Zeolites are inorganic materials that contain regular-sized pores throughout the structure of the molecules. The pores allow ion exchange with various organisms or other molecules and trap target substances for further extrusion, breakdown, release, or elimination. Zeolite substances are found in mines throughout the world or may be formed via various manufacturing processes. The molecular pore size and configuration endow these materials with unique properties, which allow zeolites to be used in a wide range of biological and manufacturing processes.
Zeolite molecules are believed to selectively isolate molecules, compounds, or toxins on the basis of their specific pore size. Zeolites are used in water purification procedures, as catalysts in the petrochemical industry, for soil treatment in the agricultural field (by providing slow release of nitrogen into the soil), as an ingredient in laundry detergents, in solar thermal collectors and hydration refrigeration, and in the production of warm asphalt concrete mixture. In the medical field, zeolites are an integral component to the production of medical-grade oxygen. Most recently, zeolites have been used in anticancer therapy1 and as an antidiarrheic drug for humans.2 Moreover, new evidence has provided insights into potential uses of zeolites as supportive treatments of animals with gastrointestinal tract diseases, including intestinal parasite infections.3
Zeolites have been fed to various production animal species, and anecdotal and published reports4–15 have indicated positive responses to their ingestion. Clinoptilolite is a naturally occurring zeolite. Inclusion of clinoptilolite in production animal diets has been associated with beneficial effects, such as mycotoxin binding, improved growth and feed efficiency, changes in concentrations of volatile fatty acid produced by rumen microbes, and alterations in milk and body fat composition.7–10,12 In 1 study13 involving lactating dairy cows, clinoptilolite was included in rations at 1.25% and 2.5% of the diet. These rations were fed to the cows beginning approximately 30 days prior to anticipated parturition and throughout the entire lactation period. No clinoptilolite-associated adverse effects on measured hematologic variables were evident in those adult dairy cows.
Clinoptilolite has also been included in colostrum fed to neonatal dairy calves with beneficial results.14 Compared with control calves that were not fed clinoptilolite, serum concentrations of IgG were greater in clinoptilolite-treated calves.14 Other investigators assessed the effects of the addition of clinoptilolite to colostrum on serum triiodothyronine and thyroxine concentrations in calves during the first 48 hours after birth.15 In that study, treated calves had lower serum concentrations of those hormones, compared with non-treated control calves.
Reports of controlled studies evaluating health and hematologic and biochemical responses to clinoptilolite feed additives in young stressed dairy calves fed milk replacer are lacking in the veterinary medical literature. Veterinarians, nutritionists, and dairy calf producers have shown an interest in inclusion of clinoptilolite in rations and its influence, if any, on the overall health, performance, and gastrointestinal tract conditions (ie, diarrhea) in young calves. The purpose of the study reported here was to identify any adverse effects on health or performance in young dairy calves fed a commercial milk replacer mixed with clinoptilolite ([Na, K, Ca]6[Si,Al]36O72•2OH2O) over a 28-day period.
Materials and Methods
This protocol for the study was reviewed and approved by the Oklahoma State University Investigational Animal Care and Use Committee.
Calves and housing—Twenty-six sexually intact male Holstein dairy calves from a single-source commercial Idaho dairy were used in the study; the calves were 1 to 7 days old. The dairy was chosen as the source of calves because of a previous relationship with the study sponsor and their veterinarian (SMF); the willingness of the dairy personnel to follow specific protocols for proper handling and care of the neonates; and the attention to details, record keeping skills, and integrity demonstrated by the staff. At birth, each calf was individually identified. No later than 12 hours after birth, each calf received a volume of high-quality colostrum that was equivalent to 10% of its body weight (divided between 2 feedings). Plasma IgG concentration was also evaluated by use of a commercial test kita 24 hours before the calves were transported to the university facility.
The calves were transported from Idaho to Oklahoma to mimic the stress of transport conditions. It was realized that the distance of transport was longer than typical field conditions16; however, during the trip (2,420 km), all calves were observed at approximately hourly intervals; any clinical abnormalities were recorded, and affected calves were treated by a practicing veterinarian.
Approximately 15.5 hours after commencement of the journey, all calves were unloaded and allowed to rest at an isolated facility in which no cattle had been present for years. At 90 minutes after unloading, 2 quarts of commercial milk replacerb was fed via a nipple bottle to each calf. The calves were then allowed to rest for an additional 9.5 hours (total rest time, 11 hours) before being reloaded and transported to the Oklahoma State University Dairy Cattle Center.
On arrival at the center's facility, veterinarians and animal caretakers examined each calf for any abnormalities or injuries. After examination, each calf was fitted with a calf collar and tether and was housed individually in a polyethylene plastic hutch (length, 2.195 m; width, 1.219 m; height, 1.359 m). Hutches were not bedded and were placed on mowed Bermuda grass at the beginning of the study. Before starting the study, the calves were allowed to rest and acclimate to their new surroundings for 36 hours. After the acclimatization period, each calf was weighed and blood samples were collected in a 4.0-mL tube containing 7.0 mg of EDTAc for a CBC and in a 10.0-mL serum tubec for serum biochemical analyses. The quantity of blood collected at each sampling was determined by the manufacturer's preset vacuum in each blood tube. A skin biopsy sample was obtained from the ear (ear notch method) of each calf and submitted to the Oklahoma Animal Disease and Diagnostic Laboratory for immunohistochemical staining to detect persistent infection with bovine viral diarrhea virus.
Treatments—After initial processing, calves were randomly assigned to 1 of 3 treatment groups. Beginning on day 0, calves (n = 8) in the control group were fed commercial milk replacerb without clinoptilolite. Calves (n = 9) in the low-dosage group were fed commercial milk replacerb with 0.5% clinoptilolited on an as-fed basis based on the weight of milk replacer solids (12% of body weight) mixed into the milk replacer mixture solution daily, which equated to 0.0074% of body weight/d. Calves in the high-dose group (n = 9) were fed commercial milk replacerb with 2% clinoptilolited on an as-fed basis based on the weight of milk replacer solids mixed into the milk replacer mixture solution daily, which equated to 0.030% of body weight/d. There is no agreement or recommendation for specific dosages of clinoptilolite in young dairy calves to the authors' knowledge. However, the FDA classifies clinoptilolite as having GRAS (ie, generally regarded as safe) status, and the maximum recommendation is 2% as fed. The 2% clinoptilolite in the milk replacer was chosen to reflect the maximum GRAS recommendation, and there is anecdotal evidence to suggest benefits of 0.5% clinoptilolite. Many commercial milk replacers include antimicrobial drugs to minimize the influence and incidence of disease in early calf development programs. We specifically did not include any antimicrobial medications in the milk replacer used in this study to eliminate that confounder. The particular milk replacer was chosen because it is commercially available and commonly used in operations raising neonatal dairy calves.
Milk replacer, with or without clinoptilolite,d was fed in 2 separate feedings such that each calf consumed approximately 12% of its body weight (based on the replacer solids in the milk replacer mixture) for 28 days. Half of the calculated amount was provided at the morning feeding, and the other half was provided at the evening feeding. The amount of milk replacer fed was adjusted weekly according to each calf's weight gain. Commercial dairy calf starter was offered to each calf (handful amount initially) within 1 week of arrival, and the amount provided was increased as individual calf intake increased.
Sample collections and measurements—On days 1, 3, 5, 7, 10, 14, 17, 21, and 28 of the experiment, the weight and rectal temperature of each calf were measured and a blood sample (4 mL) was collected via jugular venipuncture by use of a 20-gauge needle into an evacuated tube containing EDTAc for a CBC. An additional sample (10 mL) was collected in a serum tubec for serum biochemical analyses and centrifuged at 2,000 × g for 20 minutes. Serum was harvested approximately 24 hours after collection. Serum samples were stored at −20°C until analysis could be performed following the conclusion of the study. The CBCs were performed within 12 hours after the samples were collected. The biochemical analyses of serum samples included assessment of phosphorus (mg/dL), magnesium (mg/dL), calcium (mg/dL), BUN (mg/dL), albumin (g/dL), total protein (g/dL), and globulin (g/dL) concentrations and creatine kinase (U/L), aspartate aminotransferase (U/L), alkaline phosphatase (U/L), and γ-glutamyltransferase (U/L) activities.
Assessment of fecal consistency—Subjective observations of feces from all calves were made twice daily at the times of feeding in the morning and late in the afternoon for the entire study period of 28 days. Heavy rainfalls occurred during several of the scheduled fecal observation periods; therefore, veterinarians or caretakers made special efforts to observe fresh stools at other times during those days, and a score (1 score/d) was assigned to describe fecal consistency following the procedure described by Larson et al.17 Fecal consistency scores were assigned at the first time of day that fresh feces was observed. Briefly, a numeric score was assigned as follows: 1 = normal fecal consistency (ie, firm but not hard); 2 = soft (does not hold form, piled but spreads slightly); 3 = runny (spreads readily to depth of approx 6 mm); and 4 = watery (liquid consistency, splattered).
Additionally, for any feces that were not considered normal, the color (ie, white, yellowish-white, gray, yellow, brown, red [bloody], green, dark green, or very dark or black) was recorded and any additional comments (ie, foamy, sticky, presence of mucus, or abnormally dry) were noted.
Health assessments—To assess their general health, calves were monitored twice daily by the veterinarians. Clinical signs that were of specific interest included hanging of the head, glazed or sunken eyes, slow movement, arched back, difficulty getting up from a recumbent position, knuckling or dragging toes when walking, stumbling when moving, anorexia, eating less than expected, and slow eating that was considered different from the animal's normal behavior. Calves with any of the aforementioned clinical signs were assigned a daily CA score as follows: 0 = apparently normal attitude; 1 = signs of mild depression; 2 = signs of moderate depression; 3 = signs of severe depression; and 4 = moribund.
Objective health evaluations included measurement of rectal temperature, a CBC, and serum biochemical analyses. Rectal temperature ≥ 39.7°C was considered abnormally high.
During the study, any calf that was assigned a CA score of 1 or greater underwent a physical examination to determine which body systems were involved. A clinical diagnosis was made and treatment administered to the individual as deemed appropriate by the attending veterinarian. During the study, any calf allocated to a treatment group that was assigned a CA score of 1 or greater underwent a physical examination to determine whether the signs of depression appeared to be related to ingestion of clinoptilolite or to dysfunction of another body system. If the physical examination findings in a clinoptilolite-treated calf indicated that the signs of depression were a result of clinoptilolite ingestion but the calf had a CA score of 1 or 2, no medications were administered, milk replacer with the calculated amount of clinoptilolite was continued, and the calf was monitored closely; if the calf had (or progressed to) a CA score ≥ 3, administration of clinoptilolite in the milk replacer was discontinued and appropriate treatments were administered. If the calf did not improve within 48 hours, euthanasia and necropsy were performed. If the calf improved within 48 hours, administration of clinoptilolite was resumed at the dosage of the assigned treatment group. If it was determined that the signs of depression in a clinoptilolite-treated calf were originating from a body system other than the digestive system, appropriate treatment was administered and the calf continued to receive clinoptilolite and remained in the study. If that calf did not respond within 48 hours, it was euthanized and a necropsy performed.
If any control or treated calf had a CA score of 3, recovered sufficiently to be returned to its group, and was assigned a CA score of 3 on another occasion, it was euthanized and underwent necropsy. If any control or treated calf had a CA score of 4 at any time, it was removed from the study, euthanized, and necropsied. All necropsies were performed at the diagnostic laboratory.
Postmortem examination and organ collection—At day 29, calves that remained in the study were sedated with xylazine (100 mg, IV) and euthanized by use of captive bolt followed by exsanguination. A necropsy was performed and appropriate tissues were harvested for microscopic examination to determine whether any histopathologic changes may have developed in treated calves, compared with control calves. In addition, intestinal contents were removed, internal organs were dissected and individually weighed, and organ weight was recorded.
Statistical analysis—Data were analyzed as a completely random design by use of statistical software.e The individual animal was considered the experimental unit. Health and performance variables were compared among groups. For data that were collected repeatedly over time, a repeated-measures analysis was used with an autoregressive first-order covariance structure and comparison of least square means was used to detect significant differences among treatments. A value of P ≤ 0.05 was considered significant.
Results
Passive transfer of immunoglobulins—For each calf, adequate passive transfer of immunoglobulins was evaluated by measuring serum total protein concentration.18 For purposes of the study, serum total protein concentration ≥ 6.0 g/dL and PCV of 25% to 38% were considered as a reliable field marker for adequate passive immunoglobulin transfer. To obtain enough calves for the study, 3 calves that had serum total protein concentration of 5.9 g/dL were included in the study; the other 23 calves had values ≥ 6.0 g/dL. Packed cell volume was within the desired range in all calves. Plasma IgG concentration was also evaluated by use of a commercial test kita 24 hours before the calves were transported to the university facility. Results of the IgG assay were positive for 25 calves, thereby indicating adequate passive transfer of immunoglobulins. For 1 calf, the commercial test yielded a faint line, which was interpreted as adequate IgG concentration status for that animal according to the manufacturer's directions. Coincidently, the serum total protein concentration in this calf was 5.9 g/dL; the other 2 calves that had the same serum total protein concentration yielded positive results via the commercial IgG assay, confirming adequate passive transfer of immunoglobulins in those calves.
Preshipment evaluation—Prior to shipment, each calf was evaluated clinically by 3 veterinarians (DLS, SMF, and a veterinary consultant for the commercial dairy) and assigned a CA score, and rectal temperature was measured. For all calves, rectal temperatures were within reference range (37.7° to 39.3°C). Four calves had signs of mild depression (CA score, 1) and 2 calves had signs of moderate depression (CA score, 2) on the day of transport.
Evaluation on arrival—On arrival at the university facility, all calves were assigned to individual hutches and allowed to rest for approximately 1 hour before they were each fed 2 quarts of a milk replacer mixture. The milk replacer was the same formulation, containing no antimicrobials, and from the same batch shipment that was used throughout the study. All 26 calves readily consumed all of their morning feeding.
Approximately 1 hour after this feeding, the calves were reevaluated by 3 veterinarians (DLS, LBR, and SMF). Signs of depression were considered severe (CA score, 3) in 1 calf, moderate (CA score, 2) in 1 calf, and mild (CA score, 1) in 3 calves. The other calves were alert and responsive to their surroundings. At this time, rectal temperatures in all calves were within reference range (38.0° to 39.4°C).
Overall, the trip was deemed a success considering the distance (2,420 km), the duration of transit (42 hours from dairy of origin to the university facility), the ambient temperature variations (ranging from 1.7°C in Wyoming to 22.8°C in Oklahoma), and the facts that some calves started the trip at 24 hours after birth and all 26 calves were in seemingly good physical condition on arrival at the final destination. To determine whether any calf was persistently infected with bovine viral diarrhea virus, a tissue sample was obtained from the ear of each calf and submitted for immunohistochemical staining; results were negative for all calves.
Clinical observations and assessments of performance—During the examinations after arrival at the university facility, 3 calves were found to be clinically dehydrated. It was decided that the 3 calves (1 assigned to the low-dosage group and 2 assigned to the high-dosage group) would benefit from IV administration of fluids (1 L of 0.45% NaCl–2.5% dextrose solution), even though the calves had consumed their aliquot of milk replacer on arrival. The 3 calves responded well to the fluid administration. An additional 5 calves (2 assigned to the control group, 2 assigned to the low-dosage group, and 1 assigned to the high-dosage group) required IV administration of fluids (1 L of 0.45% NaCl–2.5% dextrose solution) within the first 4 days of the study. All of those calves responded favorably to the fluid administration, with the exception of 1 calf in the high-dosage group, which had required treatment on arrival. That calf developed severe diarrhea and signs of severe depression. The calf was treated with ceftiofur hydrochloridef (2.2 mg/kg, SC, daily for 2 days) but did not respond clinically to the treatments. The calf was euthanized via IV administration of euthanasia solutiong on day 13 of the study. The diagnosis was marked segmental neutrophilic necrohemorrhagic enteritis. Salmonella organisms were isolated from the affected intestinal segment, and rotavirus was detected via a fluorescent antibody technique in one of the intestinal segments.
Another control calf became tachypneic and developed signs of depression on day 15 of the study. This calf was treated with danofloxacinh (6.0 mg/kg, SC, once) but did not respond clinically to the treatment. Dyspnea and signs of depression worsened, and the calf was euthanatized via IV administration of euthanasia solutiong on day 17. The diagnosis was severe subacute fibrinopurulent bronchopneumonia. Large numbers of Arcanobacterium pyogenes and moderate numbers of Mycoplasma bovis were isolated from the lungs of this calf.
Within the first 2 weeks of the study, 4 other calves (2 in the control group and 2 in the high-dosage group) developed signs of depression and other clinical signs, including abnormal respiration patterns, weakness, nasolacrimal discharge, and fever, that were interpreted as probable respiratory tract disease. These 4 calves were treated with ceftiofur hydrochloridef (2.2 mg/kg, SC, once daily for 3 to 5 days). All of these calves responded clinically and were able to remain in the study until its conclusion.
Despite some unusual adverse weather conditions that occurred in Oklahoma during the study, calves appeared to do well overall with regard to health and performance. Some of the daily amounts of precipitation were incompletely recorded during the study period; however, 31.6 cm of rainfall were recorded during the time of the study (28 days). This amount exceeded the average total rainfall for May (13.8 cm) and June (11.0 cm) by 6.8 cm (combined total for 61 days, 24.8 cm).19 Because the duration of the study period was 28 days, the amount of rainfall during this time would have exceeded the daily average rainfall by several more centimeters of precipitation. During the heavy rainfall periods, all of the hutches were bedded with shavings for the comfort of the calves.
Body weight and average daily gain of calves in the 3 treatment groups were not significantly (P ≥ 0.12) different among treatments (Table 1). In addition, no day-treatment interaction was detected for rectal temperature (P = 0.79); however, mean rectal temperature for the control group was 0.6°C higher (P < 0.001) than values in the low-dosage or high-dosage groups. Nevertheless, all rectal temperatures were considered within the reference range (Table 2). No differences in fecal characteristics (fecal score) were evident during the first 8 days of the study (P = 0.24). Mean weekly fecal scores did not differ significantly (P = 0.76) during the entire study.
Mean body weight and average daily gain in 26 young male Holstein calves fed milk replacer without clinoptilolite (control group; n = 8), with 0.5% clinoptilolite* (low-dosage group; 9), or with 2% clinoptilolite† (high-dosage group; 9) during a 28-day period. Feedings began on day 0 of the study and were calculated to provide 12% of body weight as milk replacer solids (divided between 2 meals).
| Variable | Day or interval | Group | SEM | P value | ||
|---|---|---|---|---|---|---|
| Control | Low-dosage | High-dosage | ||||
| Weight (kg) | 0 | 44.6 | 40.8 | 41.5 | 1.54 | 0.21 |
| 5 | 46.8 | 41.9 | 44.5 | 2.10 | 0.25 | |
| 7 | 47.2 | 41.9 | 42.6 | 2.08 | 0.15 | |
| 10 | 49.0 | 43.9 | 42.3 | 2.17 | 0.12 | |
| 14 | 51.6 | 46.8 | 47.9 | 2.07 | 0.21 | |
| 17 | 52.8 | 48.4 | 50.1 | 2.12 | 0.32 | |
| 21 | 55.9 | 53.5 | 52.3 | 2.22 | 0.25 | |
| 27 | 62.1 | 57.7 | 58.8 | 3.26 | 0.61 | |
| Average daily gain (kg) | 0-7 | 0.35 | 0.15 | 0.15 | 0.23 | 0.31 |
| 0-14 | 0.48 | 0.42 | 0.39 | 0.13 | 0.54 | |
| 0-21 | 0.50 | 0.46 | 0.46 | 0.12 | 0.86 | |
| 0-28 | 0.59 | 0.59 | 0.58 | 0.17 | 0.98 | |
Commercial milk replacer with 0.5% clinoptilolite on an as-fed basis based on the weight of milk replacer solids mixed into the milk replacer mixture solution daily, which equated to 0.0074% of body weight/d.
Com-mercial milk replacer with 2.0% clinoptilolite on an as-fed basis based on the weight of milk replacer solids (12% of body weight) mixed into the milk replacer mixture solution daily, which equated to 0.030% of body weight/d.
SEM = Standard error of the least squares means.
Mean rectal temperature and fecal consistency scores in 26 young male Holstein calves fed milk replacer without clinoptilolite (control group; n = 8), with 0.5% clinoptilolite* (low-dosage group; 9), or with 2% clinoptilolite† (high-dosage group; 9) during a 28-day period. Feedings began on day 0 of the study and were calculated to provide 12% of body weight as milk replacer solids (divided between 2 meals). Rectal temperature was assessed on days 1, 3, 5, 7, 10, 14, 17, 21, and 28; fecal consistency was assessed twice daily during the entire 28-day study period.
| Variable | Group | SEM | P value | |||
|---|---|---|---|---|---|---|
| Control | Low-dosage | High-dosage | Treatment effect | Treatment-day interaction | ||
| Fecal score during the first 8 days of the study | 2.79 | 2.90 | 3.24 | 0.19 | 0.24 | 0.79 |
| Mean weekly fecal score | 2.66 | 2.67 | 2.79 | 0.14 | 0.76 | 0.62 |
| Rectal temperature (°C) | 39.1a | 38.5b | 38.5b | 0.13 | < 0.001 | 0.79 |
Fecal consistency was scored by use of a published system17 (with modifications) as follows: 1 = Normal consistency (soft but not hard), 2 = Soft (piles but spreads slightly), 3 = Runny (spreads readily to a depth of approx 6 mm), and 4 = Watery (liquid consistency).
Within a variable, values with different superscript letters were significantly (P ≤ 0.05) different.
See Table 1 for remainder of key.
Complete blood cell counts were performed on blood samples collected from each calf on days 1, 3, 5, 7, 10, 14, 17, 21, and 28 (Table 3). Among the absolute and percentage WBC counts, there were no day-treatment interactions. There was a significant treatment effect for the percentage of monocytes; the percentage of monocytes was greater in the low-dosage group, compared with values in the control and high-dosage groups (P = 0.03). Serum biochemical analyses were performed on the aforementioned blood samples (Table 4). A treatment effect for BUN concentration was evident; the value for the control group was significantly (P = 0.007) greater than values in either of the clinoptilolite treatment groups. Serum total protein concentration was significantly (P = 0.002) greater in the low-dosage group, compared with concentrations in the control and high-dosage groups. However, serum globulin concentration was significantly (P = 0.004) different among the 3 groups, being highest in the low-dosage group (2.77 g/dL) and lowest for the high-dosage group (2.36 g/dL); the value in the control group was 2.50 g/dL.
Mean absolute and percentage differential blood cell and platelet counts and other hemato-logic variables in 26 young male Holstein calves fed milk replacer without clinoptilolite (control group; n = 8), with 0.5% clinoptilolite* (low-dosage group; 9), or with 2% clinoptilolite† (high-dosage group; 9) during a 28-day period. Feedings began on day 0 of the study and were calculated to provide 12% of body weight as milk replacer solids (divided between 2 meals). Assessments were made on days 1, 3, 5, 7, 10, 14, 17 21, and 28 of the study period.
| Variable | Group | SEM | P value | |||
|---|---|---|---|---|---|---|
| Control | Low-dosage | High-dosage | Treatment effect | Treatment-day interaction | ||
| Absolute count | ||||||
| WBCs(× 109 cells/L) | 11.7 | 10.77 | 11.8 | 0.81 | 0.58 | 0.62 |
| Granulocytes (× 109 cells/L) | 5.15 | 5.39 | 5.77 | 0.07 | 0.85 | 0.21 |
| Lymphocytes (× 109 cells/L) | 6.27 | 5.61 | 5.71 | 0.32 | 0.27 | 0.95 |
| Monocytes (× 109 cells/L) | 0.28 | 0.43 | 0.34 | 0.75 | 0.36 | 0.49 |
| Erythrocytes (× 109 cells/L) | 8.68 | 9.24 | 9.24 | 0.34 | 0.38 | 0.44 |
| Platelets (× 109 platelets/L) | 617.00 | 658.00 | 637.00 | 33.1 | 0.66 | 0.19 |
| Percentage count (%) | ||||||
| Granulocytes | 40.8 | 41.4 | 45.6 | 2.69 | 0.37 | 0.92 |
| Lymphocytes | 57.1 | 54.1 | 51.6 | 2.76 | 0.34 | 0.66 |
| Monocytes | 2.00a | 3.78b | 2.79a | 0.47 | 0.03 | 0.30 |
| Platelets | 0.36 | 0.39 | 0.37 | 0.02 | 0.58 | 0.17 |
| Hct(%) | 32.0 | 34.1 | 34.6 | 1.46 | 0.37 | 0.50 |
| Hemoglobin (g/dL) | 9.05 | 10.1 | 9.93 | 0.75 | 0.52 | 0.37 |
| Mean corpuscular volume (fL) | 37.0 | 36.8 | 37.2 | 0.80 | 0.91 | 0.85 |
| Mean plateletvolume (fL) | 5.88 | 5.93 | 5.89 | 0.06 | 0.82 | 0.75 |
| RDWc (%) | 24.7 | 24.3 | 24.7 | 0.33 | 0.65 | 0.57 |
| Mean corpuscular hemoglobin (pg) | 10.4 | 10.9 | 10.9 | 0.30 | 0.43 | 0.89 |
| Mean corpuscular hemoglobin concentration (g/dL) | 28.1 | 28.9 | 28.5 | 0.23 | 0.08 | 0.49 |
| PDWc (%) | 32.9 | 33.2 | 33.0 | 0.41 | 0.88 | 0.13 |
RDWc = Red cell distribution width. PDWc = Platelet distribution width.
See Tables 1 and 2 for key.
Mean values of serum and plasma clinicopathologic variables in 26 young male Holstein calves fed milk replacer without clinoptilolite (control group; n = 8), with 0.5% clinoptilolite* (low-dosage group; 9), or with 2% clinoptilolite† (high-dosage group; 9) during a 28-day period. Feedings began on day 0 of the study and were calculated to provide 12% of body weight as milk replacer solids (divided between 2 meals).
| Variable | Group | SEM | P value | |||
|---|---|---|---|---|---|---|
| Control | Low-dosage | High-dosage | Treatment effect | Treatment-day interaction | ||
| Phosphorus (mg/dL) | 8.09 | 8.48 | 8.33 | 0.17 | 0.27 | 0.74 |
| Magnesium (mg/dL) | 2.23 | 2.28 | 2.21 | 0.03 | 0.30 | 0.47 |
| Creatine kinase (U/L) | 136 | 247 | 141 | 50.5 | 0.23 | 0.59 |
| Calcium (mg/dL) | 10.3 | 10.1 | 10.3 | 0.14 | 0.56 | 0.25 |
| BUN (mg/dL) | 9.90 | 8.52a | 8.62b | 0.33 | 0.007 | 0.27 |
| Aspartate aminotransferase (U/L) | 62.8 | 62.6 | 58.7 | 1.56 | 0.11 | 0.61 |
| Alkaline phosphatase (U/L) | 171 | 184 | 169 | 19.0 | 0.83 | 0.31 |
| γ-Glutamyltransferase (U/L) | 47.4 | 89.6 | 60.5 | 19.9 | 0.31 | 0.009 |
| Albumin (g/dL) | 3.05 | 3.65 | 3.12 | 0.32 | 0.34 | 0.40 |
| Total protein (g/dL) | 5.54a | 5.89b | 5.50a | 0.07 | 0.002 | 0.26 |
| Globulin (g/dL) | 2.50a | 2.77b | 2.36c | 0.07 | 0.004 | 0.29 |
Within a variable, values with different superscript letters were significantly (P ≤ 0.05) different.
See Table 1 for remainder of key.
Gross postmortem examination findings—Two calves were euthanized before the termination of the study: one as a result of severe fibrinopurulent bronchopneumonia (euthanized on day 17) and the other as a result of segmental necrohemorrhagic enteritis (euthanized on day 13) from which Salmonella organisms were cultured. All other calves remained in the study until day 28.
During necropsy, organs were weighed. Abomasums of the control calves weighed more than those of the calves in the low-dosage and high-dosage groups (mean abomasal weight, 0.457 kg vs 0.359 kg and 0.397 kg, respectively; P = 0.02). For the other organs examined, there were no significant (P ≥ 0.10) differences in weight or length.
No gross lesions that were interpreted as attributable to clinoptilolite treatment were detected in any of the calves. Of the calves that survived until termination of the study, several had minimal to mild bronchopneumonia (Table 5). A few calves had other lesions that commonly develop in intensely managed calves, such as tubulointerstitial nephritis, small hepatic abscesses, and abomasal ulcers. Other lesions included severe unilateral pyelonephritis, multifocal necrotizing rumenitis, and bilateral purulent otitis media (each of which was detected in 1 calf); these lesions were not associated with any treatment group. The only other pertinent change observed at gross necropsy was the accumulation of variable amounts of fine reddish-brown grit admixed with feed material in the abomasums of 12 of the calves. Calves with accumulations of gritty material in the abomasal content were equally distributed across treatment groups (4 in each group). In some calves, grit was conspicuously admixed with abomasal content but did not comprise a substantial portion of the abomasal content; in other calves, accumulations of grit comprised approximately 20% to 40% of the abomasal content and were large enough to form aggregates of mud-like material. Rumen papilla length or development was subjectively evaluated in each calf and appeared negatively affected in 7 calves; these calves were distributed across all 3 treatment groups.
Distribution of gross lesions and frequency of early termination from a study in 26 young male Holstein calves fed milk replacer without clinoptilolite (control group; n = 8), with 0.5% clinoptilolite* (low-dosage group; 9), or with 2% clinoptilolite† (high-dosage group; 9) during a 28-day period. Feedings began on day 0 of the study and were calculated to provide 12% of body weight as milk replacer solids (divided between 2 meals).
| Gross lesion | Group | ||
|---|---|---|---|
| Control | Low-dosage | High-dosage | |
| Short rumen papilla | 2 | 4 | 1 |
| Abomasal grit or mud-like material | 4 (2 [++] and 2 [+++]) | 4 (3 [++] and 1 [+++]) | 4 (3 [++] and 1 [+++]) |
| Abomasal ulcers | 1 | 0 | 2 |
| Bronchopneumonia | 7 (2 [+], 3 [++], and 2 [+++]) | 5(4[++]and1 [+++]) | 6 (all +) |
| Hepatic abscess | 2 | 0 | 1 |
| Tubulointerstitial nephritis | 1 | 2 | 2 |
| Early termination from study | 1 (bronchopneumonia) | 0 | 1 (Salmonella enteritis) |
Abomasal grit or mud-like material was characterized as follows: ++ = Obvious grit and +++ = Aggregates of mud-like material. Severity of bronchopneumonia was characterized as follows: + = Minimal, ++ = Mild, and +++ = Moderate.
See Table 1 for remainder of key.
Histologic examination findings—Sections of lungs, heart, liver, gallbladder, pancreas, kidneys, and spleen as well as specimens from each segment of the gastrointestinal tract (rumen, abomasum, duodenum, jejunum, ileum, cecum, and spiral colon) were collected from most calves for histologic examination. Definitive identification of a specific intestinal segment was not possible for 4 calves (the duodenum was not definitively identified and examined microscopically in 3 calves, and the ileum was not definitively identified and examined microscopically in 1 calf). Cecum and colon were categorized collectively as large intestine.
Examination of gastrointestinal tissue specimens revealed variable populations of neutrophils and eosinophils as well as lymphocytes and plasma cells. The number and type of inflammatory cells in the lamina propria and tunica submucosa varied among regions of the gastrointestinal tract and among calves; however, these changes did not alter the mucosal architecture, and no definitive inflammatory lesion was detected in any of the calves. Numbers of neutrophils and eosinophils were subjectively estimated in representative hpfs of sections of abomasum, small intestine, and large intestine for each calf. These estimates of inflammatory cell numbers were compared as a means of assessing relative differences among calves and treatment groups. Even with moderate variability among individual calves, inflammatory cell populations in each gastrointestinal region examined were similar among treatment groups. The only discernible difference in gastrointestinal inflammatory cell populations among groups was in the jejunal mucosa. In this tissue, calves in the high-dosage group typically had the highest number of eosinophils; calves in the low-dosage group had fewer eosinophils than calves in the high-dosage group but more eosinophils than calves in the control group.
Histologically, no lesions other than those associated with the sporadic disease processes reported previously were detected in any of the tissues examined.
Discussion
The objective of the present study was to identify any adverse effects on health or performance in young dairy calves fed milk replacer mixed with clinoptilolite. Reports of previous research on the effects of feeding clinoptilolite in milk replacer to young dairy calves are limited to our knowledge. Because there were no available data on clinoptilolite inclusion in milk replacer intended for dairy calves, it was considered that inclusion of 0.5% and 2.0% clinoptilolite on an as-fed basis would be suitable on the basis of studies in beef cattle10 and lambs.11 After analyzing the response variables in our study, there was no evidence that health or performance of dairy calves was affected negatively by ingestion of 0.5% and 2.0% clinoptilolite. Likewise, no negative effects on blood metabolites and enzymes or WBC counts were detected. Reasons for the differences in serum total protein and globulin concentrations among treatments are not readily apparent. It is interesting to note that rumen-cannulated beef steers fed 2.5% or 5% clinoptilolite had decreased ruminal ammonia concentrations,10 compared with findings in steers fed diets without clinoptilolite,10 which may partially explain the decrease in BUN concentrations detected in the calves fed clinoptilolite in the present study.
The accumulation of red-brown, finely gritty material in the abomasums of calves in our study was interesting but was not associated with the absence or presence of clinoptilolite in the ingested milk replacer. Unusually rainy and muddy conditions may have led to increased ingestion of mud (possibly from drinking out of puddles or normal grooming behavior) with secondary abomasal accumulation of silt or dirt. No gross pathologic changes that had an obvious association with clinoptilolite ingestion were evident in any of the calves. The character and intensity of inflammatory cell populations within the mucosa and submucosa of various regions of the gastrointestinal tract were considered within the range of normal variation because no architectural alterations or other lesions accompanied the inflammatory cell infiltrations. The subjective assessment that there were greater numbers of eosinophils in the jejunum of clinoptilolite-treated versus control calves was a subtle distinction, and even in the high-dosage group, no alteration in tissue architecture was associated with eosinophilic infiltrates. The cause and importance of increased numbers of eosinophils in the jejunal mucosa of calves in the high-dosage groups are not known but did not appear to be associated with an effect on intestinal function. The lack of adverse effects attributed to ingestion of clinoptilolite in milk replacer in calves of the present study appears to suggest that this zeolite may be included in milk replacer fed to young dairy calves to improve performance and reduce the incidence of diarrhea.
ABBREVIATION
| CA | Clinical attitude |
Whole blood calf IgG Midland quick test kit, Midland Bioproducts Corp, Boone, Iowa.
Advance Excelerate calf milk replacer, MSC Specialty Nutrition, Dundee, Ill.
Vacutainer, Becton-Dickinson, Franklin Lakes, NJ.
Zeolite clinoptilolite, Zeocorp Mine, Hines, Ore.
SAS, MIXED procedure, version 9.1, SAS Institute Inc, Cary, NC.
Excenel RTU, Pfizer Inc Animal Health Group, New York, NY.
Beuthanasia-D Special (euthanasia solution), Schering-Plough Animal Health Corp, Union, NJ.
A180 injectable solution, Pfizer Inc Animal Health Group, New York, NY.
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