]. The main mildew inhibitors currently used are mainly divided into three categories: chemical mildew inhibitors, Chinese herbal mildew inhibitors and compound mildew inhibitors. Chemical mildew inhibitors mainly include propionates, propionic acid, formic acid, acetic acid, acetates, etc. Propionic acid is a colorless liquid and is the most widely used at home and abroad. Its volatility makes its dosage low and its effect fast, but its volatility also makes propionic acid difficult to transport and store, and its mildew prevention time short. Propionate avoids these disadvantages and has the advantages of good stability and low irritation. The advantages and disadvantages of formic acid are similar to those of propionic acid. It has been reported that deoxyacetic acid has photothermal stability, broad-spectrum antibacterial properties, and also has a good inhibitory effect on mold[26]. Although feed mold inhibitors are currently widely used, their mold-proofing effect is affected by many factors and cannot completely control the contamination of feed with mycotoxins[26]. It is impossible to add mold inhibitors to all feed raw materials to prevent and control mold. The mold inhibitors and their mixing process will greatly increase the cost of raw materials. In addition, mold inhibitors have poor palatability (for example, sorbic acid and sorbates have a slight odor). In addition, it is difficult for mold inhibitors to inhibit all mold-causing microorganisms[27], and they are toxic to animals. For example, low doses of the mold inhibitor sodium dehydroacetate reduce the food intake of mice, and high doses can cause bleeding in tissues and organs[28].
2.2 Demycotoxins
Demycotoxins refer to a class of agents that adsorb and remove mycotoxins in feed, which mainly include adsorbents, enzymatic detoxifiers, and microbial detoxifiers.
2.2.1 Adsorbents
Mycotoxin adsorbents refer to substances that form complexes with mycotoxins, preventing the toxins from being absorbed when passing through the digestive tract and excreting them directly from the body along with the adsorbent. They can be divided into organic adsorbents and inorganic adsorbents. Currently, organic adsorbents on the market are mainly yeast cell wall extracts, which are rich in glucomannan polymers and can adsorb mycotoxins. When yeast cell walls were added to a simulated gastric fluid environment, the concentration of AFB1 decreased by nearly 20% after 30 minutes and by 80% after 180 minutes [29]. In addition, organic adsorbents such as chitosan, stachyose, and xylan have been shown to have an adsorption effect on mycotoxins. Fu Jianchao [22] found that the adsorption rate of chitosan itself for AFB1 was low (10%), but the modification of chitosan into nano-chitosan greatly improved the mycotoxin adsorption capacity, with an adsorption rate of more than 50%.
Mycotoxin inorganic adsorbents mainly refer to a class of substances rich in aluminosilicates (such as zeolite, activated carbon, montmorillonite, diatomaceous earth, bentonite, kaolin, etc.). Chen Qi[30] found that medical stone has great potential as a mycotoxin adsorbent. Medical stone is also known as longevity stone, health stone, and mineral medicine stone. Silicon dioxide (SiO2) and aluminum oxide (Al2O3) together account for more than 70% of it. It has a special porous sponge structure (2,600-2,800 pores/cm2). The surface area of 1 gram of medical stone reaches several hundred square meters, which is 20,000 times that of the same amount of activated carbon. It has strong electrostatic attraction and chemical adsorption. Fu Jianchao’s[22] experiment showed that medical stone has a strong adsorption capacity for AFB1 in vitro. His in vivo experiment also showed that adding 1% medical stone to the diet contaminated with AFB1 can reduce the residual amount of toxins in the upper liver and kidneys to a certain extent.
However, there are several important defects in the actual application of adsorbents: ① Most inorganic adsorbents have poor adsorption effect on ZEN. Since hydrated aluminosilicates are negatively charged and aflatoxin is positively charged, inorganic adsorbents have a very good adsorption effect on aflatoxin. However, inorganic adsorbents have a poor adsorption effect on negatively charged and less polar mycotoxins such as ZEN and OTA. Chen Qi’s [30] research also showed that ordinary medical stone has a poor adsorption effect on ZEN, with an adsorption rate of less than 25%. However, medical stone modified with quaternary ammonium salts has a very good adsorption effect on ZEN, with an adsorption rate of more than 90%. ② In vitro effectiveness does not guarantee in vivo effectiveness. In vivo experiments have shown that adding modified medical stone to ZEN-contaminated feed can partially restore the antioxidant status of the liver and reduce the ZEN level in the liver [31]. Chen Qi [30] found in vivo that modified medical stone significantly reduced the area of pig vulva, the interaction between ZEN and modified medical stone had a significant effect on progesterone, and the modified medical stone also significantly reduced the malondialdehyde content and ZEN residue in the liver. ③ Most adsorbents have an adsorption effect on nutrients while adsorbing mycotoxins. Some mycotoxin adsorbents do not specifically adsorb mycotoxins, but also bind to vitamins[32], mineral elements or drugs in the diet. Du Juan[33] found that medical stone has a strong adsorption capacity for iron and selenium, with adsorption rates of 50.9% and 52.6% respectively, but has a stronger adsorption capacity for cadmium, with an adsorption rate of 79.9%.
2.2.2 Enzymatic detoxification agent
The enzymatic method mainly selects certain enzymes and utilizes their degradation effect to destroy mycotoxins or reduce their toxicity. Compared with physical and chemical methods, the enzymatic degradation treatment method has less loss and impact on feed nutrients. However, there are still many problems in practical application. First, the action of enzymes is specific and highly selective, but it is precisely because of its specificity (one enzyme can only process one mycotoxin), which greatly limits the use of enzymes; there is no evidence that enzymatic hydrolysis can solve the toxicity of all mycotoxins; secondly, enzymes are very sensitive to high temperatures during feed processing and are easily inactivated; the temperature and complex environment of the gastrointestinal tract, including the changing pH, are suitable for the action conditions of the enzyme; finally, when using enzymes to degrade the toxicity of mycotoxins, some mycotoxins require a complete set of enzymes to completely degrade their toxicity, and the primary metabolites of some toxins in the body are more toxic.
2.2.3 Microbial detoxification
Many researchers have isolated microorganisms that can degrade mycotoxins from nature. Xue Beibei[34] isolated a strain of Aspergillus niger (ND-1) from various feed raw material microorganisms using coumarin as the sole carbon source. The strain can degrade AFB1, and this degradation process is enzymatic degradation. Lee et al.[35] isolated a strain named LN from a moldy corn sample, which has a high ZEN removal ability. After LN was inoculated into ZEN-contaminated corn flour culture medium for 36 hours, the ZEN concentration was reduced by 92%. Yang Xue[36] screened three ZEN-degrading bacteria from ZEN-challenged ternary pig feces, namely SY-3, SY-14 and SY-20, with an in vitro degradation rate of up to 98% for ZEN. SY-3 is a Proteus mirabilis. The removal of ZEN is mainly dominated by extracellular substances, followed by cell wall adsorption, and the role of intracellular substances is minimal. SY-14 and SY-20 are both Bacillus subtilis. SY-14 mainly degrades ZEN through intracellular substances, while SY-20 mainly reduces the content of ZEN through intracellular substances and cell walls.
However, so far, the degradation of mycotoxins by microorganisms is mostly determined by in vitro tests. Oxygen, high temperature and humidity, gastric acid, bile acid, etc. will affect the activity of microorganisms. At the same time, whether these microorganisms themselves pose a threat to animal health, which mycotoxins they can degrade, whether the products of their degradation of mycotoxins are toxic, and the detoxification mechanism of microorganisms need further research.
3.3 Antifungal agent (see Figure 1 and Figure 2)
The production of mycotoxins cannot be eliminated from the source in terms of raw materials. Detoxification agents cannot completely detoxify, exogenous detoxifiers are affected by the internal environment, and the detoxification pathways and mechanisms of endogenous detoxifiers are still unclear. Therefore, it is very meaningful to achieve the effect of removing mycotoxins by improving the liver’s own detoxification ability of toxins. The metabolism of mycotoxins in the body is divided into three stages: the first stage is biological oxidation, and the main enzymes involved are phase I metabolic enzymes (CYP450, FMO3); the second stage is conjugation, which is mainly participated by phase II metabolic enzymes (SULT2A1, UDTs, GSTs); the third stage is modification and excretion catalyzed by phase III metabolic enzymes (P-glycoprotein, OATP2, MRPs).
Under normal circumstances, mycotoxins can be biotransformed under the action of phase I and phase II metabolic enzymes in the liver, and the gene expression of phase I and phase II metabolic enzymes is regulated by the pregnane receptor (PXR) and the constitutive androstane receptor (CAR) [37]. Two nuclear receptors, CAR and PXR, are involved in the metabolism of internal and external toxins. However, after mycotoxins invade the pig body, it is still unclear how the PXR and CAR-mediated signaling pathways coordinate their functions and detoxification capabilities. Studies have shown that nuclear receptors PXR and CAR can be activated by some bioactive substances (such as vitamin C) [38-39]. Therefore, in-depth understanding of the role of pig PXR and CAR-mediated signaling pathways in mycotoxin detoxification, and the study and utilization of bioactive substances → nuclear receptors → phase I and II metabolic enzymes and transporters → ZEN detoxification pathways can not only better understand the molecular mechanism of mycotoxin metabolism in the body, but also provide new ideas and experimental basis for establishing effective intervention measures for mycotoxin metabolism in the body and strengthening the animal’s own detoxification system, opening up a new way to solve the problem of feed mold (see Figure 1).
Lou Lei[39] obtained the pgCAR target fragment from total RNA of pig liver by RT-PCR, and connected it to the eukaryotic expression plasmid pcDNA3.1, successfully constructing the eukaryotic expression vector pcDNA3.1-pgCAR; introduced the DNA binding sequence containing 5 copies of NR1 and the TK promoter into pGL3, successfully constructed the fluorescent reporter plasmid pGL3-(NR1)5-TK, and formed a dual fluorescence analysis system with pRL-TK, successfully establishing a nuclear receptor CAR high-throughput screening model. Through the high-throughput model, a number of active substances were screened in vitro, and it was found that vitamin C, folic acid and sitosterol had extremely significant activation effects on porcine nuclear receptor CAR (P<0.01). Su Yang[19] further verified through in vivo experiments that vitamin C can alleviate the redness and swelling of reproductive organs caused by ZEN and alleviate the damage of ZEN to the liver of weaned piglets by regulating the expression of nuclear receptors and their target genes, increasing the mRNA and protein levels of phase I metabolic enzyme genes (CYP1A1, CYP1A2, CYP2A6) and phase II metabolic enzyme genes (UGT1A1, UGT1A3, UGT1A6)[19]. This provides a new idea for alleviating the harm of mycotoxins to livestock and poultry through nutritional regulation.
3. Conclusion
Some people describe mycotoxins as the “hidden killer” of pig farm profits, and their harm is real. In actual production, do not be stingy with feed that is seriously moldy. In production, the use of mold inhibitors, mold removers and mold removers can alleviate the harm caused by feed mold to a certain extent, but we must also objectively analyze the pros and cons, and the cost of excessive prevention and control is also high. If a pig farm adds mold removers to feed all year round, it may not be worth the cost. Some scholars believe that pigs have a certain detoxification ability, and the harm of a small amount of mycotoxins to pigs is not as serious as the media propaganda. Therefore, they believe that more attention should be paid to the quality and storage of raw materials, and the cost of adding mold inhibitors is not as good as using them to purchase good raw materials and improve the storage conditions of raw materials. Therefore, further search for active substances that can improve the growth or health of pigs and interfere with the metabolism of mycotoxins in the body to reduce their toxicity, and flexibly use mold inhibitors and mold removers at the same time, and use three lines of defense according to local conditions, in order to achieve the best results in the battle to prevent and control the harm of mycotoxins.
