**5. Effects on health status and growth performance**

Clinoptilolite is also used as feed ingredient due to its beneficial properties as immunostimulant. One explanation of beneficial immune effects of silica, silicates and aluminosilicates could be their action as non-specific superantigen-like immunoglobulins (SAg). SAg are viral and bacterial toxins that are capable of activating a large population of T-cells. Activation occurs as a result of the simultaneous interaction between SAg, the T cell receptor (TcR) variable region *β* and the major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Consequently, SAg stimulates 10–30% of T-cells, as opposed to 0.01–0.0001% as it stimulates common antigens. Proinflammatory macrophages belonging to APC cells, CMH class II are activated by the particles of silicates [58].

An indirect action of clinoptilolite on the immune system is also achieved by its antioxidant capacity. Sverko et al. showed that administration of tribomechanically-activated clinoptilolite (12.5%) alone or together with *Urtica dioica* extract in mice *per os* for 3 weeks significantly reduced lipid peroxidation processes in the liver and significantly increased the content of superoxide-dismutase, an antioxidant enzyme. The antioxidant role of clinoptilolite is probably given by positive electrons that neutralize free radicals [59].

In weaned piglets that received 0.5% clinoptilolite for 5 weeks, it was observed that clinoptilolite was effective as an immunomodulatory agent by promoting the recruitment of circulating and intestinal immune cell subsets, even though it did not improve growth in weaned pigs, and generally failed to improve their feed conversion efficiency [60]; in heifers vaccinated against *Escherichia coli* (day 210 and 240 of gestation) the potentiating effect of clinoptilolite on the immune response was highlighted, and it indirectly improved the protection of calves [61].

In newborns, adding clinoptilolite to colostrum improves intestinal absorption of colostrum globulin, creating a good protection against neonatal diseases [18, 62–64].

Colostral period represents an important moment for the newborn ruminants, because in these species acquiring passive immunity is achieved exclusively through ingestion and absorption of adequate amounts of colostral immunoglobulins (Ig) [65]. Obtaining a good protection against neonatal diseases depends on how well this period is managed.

Scientific evidence highlights positive effects of zeolite supplementation on passive immunity [18, 21, 62] and on biochemical parameters [20, 23, 66] in newborns calves.

When we analyzed the effects of clinoptilolite supplemented in colostrum on blood serum protein electrophoretic pattern of newborn calves that received colostrum supplemented with 0.5 and 2% clinoptilolite, at 30 h after birth, the concentrations of *γ*-globulins, *β*-globulin and total protein in the group of calves that received 0.5% clinoptilolite (E1) were higher than in the control group by 42.11% (*p* < 0.05), 28.48% (*p* > 0.05) and 18.52% (*p* > 0.05), respectively, and were higher, but not significantly, in the group that received 0.5% clinoptilolite (E2), and a significantly lower albumin/globulin ratio in groups E1 and E2 (29.35%, *p* < 0.05

**129**

**Figure 2.**

*and 30 h postpartum) [67].*

*Zeolites Applications in Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.87969*

adaptation processes of the cells [68].

(**Figure 2**) [67].

and 35.87%, *p* < 0.05, respectively) was reported, compared with the control group at 30 h postpartum, which indicates an obvious increase in globulin fractions in experimental groups. Clinoptilolite was effective in improving passive transfer in newborn calves, better if added in colostrum in a dose of 0.5% than in a dose of 2%

A possible way of clinoptilolite action is explained, based on observation in Wistar rats that received zeolite for 34 days (6% of their weight) [68]. Some modifications of intestinal cells were observed, such as: the microvilli length was higher (1.2 vs. 2.0), the number of microvilli per 10 μm was higher (54.4 vs. 64.8) their diameter was smaller (0.17 vs. 0.13) compared to the control group, and also that the cellular organisms of the enterocytes, the density of mitochondrial membranes and the number of attached ribosomes were higher, which indicates a rise in the

In an experiment carried out in 20 newborn calves in order to observe the clinical effects of clinoptilolite added in colostrum (20 ml clinoptilolite/L colostrum) during the first three meals, we concluded that administration of zeolites appears to reduce the incidence of diarrhea because only two calves from experimental group had health problems, one had bronchopneumonia and the other digestive transit difficulties with symptoms appearing after 28 days (not in neonatal period) in comparison with the control group where all calves had diarrhea in the first 11 days of life. The other parameter, growth performance measured on day 0, 45 and 90 revealed that during the first 45 days body weight of calves treated with clinoptilolite was significantly higher compared with the group of calves receiving only colostrum (C) (*p* < 0.0058) (E1/C = +16.96%). This statistical difference at 45 days may be explained by the high number of calves from the control group suffering from health problems and this affected the daily gain. At 90 days, the difference between groups was not significant (*p* < 0.1035) (E2/C = +7.19%) [69, 70]. Similar results were obtained by Step et al. who found that body weight and average daily gain did not differ between treatment groups (clinoptilolite dosage was 0.5 and 2%) [23]. More

*Representative serum protein electrophoretograms observed in calves that received colostrum supplemented with 0.5% (e–h) and 2% clinoptilolite (i–l) compare with control group (a–d) at different time interval (0, 6, 16* 

#### *Zeolites Applications in Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.87969*

*Zeolites - New Challenges*

teristics [57].

that neutralize free radicals [59].

In another *in vitro* study done on mouse fibrosarcoma cells and other types of cells incubated for 24 h together with clinoptilolite researchers observed that the number of viable cells, DNA synthesis and activity of EGF-R, PKB/Akt and NFKB was reduced while apoptosis was enhanced maybe because clinoptilolite affects cellular microenvironment through mechanisms that are dependent on its charac-

Clinoptilolite is also used as feed ingredient due to its beneficial properties as immunostimulant. One explanation of beneficial immune effects of silica, silicates and aluminosilicates could be their action as non-specific superantigen-like immunoglobulins (SAg). SAg are viral and bacterial toxins that are capable of activating a large population of T-cells. Activation occurs as a result of the simultaneous interaction between SAg, the T cell receptor (TcR) variable region *β* and the major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Consequently, SAg stimulates 10–30% of T-cells, as opposed to 0.01–0.0001% as it stimulates common antigens. Proinflammatory macrophages belonging to APC cells, CMH class II are activated by the particles of silicates [58]. An indirect action of clinoptilolite on the immune system is also achieved by its antioxidant capacity. Sverko et al. showed that administration of tribomechanically-activated clinoptilolite (12.5%) alone or together with *Urtica dioica* extract in mice *per os* for 3 weeks significantly reduced lipid peroxidation processes in the liver and significantly increased the content of superoxide-dismutase, an antioxidant enzyme. The antioxidant role of clinoptilolite is probably given by positive electrons

In weaned piglets that received 0.5% clinoptilolite for 5 weeks, it was observed that clinoptilolite was effective as an immunomodulatory agent by promoting the recruitment of circulating and intestinal immune cell subsets, even though it did not improve growth in weaned pigs, and generally failed to improve their feed conversion efficiency [60]; in heifers vaccinated against *Escherichia coli* (day 210 and 240 of gestation) the potentiating effect of clinoptilolite on the immune response

was highlighted, and it indirectly improved the protection of calves [61].

In newborns, adding clinoptilolite to colostrum improves intestinal absorption of colostrum globulin, creating a good protection against neonatal diseases

Colostral period represents an important moment for the newborn ruminants, because in these species acquiring passive immunity is achieved exclusively through ingestion and absorption of adequate amounts of colostral immunoglobulins (Ig) [65]. Obtaining a good protection against neonatal diseases depends on how well

Scientific evidence highlights positive effects of zeolite supplementation on passive immunity [18, 21, 62] and on biochemical parameters [20, 23, 66] in newborns

When we analyzed the effects of clinoptilolite supplemented in colostrum on blood serum protein electrophoretic pattern of newborn calves that received colostrum supplemented with 0.5 and 2% clinoptilolite, at 30 h after birth, the concentrations of *γ*-globulins, *β*-globulin and total protein in the group of calves that received 0.5% clinoptilolite (E1) were higher than in the control group by 42.11% (*p* < 0.05), 28.48% (*p* > 0.05) and 18.52% (*p* > 0.05), respectively, and were higher, but not significantly, in the group that received 0.5% clinoptilolite (E2), and a significantly lower albumin/globulin ratio in groups E1 and E2 (29.35%, *p* < 0.05

**5. Effects on health status and growth performance**

**128**

[18, 62–64].

calves.

this period is managed.

and 35.87%, *p* < 0.05, respectively) was reported, compared with the control group at 30 h postpartum, which indicates an obvious increase in globulin fractions in experimental groups. Clinoptilolite was effective in improving passive transfer in newborn calves, better if added in colostrum in a dose of 0.5% than in a dose of 2% (**Figure 2**) [67].

A possible way of clinoptilolite action is explained, based on observation in Wistar rats that received zeolite for 34 days (6% of their weight) [68]. Some modifications of intestinal cells were observed, such as: the microvilli length was higher (1.2 vs. 2.0), the number of microvilli per 10 μm was higher (54.4 vs. 64.8) their diameter was smaller (0.17 vs. 0.13) compared to the control group, and also that the cellular organisms of the enterocytes, the density of mitochondrial membranes and the number of attached ribosomes were higher, which indicates a rise in the adaptation processes of the cells [68].

In an experiment carried out in 20 newborn calves in order to observe the clinical effects of clinoptilolite added in colostrum (20 ml clinoptilolite/L colostrum) during the first three meals, we concluded that administration of zeolites appears to reduce the incidence of diarrhea because only two calves from experimental group had health problems, one had bronchopneumonia and the other digestive transit difficulties with symptoms appearing after 28 days (not in neonatal period) in comparison with the control group where all calves had diarrhea in the first 11 days of life. The other parameter, growth performance measured on day 0, 45 and 90 revealed that during the first 45 days body weight of calves treated with clinoptilolite was significantly higher compared with the group of calves receiving only colostrum (C) (*p* < 0.0058) (E1/C = +16.96%). This statistical difference at 45 days may be explained by the high number of calves from the control group suffering from health problems and this affected the daily gain. At 90 days, the difference between groups was not significant (*p* < 0.1035) (E2/C = +7.19%) [69, 70]. Similar results were obtained by Step et al. who found that body weight and average daily gain did not differ between treatment groups (clinoptilolite dosage was 0.5 and 2%) [23]. More

#### **Figure 2.**

*Representative serum protein electrophoretograms observed in calves that received colostrum supplemented with 0.5% (e–h) and 2% clinoptilolite (i–l) compare with control group (a–d) at different time interval (0, 6, 16 and 30 h postpartum) [67].*

recently, Ural et al. observed increased total weight and mean daily gain in calves that receive clinoptilolite (1 or 2 g/kg) in colostrum at calving, 12 and 24 h [71].

In another study, the addition of 0.5 g and 1 g/kg body weight per day in colostrum and milk for 45 days reduced fecal score and its severity, probably by retarding effect of clinoptilolite on intestinal passage rate [21]. The activity of clinoptilolite on reducing signs of diarrhea could be caused by: alteration of metabolic acidosis through effects on osmotic pressure in the intestinal lumen; or through retention of the enterotoxigenic *E. coli* thus limiting its attachment to the intestinal cellmembrane receptors); and also due to water adsorption property of zeolites, the feces appear drier and more compact [32].

In human medicine there are studies that support the beneficial properties of purified natural clinoptilolite as an anti-diarrheic treatment [72]. More recent studies performed on aerobically trained subjects, who received for 12 weeks zeolite-clinoptilolite supplementation, highlighted the positive effects of zeolites on intestinal wall integrity. The results were based on decreased concentrations of zonulin, an intercellular tight junction modulator, improving in this way intestinal barrier integrity [73].

Also, clinoptilolite improve antioxidant capacity in broilers [74]; it is used as a feed additive in fish diets [75] and in turkey diets [76].

Analyzing the effects of clinoptilolite on mineral parameters in newborn Romanian Black and White calves that received 5 g/l (group E1) and 20 g/l (group E2) at parturition, and 12 and 24 h postpartum, we observed that clinoptilolite supplementation increased serum Ca (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), P (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), Mg concentration (increased in groups E1 in comparison with the control group (*p* < 0.003) and E2 (*p* < 0.009) at 30 h postpartum in neonatal calves) with the most spectacular increase in iron concentration (with +144.70% in group E1 (*p* < 0.0005) and with +126.16% in group E2 (*p* < 0.002) at 30 h postpartum) [64].

When analyzing the same parameters in other breed (Holstein) and other colostrum quantity (3 L), we observed that the most significant effect (*p* < 0.0006) was on serum iron concentrations in experimental (27.64 ± 3.78 μmol/l) vs. control group (8.93 ± 1.26 μmol/l) and it did not have negative effects on other biochemical parameters (Ca, P, Mg, GGT, ALAT, ASAT, ALP) after 48 h postpartum, one more time proving that morpho-functional processes that take place in the newborns, necessary for adapting to the new environment, were not affected by clinoptilolite [63]. A possible explanation of increased iron level could be that in duodenum and in the anterior part of jejunum, where iron absorption takes place, clinoptilolite influences iron absorption due to the ion exchange properties, altering in this way the pH or reducing intestinal transit of digesta, which could lead to a better utilization of nutrients [32, 77]. It has been shown previously that low intestinal motility and acidic pH promote iron absorption and that in the bovine neonate, the pH of the whole intestinal content ranges from 5.5 to 6.5; also, the motility of the gastrointestinal tract becomes well organized only after 2–3 days of postnatal life [78, 79]. This feature could be important in preventing iron deficiency anemia (Fe < 14.32 μmol/l) especially in veal calves fed exclusively with milk.

Short term supplementation of clinoptilolite did not affect hepatic and renal function of newborn calves and that morpho-functional changes of the newborn organism in adapting to extrauterine environment were normal, without any influence of clinoptilolite, as observed after analyzing *α*-amylase, total bilirubin, creatinine, uric acid, urea, glucose, cholesterol and triglycerides. Biochemical values were measured in the first 48 h in the newborn calves. Values recorded were physiological

**131**

in R&D.

*Zeolites Applications in Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.87969*

but also for the cow [83].

ing perspective in this field.

**Acknowledgements**

**6. Conclusion**

for the neonatal period and had no significant difference between groups, highlighting once more the safety of clinoptilolite in newborn nutrition [80].

The activity of the most important enzymes is changing very fast after the first feedings. We observed that adding clinoptilolite in the first three meals of colostrum influenced the enzymes as follows: GGT activity significantly increased in group E2 (20 g/L clinoptilolite) at 6 h after birth (E2/C: +64.83%, *p* < 0.05) and in group E1 (5 g/L clinoptilolite) at 16 h after birth (E1/C: +118.55%, *p* < 0.05) in comparison with the control group (C—received only mother colostrum); ALP increased after birth in all calves and adding clinoptilolite in colostrum influenced activity of ALP only in group E1 at 30 h postpartum; transaminases were low at birth in all calves but after feeding they increased, this coincided with the period when a morphofunctional condition of the liver is changing in a newborn calf; adding clinoptilolite (5 g/L colostrum) to colostrum determined increased ASAT (E1/C: +71.58%, *p* < 0.01) and ALAT (E1/C: +278.82%, *p* < 0.006) activity at 30 h postpartum [81]. As literature data suggests, serum GGT is the only enzyme to increase markedly as a result of its absorption from the colostrum; other serum enzymes, such as aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP), are presumably released from the tissues of the calf [82]. A good interpretation of the serum enzyme activity in newborn calves must consider the physiological increase which occurs after feeding colostrum in the first days after parturition, a period very important for the calf

Based on our research data and on continuously published literature data worldwide regarding the use of zeolites in veterinary medicine, we confirm that they can be used in animal nutrition as feed additives, mainly to reduce the gastrointestinal absorption of mycotoxins; in newborn calves, they can be used as enhancers of passive immunity during colostral period; also, to increase health status and growth performance of animals and as an adjuvant in anticancer treatment, with a promis-

This work was performed through the project "Ensuring excellence in R&D activity within BUASVM Timisoara" code 35PFE, developed with the support of Ministry of Research and Innovation, Romania, the contracting authority for Program 1—Developing national R&D, Subprogram 1.2—Performance Institutional, Institutional Development Project—Financing projects of excellence

### *Zeolites Applications in Veterinary Medicine DOI: http://dx.doi.org/10.5772/intechopen.87969*

*Zeolites - New Challenges*

barrier integrity [73].

feces appear drier and more compact [32].

feed additive in fish diets [75] and in turkey diets [76].

with +126.16% in group E2 (*p* < 0.002) at 30 h postpartum) [64].

(Fe < 14.32 μmol/l) especially in veal calves fed exclusively with milk.

Short term supplementation of clinoptilolite did not affect hepatic and renal function of newborn calves and that morpho-functional changes of the newborn organism in adapting to extrauterine environment were normal, without any influence of clinoptilolite, as observed after analyzing *α*-amylase, total bilirubin, creatinine, uric acid, urea, glucose, cholesterol and triglycerides. Biochemical values were measured in the first 48 h in the newborn calves. Values recorded were physiological

recently, Ural et al. observed increased total weight and mean daily gain in calves that receive clinoptilolite (1 or 2 g/kg) in colostrum at calving, 12 and 24 h [71]. In another study, the addition of 0.5 g and 1 g/kg body weight per day in colostrum and milk for 45 days reduced fecal score and its severity, probably by retarding effect of clinoptilolite on intestinal passage rate [21]. The activity of clinoptilolite on reducing signs of diarrhea could be caused by: alteration of metabolic acidosis through effects on osmotic pressure in the intestinal lumen; or through retention of the enterotoxigenic *E. coli* thus limiting its attachment to the intestinal cellmembrane receptors); and also due to water adsorption property of zeolites, the

In human medicine there are studies that support the beneficial properties of purified natural clinoptilolite as an anti-diarrheic treatment [72]. More recent studies performed on aerobically trained subjects, who received for 12 weeks zeolite-clinoptilolite supplementation, highlighted the positive effects of zeolites on intestinal wall integrity. The results were based on decreased concentrations of zonulin, an intercellular tight junction modulator, improving in this way intestinal

Also, clinoptilolite improve antioxidant capacity in broilers [74]; it is used as a

Analyzing the effects of clinoptilolite on mineral parameters in newborn Romanian Black and White calves that received 5 g/l (group E1) and 20 g/l (group E2) at parturition, and 12 and 24 h postpartum, we observed that clinoptilolite supplementation increased serum Ca (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), P (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), Mg concentration (increased in groups E1 in comparison with the control group (*p* < 0.003) and E2 (*p* < 0.009) at 30 h postpartum in neonatal calves) with the most spectacular increase in iron concentration (with +144.70% in group E1 (*p* < 0.0005) and

When analyzing the same parameters in other breed (Holstein) and other colostrum quantity (3 L), we observed that the most significant effect (*p* < 0.0006) was on serum iron concentrations in experimental (27.64 ± 3.78 μmol/l) vs. control group (8.93 ± 1.26 μmol/l) and it did not have negative effects on other biochemical parameters (Ca, P, Mg, GGT, ALAT, ASAT, ALP) after 48 h postpartum, one more time proving that morpho-functional processes that take place in the newborns, necessary for adapting to the new environment, were not affected by clinoptilolite [63]. A possible explanation of increased iron level could be that in duodenum and in the anterior part of jejunum, where iron absorption takes place, clinoptilolite influences iron absorption due to the ion exchange properties, altering in this way the pH or reducing intestinal transit of digesta, which could lead to a better utilization of nutrients [32, 77]. It has been shown previously that low intestinal motility and acidic pH promote iron absorption and that in the bovine neonate, the pH of the whole intestinal content ranges from 5.5 to 6.5; also, the motility of the gastrointestinal tract becomes well organized only after 2–3 days of postnatal life [78, 79]. This feature could be important in preventing iron deficiency anemia

**130**

for the neonatal period and had no significant difference between groups, highlighting once more the safety of clinoptilolite in newborn nutrition [80].

The activity of the most important enzymes is changing very fast after the first feedings. We observed that adding clinoptilolite in the first three meals of colostrum influenced the enzymes as follows: GGT activity significantly increased in group E2 (20 g/L clinoptilolite) at 6 h after birth (E2/C: +64.83%, *p* < 0.05) and in group E1 (5 g/L clinoptilolite) at 16 h after birth (E1/C: +118.55%, *p* < 0.05) in comparison with the control group (C—received only mother colostrum); ALP increased after birth in all calves and adding clinoptilolite in colostrum influenced activity of ALP only in group E1 at 30 h postpartum; transaminases were low at birth in all calves but after feeding they increased, this coincided with the period when a morphofunctional condition of the liver is changing in a newborn calf; adding clinoptilolite (5 g/L colostrum) to colostrum determined increased ASAT (E1/C: +71.58%, *p* < 0.01) and ALAT (E1/C: +278.82%, *p* < 0.006) activity at 30 h postpartum [81]. As literature data suggests, serum GGT is the only enzyme to increase markedly as a result of its absorption from the colostrum; other serum enzymes, such as aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP), are presumably released from the tissues of the calf [82]. A good interpretation of the serum enzyme activity in newborn calves must consider the physiological increase which occurs after feeding colostrum in the first days after parturition, a period very important for the calf but also for the cow [83].
