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INTRODUCTION

 

Between 1990 and 1999, the average sales of farmed mink and fox pelts to Western markets were 25.6 and 3.6 million pelts, respectively, of which 1.8 million mink and 1.9 million fox pelts were produced in Finland (Anon., 1999a). Compared with the national meat industry, fur farming is a sufficiently large business in Finland to be able to utilize all suitable by-products that become available and therefore, plays an important role in the utilization of by-products and recycling of nutrients.

Because biologically and hygienically edible parts of cattle and pigs form only 55 and 63% of the live weight, respectively, great quantities of different by-products not qualified for human consumption are produced by meat industry in every country (Bengtsson & Holmqvist 1984). The percentage of by-products that can be reused as an animal feed varies from one country and culture to another, but on average it is 40% of carcass weight in cattle, over 10% in pigs and over 30% in poultry (Miller & De Boer, 1988). Based on a national meat production of 340 million kg in 1999 and information provided by a rendering plant (Lahtinen, personal communication), production of slaughterhouse by-products (including bones) in Finland exceeded 150 million kg. Fur animals consumed approximately 95 million kg of those by-products, representing 25% of total feed for fur animals. In addition, suitable low bone by-products were also imported, which increased total percentage of by-products in total fur animal feed to over 30% (Anon., 1999a; Valkosalo, personal communication).

High moisture and nutrient content combined with enzymes and temperature favoring growth of microbes make slaughter by-products perishable and if not properly treated, can be even dangerous to the environment and human health. Due to a continuous offal output from slaughterhouses, but irregular consumption by fur animals leads to fur farmers having to preserve and store these materials for several months. Even though ensiling of by-products with formic acid is well documented, primarily in fox farming (Backhoff, 1943), a break-through for conservation of offal has not been achieved and great quantities of feedstuffs are still stored frozen. Use of formic acid among mink farmers was considered to be associated with a lowering fur quality and decreased male reproductive performance. Its use for ensiling was also decreased due to unreliable information concerning the toxicity of formic acid, an effective organic acid preservative (Näveri, 1983). Furthermore, due to a one-feed system and due to the use of mink as an animal model also for both the blue ( Alopex lagopus ) and silver ( Vulpes vulpes ) fox, inter-species differences and performance of foxes with different feeds were not emphasized. Furthermore, since homogenous fish products better suited large scale ensiling applications, research was focused on the preservation of these materials (Møller Jensen & Jørgensen, 1975).

The possibility to use nonheated low risk slaughterhouse by-products in fur animal feeds is based on the Council Directive 90/667/EEC (Anon., 1990) which is implemented in Finland by a ministerial decision on the 7 July 1994. The decision defines conditions for the use of by-products in fur animal feed. Provisions of the decision cover transportation and storage including an official registration of fur animal feedingstuff processing plants that manufacture fur animal feed for sale. The basic principle is that the use of by-products must not threaten the environment, human or animal health. As far as BSE is concerned, by Commission decision (2000/418/EC) specified risk material of bovine animals has to be removed and destroyed after 1 October 2000 in all Member States. Optionally to immediate incineration the risk material can be pre-processed in specified process lines of high risk plants approved for this purpose. It is yet unclear if this dehydrated product will be allowed in fur animal feed in a Member State. In Finland BSE specified risk material indicates a removal of approximately 30% of bovine by-products conventionally used as fur animal feed (Lahtinen, personal communication), which is less than 10% of all meat industry by-products utilized by fur animals.

In Finland the proper treatment of slaughterhouse by-products is nationally important because of the very low level of zoonooses in the country (Anon., 1999b; Anon., 1999c). In order to maintain this level low future (below 1%), the Finnish Ministry of Agriculture and Forestry created a national salmonella control program (Anon., 1994b) which the Commission approved during EU membership negotiations in 1994 (94/968/EC). The Finnish Ministry of Agriculture and Forestry in 1998 also banned the use of antibiotics in fur animal feed as a preventive medication (Anon., 1998), highlighting the importance of hygienic quality of slaughterhouse by-products. There is also special legislation under preparation in order to prevent the spreading of zoonooses via slaughterhouse by-products that are fed to fur animals, which will require immediate treatment of by-products, in a special section within the slaughterhouse. Heating or ensiling will be the two alternatives that all by-products designated for fur animal feed have to go through before leaving the plant.

Salmonella is not a large problem with fur animals, although pregnant mink and foxes and young fox cubs are vulnerable (Jørgensen, 1985; Kangas, 1982). Instead, the main concern deals with the fact that contaminated fur farms are in contact with food producing area. Hence, without being the initial source of the disease, fur farms may maintain and spread the disease in a particular area. Furthermore, some farms also produce meat or milk in addition to furs. Therefore, it is important that feed manufacturing practices of fur farming do not contradict, but are in accordance with other fields of animal production and contribute to the common national goal.

 

Central substances of the study

Formic acid

Formic acid (HCOOH) is an organic acid with a molecular weight of 46.03 and pK a of 3.75. It is colourless, transparent liquid with a pungent odour, an irritant to eyes, skin, and mucous membranes and is miscible with water. Formic acid occurs naturally in a variety of plants and fruits, mammalian tissues and insect venoms. Formic acid is a normal constituent of the body and is metabolically important in the transfer of one-carbon substances which primarily come from amino acid metabolism, and act as a substrate for nucleic acids (Mathews & van Holde, 1990; Stryer, 1988). Formic acid is used in industry during the preparation of a variety of drugs, dyes, and chemicals. Formic acid is produced by heating carbon monoxide and sodium hydroxide under pressure and then treating the resulting sodium formate with sulphuric acid.

Even though some of the antimicrobial action of formic acid is based on its pH decreasing effect, inhibition of microbes is mainly associated with the undissociated molecule under acidic conditions ( Figure 1) . Being the strongest organic acid and having a relatively low pK a , formic acid acts both as acidulant and preservative ( Figure 2 ). Formic acid inhibits the growth of yeasts and bacteria and can also eliminate salmonella from feeds (Lueck, 1980; Frank, 1994). In animal feeds, formic acid has been used for a long time to ensile grass for ruminants, but it has now become an established feed additive for monogastric animals. Formic acid improves the digestibility of dietary protein and the growth of young piglets, and is beginning to replace antibiotic growth promoters in Europe (Partanen et al., 1998; Partanen & Mroz, 1999).

 

Figure 1. Schematic representation of the mode of action of preservative acids and their salts. Undissociated molecules permeate cell membrane, anions accumulate in the cell and disrupt cell functions. Cell energy sources are depleted in transporting protons from cytoplasm in order to re-establish pH-balance.

 

Formic acid is readily absorbed from the gut or through skin and mucous membranes. It is also a breakdown product of methanol and formaldehyde. Regardless of being a natural metabolite, formic acid is toxic if accumulated in free form in the body. Primates, particularly humans, are more sensitive to accumulation than nonprimates (Clay et al., 1975). Symptoms include metabolic acidosis, ocular pathological changes and death (Frenia & Schauben, 1992; Clay et al., 1975; McMartin et al., 1977). The rate at which formic acid is oxidized into CO 2 depends on hepatic levels of folic acid vitamer THF and on species-specific activities of involving enzymes (Johlin et al., 1987) ( Figure 3 ). The toxicity of formic acid is based on its biological activity. It is an inhibitor of cytochrome-oxidase complex at the terminus of the respiratory chain in mitochondria (Moody, 1991).

Figure 2. Percentage of formic and benzoic acid molecules that exist undissociated in media of various pH. The pH where half of the preservative is dissociated and half undissociated is indicated by the pKa value, 4.2 and 3.75 for benzoic and formic acids (and their salts), respectively.

 

Even though formic acid has been known for decades within fur animal feeding (Backhoff, 1943), its use has also been the subject of some dispute, partly due to a lack of knowledge concerning formic acid metabolism (Näveri, 1983), and partly due to confusion caused by several other concomitant dietary factors, such as anemiogenic fish and sulphuric acid present in fish silage (Kangas, 1977; Näveri, 1983) and peroxidized feed fat (Havre et al., 1973). Furthermore, formic acid was suspected to deliteriously effect reproductive performance in mink (Näveri, 1983; Näveri, 1984).

Because trimethylamine oxide (TMAO) and its breakdown product, formaldehyde that exists naturally in anemiogenic fish were identified to render dietary iron unabsorbable (Ender & Helgebostad, 1968; Costley, 1970), doubts concerning potential anemiogenic agent were raised (Näveri, 1983). Even though detrimental effects of formic acid on foxes were not experienced or verified with experiments, due to a one-feed system for all fur animal species, use of formic acid as feed preservative declined. In Denmark, acetic acid was substituted for formic acid in sulphur acid-formic acid ensiled fish because of its limited palatability in mink kits of 4-6 wk of age (Møller Jensen & Jørgensen, 1975; Jørgensen, 1981). Therefore, one of the objectives of this study was to investigate and assess fur animal species-specific uses of formic acid.

 

Figure 3. Structure of folic acid. A biologically active form of folic acid is 5,6,7,8-tetrahydrofolate (THF), a highly versatile carrier of one-carbon units (methyl, methylene, formyl, formimino, methenyl) that are bonded to the N 5 and N 10 nitrogen atoms. In the oxidation of formate, THF receives formyl group (catalyzed by N 10 -formylTHF synthetase, EC 6.3.4.3.) and donates CO 2 (catalyzed by N 10 -formylTHF dehydrogenase, EC 1.5.1.6.). Because vitamin B 12 is required for generation of THF from N 5 -methylTHF, a B 12 deficiency leads to the accumulation of N 5 -methylTHF and hence, to a deficiency of the functional form of folic acid, THF.

 

Folic acid

Folic acid, or folates as a generic name, are available from a large variety of sources such as green leafy vegetables, liver, beans and fermented dairy products (Vahteristo, 1998). A disease later discovered to be folic acid deficiency was first described by Wills in 1931 as a "tropical macrocytic anemia" observed in India, that was often associated with pregnancy, and prevented or relieved by extracts of autolyzed yeast and liver (Scott et al., 1982). In 1940 this unidentified factor was concentrated from spinach and named according to requirement by Streptococcus faecalis . Folates are necessary for red blood cell formation, metabolism of fats and amino acids, cell division, protein synthesis, DNA and RNA synthesis, and thus for the growth and reproduction of all body cells. The importance of folates is based on their requirement as coenzymes in the transfer and utilization of one-carbon units in a variety of biosynthetic reactions. Therefore, during periods of rapid cell regeneration and growth, such as pregnancy and infancy, increased amounts of folate are required. In addition, folate has further roles in the prevention of diseases such as neural tube defects and accumulation of homocysteine, a risk factor for cardiovascular disease (Scott, 1997).

By nomenclature folates are a group of heterocyclic compounds based on 4-[(pteridin-6-ylmethyl)amino]benzoic acid skeleton conjugated with one or more, usually 5 to 8, L-glutamic acid residues. Folic acid ( Figure 3) (mol. wt. 441.4 g) is not present in biological systems but is the form generally used in pharmaceutical and fortified food products.

Even though the requirement of folic acid in animal feeds is, markedly lower than 1 mg kg -1 DM, and it takes several weeks before deficiency symptoms appear even in growing animals (Thenen & Rasmussen 1978; Kim et al., 1994), there is evidence that folate status for optimal performance and development of production animals is achieved with additional supplementation as high as above 10 mg kg -1 feed DM (Matte et al., 1999).

 

Benzoic acid

Benzoic acid (E 210), C 6 H 5 COOH (mol. wt. 122.1) is a granular or crystalline powder with a sweet or astringent taste and is generally known as a preservative. It is mainly used in the form of sodium benzoate (E211), C 6 H 5 COONa (mol. wt. 144.1), because the sodium salt has a higher solubility in water (500g L -1 ) than the acid (3.4 g L -1 ). Benzoic acid occurs naturally in many acidic fruits and berries, such as cranberries, plums, cinnamon, and ripe cloves.

Benzoic acid is one of the oldest chemical preservatives used in the cosmetic, drug and food industry. Sodium benzoate was the first chemical preservative permitted in food for human consumption in the U.S. in 1908, and continues to be used in a large number of foods (Jay, 1992). Benzoate inhibits yeasts more than it inhibits moulds or bacteria. As with other lipophilic acids, the undissociated form is essential to its antimicrobial activity ( Figure 1 ). In undissociated form, benzoic acid and sodium benzoate are soluble in cell membranes and facilitate proton leakage into cells, increasing cellular energy requirements to maintain internal pH. Due to a relatively low pK a , 4.2, the antimicrobial activity of benzoate is rather low at higher pHs ( Figure 2) . Instead, alkyl esters of p-hydroxybenzoic acid, parabens, with a pK a 8.5, are used to extend the activity spectrum to pH 8 (Jay, 1992; Lueck, 1980). In animal feeds the use of benzoic acid has increased during recent years. Benzoate inhibits fungal growth in formic acid treated grass silages (Aronen et al., 1987). In liquid pig feeds benzoate has been used to inhibit yeast fermentation (Rantanen, personal communication) and according to Mroz et al. (1998) Ca-benzoate in pig feed increases urine acidity and reduces ammonia emissions.

Like other aromatic carboxylic acids, benzoic acid is xenobiotic and is eliminated from the body, mainly by conjugation with glycine, and to a lesser extent with glucuronic acid (Bridges et al., 1970). Conjugation occurs mainly in the liver and kidneys, and the product benzoylglycine, also called hippuric acid, is excreted in urine (Hutt & Caldwell, 1990) ( Figure 4 ). As an exception to all other studied species, the cat (family Felidae ) is rather sensitive to benzoic acid, due to an inability to produce benzoyl glucuronide (Bedford & Clarke, 1972).

 

Figure 4 . Congugation of benzoic acid with glycine. In many animal species conjugation with amino acids is an important route in the biotransformation of xenobiotic carboxylic acids before elimination in urine.

 

 


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