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Сельскохозяйственная биология, 2011, № 2, с. 21-31.

УДК 636.5.033:636.084/.087:615.372:577.121.2

METHYL GROUP DONORS — PROMISING MEANS FOR PREVENTION OF CHRONIC MYCOTOXICOSIS

S.Yu. Gulyushin, R.A. Zernov

The investigation, made on broiler-chicken of the Cobb Аvian 48 cross, shown, that the application of biological active substances — methyl group donors (methionine, choline, betaine) against the background of experimental chronic mycotoxicosis of multisystem type promotes to reduction of negative consequences of disease no less than 60 %. The preparations in doses, which exceed a physiological requirement to 3-5-fold, have the most activity. They ensure both a replenishment of methyl group pool, essential for one of conclusive stage of detoxication (methylation), and an elimination of symptoms of liver toxic damage and recovery of liver role in general metabolism. The periodical addition of studied substances to ration is more reasonable physiologically since decreases significantly their side effect (inhibition of productivity) without appreciable limitation of active potential.

Keywords: broilers, mycotoxicoses, ochratoxins, T-2 toxin, fumonisin, methylation, methionine, choline, betaine.

 

Today, poultry farming has reached the genetic potential of meat productivity that was unbelievable 10 years ago - daily increase in live weight exceeds 60 g while the growing period of 35-38 days. Implementation of this potential becomes possible under growing conditions best close to the established standard. Primarily, this is achieved by minimizing the impact of unavoidable stresses of industrial technology and adverse environmental factors. The presence of mycotoxins in feed raw is an important alimentary factor providing highest impact on bird health. High content of mycotoxins (enhanced by synergistic effects) can lead to livestock deaths, low quality products and resulting direct damages to the company (1, 2).
Mycotoxicoses providing multiorgan adverse effects are widespread in Russia, and the peak in number of reported cases is recorded in winter-spring period. In recent years, this becomes increasingly originated from low feed quality and often occurs in a latent form, so the true scale of bird poisoning with toxic fungal metabolites is revealed very late, when applying urgent measures is difficult or inefficient. This fact requires prevention strategy based on the feed quality control and time-accurate use of antitoxic preparations necessary for effective functioning of the industry (3-5).
The data of own long-term observations and literature reports indicate that chronic mycotoxicoses most often (over 60-90% cases) involve liver disorders such as steatosis and/or steopathite, while the rate of such cases increases at the content of xenobiotics corresponding to upper limit of maximum allowable concentration (MAC) and further grows as it exceeded (6). In other words, liver condition (not as a specific marker, but the one reflecting damage degree) can serve as a reliable diagnostic criterion of mycotoxicosis expression and effectiveness of curing it. It should be noted that the improper use of certain recommended bioactive feed additives can cause "fatty liver" in animals. However, the presence in feed of foreign chemicals and toxins combined with total dietary imbalance almost always damages the function of hepatocytes (up to their necrosis) and contributes to cholestasis, fibrosis, inflammatory, allergic and idiosyncratic reactions, as supported by results of autopsy of control animals (6-8).
There is another important fact that liver dysfunction is accompanied by violation of detoxication from mycotoxins metabolized in the liver, which enhances mycotoxicosis effects owing to interactions with cytochrome P-450 enzyme system and results in further deaths of hepatocytes. Such complex abnormalities initiate further metabolic disorders along with fatty degeneration of the liver and reducing its role in the interstitial metabolism, which often ends in severe metabolic decompensation, coma and death from paralysis of the respiratory system. That's why during the liver dysfunction caused by feeding low-quality feeds, it is preferable to use corrective hepatotropic preparations combined with conventional means to strengthen their positive effects (9, 10). There are promising chemicals containing a mobile methyl group in the molecule – methionine, choline, betaine – included in the composition of balanced combined feeds (11-14). At the same time, there are significant dose-dependent differences between increased biological value of diets and their therapeutic use.
This group of substances provides a direct antitoxic action and accelerates recovery through activation of certain specific reactions in the organism. It is known that all biochemical processes of utilizing xenobiotics include two phases. The first one is oxidation, after which mycotoxins lose lipophilicity and become transformed into compounds with high capacity for excretion. The effect is greatly enhanced should the first phase hydrophilic products be added with acetate, sulfate, glucuronic acid, glutathione, methyl groups, etc. The obtained conjugates (second phase) are chemically stable, in most cases – less toxic, and even more actively excreted from the organism (15-17).
Methylation – the transfer of a methyl group (CH^3-)from S-adenosyl-1-methionine molecule (SAM) on heterogeneous substance – terminates detoxification of many xenobiotics such as mycotoxins and their metabolites. The reaction is catalyzed by transmethylazes having different substrate specificity.  However, this process occurs in the liver only at the required quantity of free methyl groups and it significantly slows down when they lack, which often takes place during the abovementioned pathology. At the same time, SAM is the main factor protecting the liver from steatosis. Thus, chronic mycotoxicoses involve highly correlated processes with synergistic adverse effects – development of fatty liver and inhibition of endogenous detoxication of xenobiotics and protein biosynthesis (as a result of a profound methionine deficit). At the same time, inclusion into the diet of methyl donors in doses greatly exceeding physiological need can reduce harmful effects of mycotoxins (18-21).
Over a quarter of a century, the synthetic amino acid – D,L-methionine (D,L-α-amino-γ-methyl butyric acid) is used in veterinary practice as highly effective preparation for curing lipid dystrophy. Treatment of this disease was revolutionized by this substance prescribed for normalization of lipid metabolism, obesity prevention, as well as for more active neutralizing in the liver of various toxins coming from food and circulating in the body during metabolism (22, 23). However, methionine is insufficiently used to prevent mycotoxicoses in poultry farming; this essential amino acid necessary for growth and nitrogen metabolism is often applied just as a component of diets balanced for amino acid composition (24). There’s still no clear data about effective therapeutic doses and optimum conditions for using this substance.
Similar results can be expected from applying other hepatoprotectors - betaine and choline, which are participants of methionine metabolism and methyl donors for the alternative pathway of homocysteine re-methylation to methionine (16, 18). Along with it, they are a part of the phospholipid lecithin kept in cells at high contents and playing an important role in metabolism of phospholipids and digestion.
The authors didn’t find any available scientific reports about such studies on poultry, so the purpose of this work was studying the effectiveness of methionine, choline and betaine as preparations for prevention of chronic mycotoxicoses and steatosis during the experimental injury of the liver with a mixture of mycotoxins.
Technique. The work was performed in the vivarium of SUE "Zagorskoe the Experimental Production Enterprise of the All-Russia Research and Development Institute of Poultry Farming (VNITIP) of the RAAS”(Moscow province, 2009) during the science-industrial experiment on broiler chickens the cross Cobb Avian 48. 8 groups were formed by the principle of analogues – 2 control and 6 experimental, 30 birds in each group. The bird was fed ad libitum with dry balanced maize-type mixed fodder with nutritive value corresponding to VNITIP standards (2006). Farming conditions were consistent with established zootechnical regulations, duration of experiment – 35 days (25).
Zero diet was fed to chicks up to the 7-day-old age, and after the 8th day – the experimental fodder. Group I (control) were given mycotoxins-free feed (standard diet – SD). Group II (negative control) were given the fodder containing mycotoxins significantly reducing productivity of poultry: ochratoxin A - 117 ug/kg food, T-2 toxin - 296 ug/kg, fumonizin B1 – 10,2 mg/kg and DON (4-dezoxynivalenol - vomitoxin) – 3,1 mg/kg (total toxicity 8,9 MAC). Mycotoxins were introduced into the feed as fungal biomass based on maize contaminated with four fungi of Fusarium and Aspergillus genera and toxic products of their activity (experimental diet 1 - ED1). The rest of the bird was given the same feed as group II (fodder with mycotoxins) added with donors of methyl groups: group III - D,L-methionine (98%; 2,5 kg/t), group IV - cholinechloride (60%; 6,3 kg/t) and group V - betaine (96%; 1,8 kg/t). Chickens from groups VI, VII and VIII were fed the same supplements during the 2nd and 4th weeks of life (7 days after 7 days, i.e. with pauses during the 3rd  and 5th weeks), which reduced the costs of preventive action and optimized the use of tested preparations.
The effectiveness of tested bioactive preparations against mycotoxicoses was assessed by the range of biochemical parameters characterizing some processes of lipid metabolism and the activity of enzymes and metabolites involved in methylation (26-28). Ready-made fodders were analyzed for nutrients, mycotoxins and dynamics of their deposition in the liver or broilers (29, 30).
The data were processed by the method of variation statistics (t-Student test) in Microsoft Excel 9.0.
Results. Doses of tested preparations were selected upon the data on chemical purity of substances and their biological activity conditionally equaled to that of D,L-methionine at its content in a diet of 2,5 kg/t (based upon the proved data on its efficiency when administered to a diet at this quantity) (31-32). Knowing biochemical efficiency of endogenous transformation of substances containing methyl groups and formation of SAM, 1 kg D,L-methionine (98%) was considered to be equivalent to 0,72 kg betaine (96%): the active role of 2nd and 3rd methyl groups of betaine can increase methylation of homocysteine via the formation of folic acid, 5-methyl-tetrahydrofolate and vitamin B12, and, therefore, it raises the efficiency of trimethylglycine. Therefore, equal activities of betaine (96%) and D,L-methionine at standard dose (2,5 kg/t) can be provided by betaine content in feed of at least 1,8 kg/t. Then, 1,155 kg choline chloride (100%) is chemically equivalent to 1 kg betaine (96%), while biological efficiency of choline chloride conversion in bird organism is equal to 55% (33), so 1 kg betaine ( 96%) is biologically equivalent to 3,5 kg choline chloride (60%), or its content in feed of 6,3 kg/t.
Zootechnical observations (data not shown) indicated positive impact of donors of methyl groups on growth of experimental bird. Detailed studies revealed physiological, biochemical and morphological parameters allowing to analyze the development of metabolic disturbances and peculiarities of therapeutic action of tested bioactive preparation.  
For example, in group II fed the diet with high content of mycotoxins, 100% bird exhibited pathologic changes of the liver and 30% of these cases were very severe (Table). Anatomical investigation of the liver showed specific changes in color, histological structure and architectonics of the body – it was yellow or brown in color, loose, with numerous hemorrhages and necrotic spots on ventral surface; relative weight of the liver was reliably increased by 24,6% compared to that of group I (control). Histological dystrophy was characterized by degenerative changes in liver cells along with enlarged fat vacuoles, which resulted in increased size of the liver, its flabby easily torn texture and abnormal color due to excessive fat deposition. Liver disease is one of the most important risk factors leading to significant modification of all functions of the organism. This fact was proved by close correlation (r= - 0,64 ...-0,75) found between the rate of fatty liver in the experimental groups, on the one hand, and vitality of birds, growth rate and efficiency of consumption feed during growth period - on the other hand.
Inclusion into broilers’ diet of methionine (2,5 kg / t) in continuous regime (group III) contributed to approximately 50% lower rate of liver infiltration and inflammatory changes peculiar to chronic intoxication and general pathological state. At the same time,  cholesterol content in the blood of experimental birds was slightly decreased while the increased concentration of phospholipids, which resulted in raise of cholesterol index (the quantitative ratio phospholipids : cholesterol) by 25-30% relative to the group II (negative control). These results confirm the capability of methionine to cause lipotropic effect by removing excessive fat from the liver. It donates a methyl group and promotes choline synthesis. Should choline synthesis be insufficient, it leads to violations of phospholipid synthesis from fat and deposition of neutral fat in the liver. At the same time, when methionine was added to contaminated mixed fodders, some researchers have established a significant stimulation of transsulfatation and compensation of protein deficiency, which suggests using this amino acid as very promising mycotoxicosis-effective means (7, 27).
This conclusion was indirectly confirmed by mycotoxicological studies: the liver of chickens from group II contained 1,2-2,0 times lower quantity of residual untransformed mycotoxins (T-2 toxin, fumonizin B, ochratoxin A) (p £0,05). However, it is significant that intense transformation of fungal toxic metabolites proceeded against the background of raising activity of the key enzyme of methylation – methionine adenosyl transferase (EC 2.5.1.6) directly involved in synthesis of SAM providing detoxification of xenobiotics.
Periodic inclusion of methionine in contaminated feed (group VI) resulted in the 40% total economic saving and even more pronounced positive effect on basic physiological and biochemical parameters. Thus, total lipid content in chicken liver was slightly lower (by 1,6%, or 35 rel.%) than in birds of the same age fed methionine constantly. Along with it, total and relative activity of AST and ALT (enzymes of hepatotropic origin indicating the integrity of cell membranes of hepatocytes) decreased by 20-25% (p£ 0,10), so there was general stabilization and significant relief of negative effects of mycotoxins, which are usually manifested as severe hyperenzymemia. It is also important to emphasize that both methylation activity  and residual content of untransformed mycotoxins in the liver of chickens were slightly lower (by 10-18%) in this group than in that constantly fed methionine in a diet, but these differences were not statistically reliable. Thus, the obtained data can be regarded as the fact indicating capability of methionine to provide a prolonged antitoxic effect.
Constant regime of feeding methionine resulted in greater increase of ammonia and uric acid contents in blood plasma (15%, p £ 0,02) than at periodic inclusion it into a diet. In this regard, it should be highlighted that using methionine as a donor of methyl groups is reasonable at mycotoxicoses and quite risky as well: high content of methionine is accompanied by total imbalance of amino acid composition and excessive formation of ammonia – the quite toxic substance for animals. Utilizing ammonia in the organism is associated with extra costs of energy and functional reserves of the liver, which adversely affects the animals’ productivity.

Such assumptions are relevant when constant feeding betaine (3-methyl-aminoacetic acid) as well: this substance is a source of amino nitrogen not involved in protein synthesis. Furthermore, high intensity of protein catabolism aggravates mycotoxicosis. So, periodic use of these bioactive substances has obvious advantages, because activation of methylation is not accompanied by excessive ammoniemia, which reduces the risk of hepatic-portal deficiency and deaths.
The analysis of literature data shows the following sequence of reactions during methionine activation and conversion in the organism: methionine →  S-adenosyl methionine → S-adenosyl homocysteine → cysteine. However, methylation can be promoted without increasing methionine content in the diet, because homocysteine along with transformation into cysteine can capture the missing methyl group from a certain donor to form methionine capable to participate methylation repeatedly. As noted above, these donors can be choline and betaine containing more methyl groups (n = 3) than methionine molecule (n = 1). For example, dehydrogenation of choline contributes to betaine, and betaine, in turn, gives one methyl group during the cyclic reaction of formation methionine from homocysteine (16, 23, 28).
Therapeutical effectiveness of these preparations is evidenced by the fact that free homocysteine content in blood plasma  decreases (by 30-40 %, p < 0,02-0,001) – negative effects of this strong toxicant were relieved owing to  the appearance of additional donor of methyl groups and active participation in biochemical processes. The bird of group II (negative control) didn’t manifest this feature. In the liver of broilers not given methionine at treatment doses (groups IV, V, VII and VIII), there was some increase in contents of essential amino acids (by 6-11% of dry weight) indicating the effect of “protecting” the liver when using choline chloride and betaine – it was excluded from catabolism and accumulation providing enhanced protein biosynthesis. Thus, it can be concluded that cholinechloride and betaine given as a part of animal feeds contaminated with mycotoxins provide detoxication activity and significantly improve general condition of bird already on the 2nd-3rd day after the start of use.
Choline (2-hydroxyethyl hydroxide trimethylammonium) presents at domestic market as a stabilized salt with chloride ion (choline chloride); in this study, it was found to be the lipotropic substance effectively preventing infiltrates when fatty liver in broilers (over 50-60%). Choline is a nitrogen ammonia-like compound, but not a toxic ammoniagenic substrate due to substantial differences in molecule structure. Cholinechloride is capable to be diffusely absorbed into the intestinal wall unlike two other analogs entering the body mainly through the active transport. This fact makes applying it very effective against the background of disorders of neurohumoral regulation mechanisms and severe injury of intestine mucosa during mycotoxicoses. In this experiment, the ratio between methylation activity and accumulation of ammonia in the blood when feeding choline chloride was optimum even in the variant with constant adding it to a diet at the quantity of 6,3 kg/t (group IV).
Discussion on the problem of chronic mycotoxicoses must consider high intensity of lipid metabolism in bird organism (compared with other types of farm animals); its general oppression includes a complex network of metabolic events in which outcomes of certain mechanisms are the activators for other links. During the intoxication, growth rate is suppressed and energy costs are significantly reduced, so fatty acids coming into the liver are being utilized via the synthesis of triglycerides. The development of hyperlipidemia with predominant rise of cholesterol and triglycerides contents in the blood plasma and metabolically active tissues contribute to  35-36% significant effects on bird health (p < 0,10). Human can be the next target: negative effects of xenobiotics result mainly in increased fat content in chicken meat (commonly considered as a dietary food product) while the excessively (in 1,5-2,0-fold over control) and the increased content of VLDL – the stimulator of atherogenesis and atherothrombosis).
The inclusion into contaminated feed of all tested preparations (methionine, cholinechloride, betaine) provided total  beneficial effect on metabolism in experimental animals. These means partially corrected violations of carbohydrate and lipid metabolism: concentrations of glucose and HDLP increased by, respectively, 5-14 and 10-20%, while contents of  total cholesterol and triglycerides reduced by 9-16%. So, the negative effects of experimental poisoning with fungi-contaminated feed were indirectly relieved, which was also confirmed by zootechnical observation.
However, there was a question about the discrepancy between significant correction of metabolism and insufficiently high growth rates of chickens in the variants with choline chloride. This fact was hypothesized to be the result of increased consumption of chlorine when feeding this substance in a diet. Today, most of the experts in farm animals’ nutrition have a negative opinion about chlorine-containing feed supplements (up to considering the excessive use of them as a serious mistake). It was highlighted the reduced immunity and low total resistance when the high Cl- load with; though, these facts need to be  proved. However, the authors’ findings about the content of chlorides in blood plasma of experimental birds gave rise to additional questions.
Indeed, chicks from group IV showed 12-13% higher content of chlorine anions in the blood (p £ 0,01) than the control group I. The observed levels were consistent with the fact that total content of chlorine in the diet when feeding choline chloride at the dose of 6,3 kg/t grows by 30 rel.% (from 0,30% to 0,39%). The performed calculations show more than 34,0% increase in relative consumption of  chlorine per 1 kg of a chicken live weight for the entire growth period. In the variant with periodic feeding choline chloride (group VII), these levels were somewhat lower: chlorine content was 7,2% higher in the blood plasma and by 20 rel.% - in the diet relative to control (group I). These data seem to agree with the proposed concept, but there’s still no a confident proof for negative effects of excessive contents of Cl- anion. The close increased levels in blood plasma (by 10-12%, p < 0,10-0,01) were recorded in groups II, III , V, VI and VIII while the content of this macronutrient in a diet was comparable with control (0,30%).
It is known that mineral substances are capable to enter the body via passive transport and pinocytosis with almost no obstacles. During a chronic mycotoxicosis (at high feed consumption and low live weight of bird), contents of inorganic macro- and micronutrients in the blood can be greatly increased depending on intoxication degree. There’s no doubt that such redistribution and high content of chlorides in the blood can be connected with disturbance of renal function and water metabolism (accompanied by high hydration of internal organs) along with a number of other factors induced by mycotoxins –  elimination of hydrocarbons at diarrhea, suppression of hydrochloric acid synthesis prior to digestion disturbance etc.. However, a diet composition (chloride content in feed) seem to have no decisive influence on this matter. When mycotoxicosis (like any other growth inhibition in animals), the kinetics of mineral salts should be of a serious attention as it must be corrected by dosage or application modes. For choline chloride, this study have shown generally positive results and effectiveness of using it.
Another fact related to specific effects of donors of methyl groups is regular behavioral changes of experimental birds. The chicks from group II (negative control) were depressed, disheveled, unstably standing on their feet and moving reluctantly in a cage thereby reflecting poisoning effects of mycotoxins, while other groups didn’t exhibit such symptoms. On the contrary, the birds of groups III-V demonstrated the aggressiveness sharply contrasting even to the control group I having an extremely high mobility, good appetite and a very clean feather cover: birds of these groups used to shout loudly and often bite the staff during a hand feeding, though not hurtling themselves (without rise of cannibalism rate). Such expressive behavior is easily explained: choline is an essential part of the excitatory neurotransmitter acetylcholine involved in synaptic transfer of momentum, while methionine and betaine (as shown above) indirectly participate choline synthesis. So, all these data suggest using the tested preparations in zootechnical practice as means improving adaptive capabilities of animals and birds.
At the same time, the abovementioned complex chain of transformations passed in the organism by donors of methyl groups indicate that effectiveness of a therapy is highly determined by activity of liver enzymes providing activation and utilization of methyl-keeping preparations as well as by doses of these substances. Therefore, the preventive use of them (at first suspected poisoning) combined with improved biological value of a diet are key factors of rapid and full recovery of the bird.  Antitoxic activity of these substances increases in the line: choline chloride " betaine " methionine. Should the further pathogenesis involve liver lesions, high doses of these preparations can be low-effective and even contribute to adverse metabolic consequences. During the prolonged mycotoxicosis and complex pathology, its reasonable to avoid high doses and constant feeding methyl donors in order to prevent hepatotoxicity symptoms. In such cases, the best is applying separate courses (pulse therapy) providing a stable result and general normalization of physiological functions by reducing excessive load of the liver. The regime of inclusion these substances in fungi-contaminated feed can be determined and optimized during the production testing according to feed toxicity, which greatly increases product quality. In some farms,  biological feasibility of a large-scale preventing mycotoxicoses can be limited by technological capabilities, so the proposed scheme 7 days/7 days (feeding supplement/interval) can be the noteworthy compromise variant of feeding methyl-containing preparations.
Thus, using donors of methyl groups - methionine, choline chloride and betaine – was found to be biologically and economically feasible mean that relives negative effects of chronic mycotoxicoses. These substances positively affect zootechnical characteristics of the bird and help to restore by 60% its productivity even against the relatively high level of fungal contamination. These preparations are accessible, convenient and safe in use when feeding with moderately toxic feeds, so overdoses are almost completely excluded. Besides, these substances are always easily combined with traditional antitoxic means  to amplify their beneficial effects.

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All-Russia Scientific Research and Technological Institute of Poultry, RAAS, Moscow province, Sergiev Posad 141311, Russia e-mail: micotox@newmail.ru, rzernoff@yandex.ru

Received January 17, 2011

All-Russian Institute for Selection and Seed-Breeding of Vegetables LIK Cosmetics Institute of Agricultural Biotechnology Joint Stock Company "GosNIISyntezBelok"