Alternatives to antibiotics for farm animals

For several decades, the use of sub-therapeutic levels of antibiotics in animal feeds has been a common practice in many countries in order to improve growth performance and prevent from the adverse effects of pathogenic and non-pathogenic enteric microorganisms. However, there are increasing concerns for the public health about the consequences from the use of antibiotics in livestock [1]. The risk of developing cross-resistance and multiple-antibiotic resistance in pathogenic bacteria both in human and farm animals, has been strongly linked to the therapeutic, metaphylactic or prophylactic uses of antibiotics in human and veterinary medicine, as well as growth promoters in animal feed [2].

The use of antibiotics as growth promoters has been complete banned by the European Union (EU) since 2006 (EC Regulation No. 1831/20031), based on their possible negative consequences for animal health and food safety [3, 4]. This ban has led to animal performance problems and a rise in the incidence of certain diseases [5, 6]. Thus, there is an urgent need to develop alternatives to antibiotics, especially in EU. As a consequence of the public health concerns and the demand of the farmers to prevent the economic losses, non-antibiotic additives have been developed for prophylactic use against pathogens or as growth promoters.

The aim of this review is to present the beneficial effects of some currently used alternatives to in-feed antibiotics, i.e. probiotics, prebiotics, organic acids, phytogenics and zeolites on health as well as on growth performance of farm animals. 

Organic acids are considered to be any organic carboxylic acid of the general structure R-COOH. They are widely distributed in nature as normal constituents of plants or animal tissues and also formed through microbial fermentation of carbohydrates, mainly in the large intestine. They are sometimes found as their sodium, potassium or calcium salts. Most organic acids with specific antimicrobial activity are short-chain acids (C1–C7, SCFA) and they have a pK_a – between 3 and 5.

The most common organic acids (also called acidifiers) that are used in farm animal feed are formic, acetic, propionic, butyric, lactic, sorbic, fumaric, tartaric, citric, benzoic and malic [7]. According to their effects, they can be categorized into two groups: (a) the first group (lactic, fumaric, citric) is characterized by indirectly reduction of bacterial populations by decreasing pH in the stomach, and (b) the second group (formic, acetic, propionic and sorbic) is characterized by a direct effect of lower pH in the gastrointestinal (GI) on the cell wall of Gram-negative bacteria [8–10].

The mechanisms of their action include reduction of gastric pH or buffering capacity of diets, increase of proteolytic enzymes activity and nutrient digestibility, improvement of pancreatic secretions, stimulation of digestive enzymes activity, balancing the microbial population and promotion of beneficial bacterial growth, reduction of pathogens survival through the stomach and direct killing of bacteria [11–15].

Their effects depend on several factors as: type and pK_a of acid, inclusion rate of supplemented acids, composition of diets and their acid–base or buffering capacity, level of intraluminal production of acids in GI tract by inhabiting microflora, feed palatability, intrinsic acid activity, receptors for bacterial colonization on the epithelial villi, maternal immunity by vaccinations, hygiene and welfare standards, age of animals [11, 16–18].

Many studies proved benefits from the use of dietary acidifiers in swine, including positive effects on growth performance as well as on prevention and control of diseases. Their antimicrobial effects depend on their concentration and pH [19]. For example, lactic acid is more effective in reducing gastric pH and coliforms [20–22], whereas other acids (e.g. formic, propionic) have broader antimicrobial activities and they can be effective against bacteria (e.g. coliforms, clostridia and Salmonella), fungi and yeast [11, 22–24]. Several studies have reported reduction of coliforms burden along the GI tract, decrease of piglet scouring or mortality as well as effective control of post-weaning diarrhoea and oedema disease in piglets [21, 25–27].

Acidifiers have received much attention in pig production owing to their beneficial effects on growth performance by improving digestive processes through several mechanisms [12, 18, 25, 27–33]. They can improve gut health by promoting the beneficial bacterial growth, while inhibiting growth of pathogens (through reduction of pH and buffering capacity of diets). A reduced buffering capacity of diets containing organic acids is also expected to slow down the proliferation and/or colonization of undesirable microbes, e.g. Escherichia coli, clostridia, Salmonella spp. in the gastroileal region [11, 20, 24, 26, 33–36]. Acidifiers can also stimulate pancreatic secretions [37], which increase the digestibility, absorption and retention of protein and amino acids [38, 39] and minerals (e.g. Ca, P, Mg and Zn) [29, 32] in the diet.

Organic acids are formed through microbial fermentation of carbohydrates predominantly in the caeca of poultry [40]. The mechanism of their action probably reflects their antibacterial nature, such as decreasing the pH of drinking water and reducing the buffering capacity of the feed with subsequent effect on the physiology of the crop and proventriculus [41, 42].

Acidifiers reported to have beneficial effects on poultry performance or health. For example, some (e.g. butyric acid) also decrease the incidence of subclinical necrotic enteritis caused by Clostridium perfringens, which is highly relevant for the poultry industry [43]. Butyric acid has also anti-inflammatory effects [44] and has been shown to strengthen the gut mucosal barrier by increasing production of antimicrobial peptides in mucous and by stimulating the expression of tight junction proteins [45–48]. Moreover, there is some evidence of increased growth of the GI mucosa in the presence of organic acids, particularly fatty acids such as butyric acid. Indeed, butyric acid has been shown to be an important energy source for gut epithelial cells and to stimulate epithelial cell proliferation and differentiation [49].

Organic acids establish their antimicrobial effect in the intestines by suppressing fungal activity and maintaining an acidic environment [7]. The main organic acids of interest in ruminants are malate, fumarate and aspartate. Acidifiers are reported to improve rumen fermentation, like ionophor antibiotics and maintain the rumen pH even after consuming carbohydrate-rich feeds through, which increased growth performance, are achieved [50]. In addition to buffering effect in rumen, acidifiers might increase energy-efficiency and digestibility of crude protein, Ca and P by lowering methane production and decreasing the numbers of harmful bacteria attached to the intestinal wall [51].

Limited in vivo research has been conducted to evaluate the effects of organic acids on ruminant performance. Malate supplementation reported to increase nitrogen retention in sheep and steers and improve growth performance in bull calves [50, 52, 53]. Additionally, acidifiers such as malate and fumarate can improve milk production [54–57].

The interest in phytogenic feed additives has considerably increased during the past years. Phytogenic feed additives are commonly defined as plant-derived compounds incorporated into farm animals’ diets, such as herbs, spices and essential oils [58, 59]. They have bene- ficial effects on farm animals, including improvement of growth parameters through amelioration of feed properties, promotion of the animals’ production performance, and improving the quality of food derived from those animals [58].

The mode of their action as feed additives is still not fully understood. However, many studies reported antimicrobial, antioxidative and growth-promoting effects [58–60]. The potential mechanisms of their action include: (a) antimicrobial effects: Oregano and thyme are among those which have received a great deal of interest [58, 60–62]; (b) antioxidative effects: phytogenic feed additives derived from plants high in terpenes (e.g. rosemary, oregano and thyme) have anti-oxidative properties, mainly due to their phenolic terpenes [58, 63–65]; (c) growth-promoting effects (increased feed intake, improved gut function and dietary palatability): their stimulatory effect on feed intake is probably due to the improvement in the dietary palatability of resulting from the enhanced flavour and odour, especially with the use of essential oils [65, 66].

Recent studies indicated stabilizing effects (essential oils and oleoresins) on the ecosystem and the activity of GI microbial flora of swine [51, 66–68] associated with a decrease in microbial activity of the GI gut. Improvement in gut function is mainly attributed to the possible stimulatory effect of phytogenic substances on digestive secretions, such as digestive enzymes, bile and mucus [69]. Based on Greek experience, the use of phytogenics can have significant antimicrobial activity against Gramnegative bacteria (mainly E. coli), antioxidative action, enhance dietary palatability, improve the gut functions and promote growth performance and carcass quality of pigs [60, 70–73].

The phytogenic additives are a proven dietary supplement for poultry, containing a proprietary blend of plant extracts (essential oils, bitter substances, pungent substances and saponins). Some of these compounds stimulate appetite (e.g. menthol from peppermint), provide antioxidant protection (e.g. cinnamaldehyde from cinnamon) or suppress microbial growth (carvacrol from oregano). Because of possible ‘synergy’ between constituents, it remains unclear which components of etheric oil products may stimulate the endogenous digestive enzymes, act as an antioxidant, antimicrobial agent or immunomodulator. In vitro studies indicated antimicrobial effects with respective minimum inhibitory concentration (MIC)- values and spectrum of activity [74–76]. The antimicrobial activity is rather weak for ginger and pepper, medium for cumin (p-cymene), coriander (lialol), oregano (carvacrol), rosemary (cineol), sage (cineol) and thyme (thymol) and strong for clove (eugenol), mustard (allylisothiocyanate), cinnamon (cinnamaldehyde) and garlic (allicin) [77].

The essential oils stimulate the intestinal endogenous enzymes. Essential oils from oregano are showing the greatest potential as an alternative to antibiotic growth promoters. Oregano contains phenolic compounds (e.g. carvacrol) that have antimicrobial activity [78]. Oregano essential oils can modify the gut microflora and reduce microbial load by suppressing bacteria proliferation. There are some claims that oregano oil can replace anticoccidial compounds, not because they inactivate coccidia, but because they increase the turnover of the gut lining and prevent coccidial attack by maintaining a more healthy population of gut cells [79]. This mode of action would increase the animal’s maintenance energy requirement because enterocyte turnover is a major proportion of the basal metabolic rate.

Bitter substances are found in herbs and stimulate the secretion of gastric juices. The pungent substances are found in plants such as paprika, garlic and onion, and are purported to function by increasing blood circulation, leading to faster detoxification of the whole metabolism. Saponins enhance the permeability of the gut wall and reduce ammonia. Flavonoids are plant polyphenols with anti-inflammatory effects and they also help to maintain the health of small blood vessels and connective tissue.

Tannins: Two categories of tannins exist: condensed tannins or proanthocyanidins and hydrolysable tannins. As they can form chemical complexes with proteins, they slow dietary protein ruminal breakdown, enhance small intestine amino-acid bioavailability and reduce ruminal NH₃ production and nitrogen (N) excretion in urine. The prevention of ruminal protein degradation improves the nutritional status and reduces the amount of N released environmentally [80–82].

Ruminal and intestinal feed digestion is modified by tannins’ antimicrobial effects, which lower bacterial concentrations in the rumen and decrease the bacterial proteins’ amounts flowing to the intestine [83]. Condensed tannins increase the microbial protein synthesis efficiency by redirecting a higher proportion of fermented nutrients to microbial mass synthesis at the expense of volatile fatty acids (VFA) production [80, 84–88].

The methane produced per unit of digestible dry matter is reduced (20–30%) when ruminants consume forages rich in tannins (Lotus pedunculatus, Lotus corniculatus etc.) [89–91]. Severity of bloat legumes is lowered when condensed tannins are present, since gas formation and microbial protein degradation decrease [92–94].

Tannins can substitute chemical anthelmintics in controlling gastrointestinal parasitic nematodes [95–97]. Tannins have also exhibited a direct antiparasitic activity, while stimulating host resistance as a result of an increase in intestinal protein supply [98].

Saponins: Lucerne and soybeans are the main examples of saponin-rich plants used in ruminant diets [99]. They have hypocholesterolaemic, anticoagulant, anticarcinogenic, hepatoprotective, hypoglycaemic, immunomodulatory, neuroprotective, anti-inflammatory and anti-oxidant activities [82, 100]. Their action depends on dosage rate and rumen pH [101–103].

Dietary supplementation of ruminant diets with saponins supposedly improves growth, feed efficiency and health [104]. Their effects based on their effect on ruminal microbes, result in a decrease in fed proteins’ degradability in the rumen in conjunction with an increase of microbial protein synthesis, which in combination increase the intestinal flow of amino acids [105]. Eventually, saponin administration improves nitrogen digestion, since less NH₃ is produced in the rumen and less urea is eliminated in urine [85, 102]. Ruminal NH₃ concentration and methane production are also significantly decreased [88, 102, 106–109]. Saponins can alter the cell wall structure of Gram-positive bacteria and because of their strong inhibiting effect on Saccharomyces cerevisiae [110], it is strongly recommended that they are not used associated with yeast-based probiotics. They can also exhibit antimicrobial action by increasing bacterial membranes’ porosity [110, 111]. Additionally, bacterial growth inhibition may be caused by complication of essential minerals and steroids with saponins, consequently limiting their bioavailability for bacterial metabolism [112]. Moreover, the antiprotozoal property of saponins could be exploited in the treatment of protozoal infections in ruminants [113, 114]. Finally, saponins (e.g. extracted from Sesbania sesban leaves or lucerne roots) can reduce protozoal numbers [81, 115–117].

Essential oils: Because of their lipophilic nature, essential oils interact with the cell membrane of bacteria, thus acquiring their toxic and antimicrobial effects, especially against Gram-positive bacteria. The external capsule of Gram-negative bacteria can protect them against essential oils [118, 119], but some are small enough to enter the inner membrane and damage it. They can also cause coagulation of cytoplasmic material [120] and impair fungal, protozoal and viral growth [119, 121–125].

The effectiveness of commercial blends of essential oils depends on the protein source [126, 127]. Garlic oil, cinnamaldehyde, eugenol, carvacrol and thymol, are more active on rumen fermentations, such as depress NH₃ and methane production and improve propionate production at the expense of acetate. Most depress NH₃ and methane production and improve propionate production at the expense of acetate [128–133]. Garlic oil could have the potential to inhibit methanogens without affecting other rumen micro-organisms [134]. Optimal doses of essential oils are difficult to assess, because of vast differences in chemical composition between preparations. Differences in efficacy of the same essential oil mixture may be explained by their ability to adsorb on the surface of some dietary ingredients and more specifically affect the microbes attached to them [135]. Thus, ration composition may modulate the response of rumen microbes to essential oil addition. The simplest and most economically efficient method of delivering bioactive plant secondary metabolites to farm animals would be to feed them with a fresh or dried plant.

Probiotic comprised of individual species or mixtures of lactic acid bacteria, yeasts or their end products. Probiotics for use in farm animals are typically divided into the following categories: (1) live cultures of yeast or bacteria, (2) heat-treated (or otherwise inactivated) cultures of yeast or bacteria or (3) fermentation end products from incubation of yeast or bacteria. The mechanisms of their action include (a) competition between yeast or bacteria of probiotics and pathogenic micro-organisms in the intestinal mucosa [136–138], (b) nutrient availability [138, 139] and (c) total inhibition of pathogen growth by production of organic acids and antibiotic-like compounds [138–141].

The most commonly used probiotic bacterial strains are Bifidobacterium (B. bifidum, B. pseudolongum), Lactobacillus (e.g. L. acidophilus, L. casei, L. rhamnosus), Bacillus (e.g. B. subtilis, B. cereus, B. toyoi, B. licheniformis), Lactococcus (e.g. L. lactis), Enterococcus (e.g. E. faecium), Streptococcus (e.g. S. thermophilus), Pediococcus and Saccharomyces (e.g. S. cerevisiae). Many studies demonstrated their beneficial effects on health and growth performance of farm animals [60, 142–144]. In particularly, probiotics have positive effects on: (a) the digestive process by increasing the activity of microbial probiotic enzymes and the digestibility of food [145], (b) immunity by stimulating the immune system and the regeneration of intestinal mucosa (e.g. macrophages and natural killers cells, increase of immunoglobulin production, regulate anti- and proinflammatory cytokine production) [145–148].

The effects by the use of probiotics in animals’ feed depends on the combination of selected bacteria, doses in feed, and on their interactions with pharmaceuticals, feed composition, storage conditions and feed technology [149–151].

Probiotics in swine can inhibit pathogenic micro-organisms, improve the intestinal microflora and stimulate immune by modulating intestinal microflora and/or lowering the pH value in the small intestine and producing organic acids and antibacterial substances [140, 141, 144, 152–155]. For example, members of the genus Bacillus support natural intestinal microflora, compete with undesirable microorganisms, and reduce the numbers of Enterococci, Bacteroides and coliforms [149, 152, 156–159] the bacteria E. faecium were found to be able to prevent the K88 positive ETEC strain from adhering to the intestinal mucous membrane of piglets [158, 160] or regulating intestinal microbial balance by increasing the activity of microbial digestive enzymes [138].

Beneficial effects of probiotics have reported in the health status and growth performance in newborn and weaned piglets [151–155, 158, 161]. In the post-weaning period, probiotics could be used for the prevention of post-weaning diarrhoea caused by enterotoxigenic E. coli strains [138, 140, 141, 145, 162].

The most well-known group of probiotics are lactic acid bacteria. It has been shown that lactic acid produced in vitro by lactic acid bacteria is used by the strictly anaerobic butyrate producing bacteria of clostridial clusters IV and XIV for the production of large concentrations of butyric acid [163]. This mechanism is called cross-feeding and is a further reason why lactic acid bacteria administrations have beneficially performance. The intestinal microbiota have a specific multifactorial ‘barrier’ impact, such as (1) induction of anatomical and physiological changes in the intestinal cell wall structure, (2) immunological modifications in the gut and (3) enhancement of the bird’s resistance to enteropathogenic bacteria, such as C. perfringens [164–167]. Depending on the probiotic strain, the mode of action probably involves production of specific metabolites (short organic fatty acids, H₂O₂, intermediary metabolites with antimicrobial activity), interaction with receptor sites, stimulation of the immune system and some others [168, 169].

Probiotics are generally recommended in ruminants’ nutrition whenever a risk of rumen dysfunction exists, in order to improve anaerobiosis, stabilize pH and supply nutrients to microbes in their microenvironment. Probiotics are recommended in young ruminants [170, 171] to prevent diarrhoea caused by enterotoxigenic bacteria in the gut and also during weaning period to enhance the rate at which rumen flora and fauna become established. L. acidophilus alone or in combination with other lactobacilli has been shown to reduce scouring and increase growth rate of calves in some trials [172, 173].

Bacterial probiotics have been predominantly promoted to prevent ruminal acidosis. Lactic acid-producing and lactic acid-utilizing bacteria are used, sometimes combined, to reduce the negative impact of rapid fermentation of high-starch feeds in the rumen. Lactate utilizers such as Megasphaera elsenii or Selenomonas ruminantium have been reported to prevent lactate accumulation and alleviate the drop in ruminal pH when animals are fed high-starch or high-sugar diets [174]. Propionibacteria are also used for their lactate-utilizing activity and high production of propionate. The rationale for the utilization of lactate producers, such as Lactobacillus and Enterococcus sp., is that by maintaining a low and constant level of lactic acid, they sustain an active population of lactate utilizers that in turn will prevent lactate accumulation and ruminal pH drop [175]. The most commonly used probiotics in adult ruminants, however, are those based on yeast preparations of Aspergillus orizae and (or) S. cerevisiae. Live yeasts are mainly used because they act as regulators for rumen pH and prevent its drop when diets rich in fermentable carbohydrates are fed [176]. They may also exert an effect in the post-rumen digestive compartments as about 17–34% of administered yeasts remain alive during transit along the gut of ruminants [177].

The addition of probiotics in lambs, calves or dairy cattle diets seems to have beneficial effects on their performance (e.g. average daily gain, feed intake) [178–181] and especially in milk production (e.g. higher production numerically, increased milk fat and protein) [182–186].

Prebiotics are dietary short-chain carbohydrates (oligosaccharides). They have beneficial effects on health and growth performance in farm animals, stimulating the growth and/or activity of one or more of beneficial bacteria. The non-digestibility of prebiotics ensures that they can reach the colon and act as an energy source for bacteria, unlike normal sugars, which get digested directly by the host [187]. As a result, the composition and/or the activity of the microbiota are altered, leading to secondary effects such as increased gas production and a drop in pH. Prebiotics can also prevent the adhesion of pathogens to the mucosa, by competing with its sugar receptors and several studies have shown that supplementing feed with various oligosaccharides have led to reduced susceptibility to Salmonella and E. coli colonization [188–191].

The most common non-digestible oligosaccharides (NDO), which are used as prebiotics in farm animals, are the following: mannanoligosaccharides (MOS), galactooligosaccharides, fructooligosaccharides (FOS), soybeanoligosaccharides, isomaltooligosaccharides, xylooligosaccharides, lactulose and inulin [192–195].

MOS have beneficial effects on the intestinal microflora by modifying the microbial gut ecology and preventing the colonization of bacterial pathogens (e.g. stimulate the growth of non-pathogenic bacteria such as B. longum, L. casei, L. acidophillus or L. delbru¨ckei and suppress the growth of pathogenic bacteria such as E. coli, Salmonella typhimurium, Clostridium botulinum and C. sporogenes) [193, 196, 197].

The adding of indigestible NDO to the animal feed based on either fructose or mannose sugars derived from yeast-cell wall can be used to attract pathogenic bacteria to attach to these dietary particles rather than the intestinal cells. Bifidogenic effects of galactooligosaccharides, FOS and soybeanoligosaccharides have been reported in many in vitro and in vivo studies [162, 194, 198].

Lactulose is formed by lactose isomerization and it cannot be absorbed from the small intestine. Therefore, it passes down to the large intestine, resulting in lactic and/ or acetic acid production by the resident microflora [187]. Therefore, it stimulates the growth and/or activity of indigenous intestinal microflora, especially of the genera Bifidobacterium and Lactobacillus, and reduces the activity of proteolytic bacteria. The dosage of 1% lactulose is usually adding in swine diets for the prevention or control of enteric infections [141, 187].

Inulin is present in many vegetables (e.g. onion, garlic, asparagus and banana) [190]. Its supplementation has positive effects on SCFA production, sufficient height of intestinal villi, stimulation of natural microflora and improvement of performance parameters [199].

Two of the most commonly studied prebiotic oligosaccharides in poultry are FOS and MOS. The supplementation of poultry feed with MOS resulted in an improvement in intestinal morphology and intestinal enzyme activity, yet the growth performance of the broilers was not up to the level of including an antibiotic growth promoter to the feed [200]. Mannose, the main component of MOS, is a unique sugar because many enteric bacteria have receptors that bind to it. These receptors, called Type 1 fimbriae, are involved in attachment of the bacteria to the cells of the host. Attachment is critical for the bacterium to be able to cause disease in the host. Chickens likely have receptors for Type1 fimbriae in their small intestine [201]. MOS functions as a competitive binding site; the bacteria bind to it and are carried out of the gut rather than binding to the intestine. In a study that supports this theory, it was found that supplementing the drinking water of broilers with 2.5% mannose reduced S. typhimurium colonization of the intestines [202].

Studies with adding FOS in poultry diets reported significant reduction in Salmonella carriage in the ceca [203] significant improvements on growth performance [204]. Results obtained from synthetic materials suggest some benefits using inulin and FOS that act as substrates for ‘desired’ micro-organisms, for example Bifidobacteria [188, 205–207], whereas MOS have receptor properties for fimbriae of E. coli (sensitive to mannose) and Salmonella spp., which leads to elimination of these bacteria with the digesta flow instead of binding a mucosal receptor [150, 208–210].

Oligosaccharide b-glucans of yeast cell wall origin are thought to stimulate performance because of their immunomodulatory effects. Recent reviews elaborate on the action of glucans on immune stimulation [211, 212].

Zeolites are crystalline, hydrated aluminosilicates of alkali and alkaline earth cations, with infinite structures which are three-dimensional. Based on their unique properties, zeolites (especially clinoptilolite), have been used as feed additives in order to ameliorate mycotoxicosis and improve animals’ performance. Recently, clinoptilolite has been approved as feed additive in EU at the highest inclusion rate of 2% of dry matter. Its effectiveness on mycotoxins’ binding as well as the increased interest for organic products that favours the use of feed additives, which have no residuals on animal products, are expected to increase the use of clinoptilolite as feed additive.

The addition of clinoptilolite in swine rations has positive effects on growth performance of growing and fattening pigs [212] and carcass characteristics [213, 214]. It is believed that enhanced swine performance by clinoptilolite results from its direct binding effects to some harmful by-products of the intestinal flora (e.g. ammonium ions, p-cresol) [215].

Additionally, the use of clinoptilolite as feed additive during pregnancy has beneficial effects on reproductive traits of sows, increasing litter size and body weight (BW) at birth and weaning [212, 216, 217] and reducing the interval between weaning and mating [212].

In laying hens, the administration of clinoptilolite improves feed conversion rate [218], increases the number of eggs laid [218, 219] and improves their quality characteristics [218–220]. In broilers, clinoptilolite accelerates their growing rate by increasing feed consumption [221, 222] and feed conversion rate [222, 223] and improves carcass quality by lowering fat percentage [223, 224].

In ostriches, it has been reported that clinoptilolite affects the total bacterial counts of the eggshells. Dedousi et al. [225] observed that its use as nest material in ostriches reduces the total bacterial counts of eggshells compared to river sand. This finding was attributed to the fact that clinoptilolite adsorbed and immobilized the bacteria from nest environment, resulting in a net reduction of their number. As a consequence, the number of free micro-organisms able to infect the eggs laid in nests with clinoptilolite, was less than those in the nests with other materials.

Clinoptilolite acts as a regulatory factor when added to acidic or basic aqueous solutions [226]. Recent studies proved that the administration of clinoptilolite in dairy cows (200 g daily or 1.4% dry matter) resulted in significantly higher pH values [227–229]. Dschaak et al. [229] further observed that the pH values of cows fed clinoptilolite were comparable to those obtained from cows consuming equal amounts of sodium bicarbonate, concluding that clinoptilolite can cost-effectively replace sodium bicarbonate as ruminal buffer.

Recent studies have shown that the use of clinoptilolite as a feed additive can prevent ETEC diarrhoea by increasing intestinal immunoglobulin absorption in newborn calves. The administration of clinoptilolite (5 g/kg BW) along with colostrum can increase the degree of absorption of colostral IgG, as well as blood serum concentrations of IgG in dairy calves [230, 231]. Moreover, the use of 25 ml of clinoptilolite suspension (20% in distilled water) in the colostrum can increase the apparent intestinal absorption of colostral IgG and blood serum concentration of IgG in newborn calves [232]. Recently, Pourliotis et al. [233] proved that the administration of clinoptilolite with colostrum initially, and milk afterwards (1 g/kg BW and 2 g/kg BW/day during the first 10 days) is associated with: (a) significantly higher antibody titres against E. coli in blood serum of calves and the incidence of ETEC diarrhoea was significantly lower in calves that were receiving clinoptilolite, (b) increase of the intestinal absorption of immunoglobulins either by increasing the pinocytotic activity of intestinal epithelial cells or by retarding the intestinal passage rate, (c) increase the time that immunoglobulins are available to the specific receptors of the epithelial cells, (d) bind some degradation products of the colostral proteins in the intestine that have negative effect on the intestinal epithelial cells, such as NH₃. The shorter duration of ETEC diarrhoea incidences in experimental calves was further attributed to the alteration of metabolic acidosis, through clinoptilolite effects on osmotic pressure in the intestinal lumen and to the absorption by clinoptilolite of bile acids (endogenic cause of diarrhoea) and glucose (high content in intestinal fluid acts as an irritant factor).

The administration of clinoptilolite in sheep can be beneficial for the prevention of certain parasitic infections. In ewes, its dietary inclusion (2.5% of the concentrates) during the transition period can reduce Eimeria oocyst output [234]. In addition, the use of clinoptilolite supplementation in lambs can decrease their total worm burden and faecal egg counts per capita and reduce the establishment of GI nematodes [235]. Moreover, the use of clinoptilolite as feed additive in dairy goats seems to improve their energy status. Its dietary inclusion (2.5% of the concentrates) during the transition period can reduce the blood serum concentration of b-hydroxybutirate and increase the BW of triplets and quadruplets kids at birth [236]. Moreover, the administration throughout lactation can increase the milk fat content and reduce the somatic cell counts in milk [236]. 

Modern animal production is trapped between concerns on risks for public health and an increasing demand for animal origin products. The increased concern about the potential for developing antibiotic resistant strains of bacteria within the food chain, especially after the ban of non-therapeutic antibiotics in animal feed in EU leads to an increased development and research on alternatives to antibiotics for use as feed additives in livestock.

Alternatives to antibiotics could be important tools for veterinary practice in case they can improve growth performance of farm animals at levels comparable to antibiotics. For this reason, new strategies and commercial products must be developed, based on their costeffectiveness as well as on their efficacy to minimize or eliminate the pathogen load in the livestock and food chain. The future studies should be focused on strategies to develop commercial products not only in agreement with the modern consumer demands for more environmental friendly animal production (e.g. organic farming), but also supporting the farmers needs for higher livestock production. In conclusion, useful tools for farmers and veterinarians’ to improve the animal health and performance could be products such as probiotics, prebiotics, organic acids or zeolites. 

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