Evaluation of Four Dry Commercial Gut Loading Products for Improving the Calcium Content of Crickets, Acheta domesticus

Transcription

1 N u t r i t i o n N o t e s Evaluation of Four Dry Commercial Gut Loading Products for Improving the Calcium Content of Crickets, Acheta domesticus Mark D. Finke1, PhD, Shari U. Dunham2, PhD, Christabel A. Kwabi2 1. Mark D Finke Inc., 6811 E Homed Owl Trail, Scottsdale, AZ 85262, USA 2. Colby College, 5762 Mayflower Hill, Waterville, ME 04901, USA Abstract: Crickets, Acheta domesticus, are commonly fed special diets to alter their nutrient content (especially with regard to calcium) to make them a more complete diet for insectivorous reptiles and amphibians. Calciumfortified dry diets are used by zoos to increase a crickets calcium content and a variety of products are available in pet stores for the hobbyist. In this experiment we compare the moisture, calcium and phosphorus content of small cricket nymphs and adult crickets offered an experimental dry calciumfortified gut loading diet with those fed four commercial dry diets. An unfortified dry diet served as a negative control. Crickets fed the experimental calciumfortified dry diet and one of the commercial products (TRex Calcium Plus Food for Crickets) contained sufficient calcium to meet the estimated requirements of insectivorous reptiles and significantly more calcium than those fed the other treatments. Crickets fed the other three commercial products contained no more calcium than those fed an unfortified diet and would likely be considered calcium deficient when used as food for insectivorous reptiles. Cricket nymphs contained significantly more moisture (as is basis) and phosphorus (dry weight basis) than adult crickets but neither were affected by diet. Despite marketing claims on three of the four products suggesting their use as a calcium source for gut loading crickets, only one of the commercial foods tested was effective in increasing the calcium content of crickets. K ey words: crickets, Acheta domesticus, calcium, gut loading. I n t r o d u c t io n Crickets, Acheta domesticus, are an important food source for many insectivorous reptiles and amphibians kept by pet owners but unless treated, contain inadequate levels of calcium to meet the animal s requirement (Allen and Oftedal 1989, Pennino, et al, 1991, Barker, et al, 1998, Finke, 2002). There are a variety of commercial products designed to enhance the calcium content of crickets before being used as food for reptiles. These products fall into one of three broad categories. The first group of products are dusts (typically composed primarily of calcium carbonate) which are used to coat the cricket just prior to being used as food. While effective, this method can provide variable results since the amount that adheres to the insect depends on the characteristics of the powder (calcium content, particle size and electrostatic properties), the size of the cricket, and the ability of the cricket to groom itself and remove the added calcium (Trusk and Crissey, 1987, Winn, et al, 2003). A second group of products consists of calcium fortified highmoisture cricket waters (typically hydrated polyacrylamide gels containing 95% or more water) or high moisture foods designed to provide crickets with both food and water. While seemingly popular based on their presence in pet stores these products are ineffective at increasing cricket calcium content (Finke, et al, 2004). The third group of products are highcalcium dry diets. They are often called gut loading diets since they increase the cricket s calcium content, not because the calcium is absorbed by the cricket but rather due to the food retained in the crickets gastrointestinal tract (Allen and Oftedal 1989). These high calcium diets are usually fed hr prior to using the crickets as food since high calcium diets are not suitable for growth and reproduction of crickets. When these diets are used to gut load crickets factors other than diet calcium can also affect cricket calcium content. These factors include diet palatability, diet particle size, and the size of the cricket (Allen and Oftedal, 1989, Anderson, 2000, Hunt, et al, 2001, Finke 2003, Winn, et al, 2003). As such, a simple analysis of dietary calcium may not be effective in predicting cricket calcium content. Recently Anderson (2000) evaluated a number of commercial products available for use by zoos, however no data are available concerning the effectiveness of diets available to hobbyists. Because commercial products range widely with respect to formula, physical form and calcium content, this experiment was designed to test the effects of calciumfortified dry commercial products on the calcium content of both cricket nymphs and adult crickets. Journal of Herpetological Medicine and Surgery 7

2 M a t e r ia l s and M ethods Treatments The experiment consisted of six treatments (Table 1). Treatment 1 has previously been shown to result in crickets that contain sufficient calcium for insectivorous reptiles and thus served as a positive control, while treat ment 2 served as a negative control (Finke, 2003, Finke, et al, 2004). Treatments 3 through 6 were four commercial gut loading diets labeled for use with crickets. Animals Small cricket nymphs and adult crickets were obtained from Industries ( Industries Inc, Marion, IL) and used as received. Food Sources The diet for treatment 1 was Food mixed with 15% calcium carbonate (HuberCal, JM Huber Corporation, Edison, NJ) to achieve an estimated 65 g Ca/kg diet (Finke, 2003, Finke, et al, 2004). The diet for treatment 2 was Cricket Power Food (a diet designed for cricket growth and repro duction) without any additional supplementation. The diets for treatments 3 through 6 were commercial dry gut load ing cricket foods (treatment 3 Fluker s HighCalcium Cricket Feed (Fluker Laboratories, Port Allen, LA); treat ment 4 ESU Reptile Gut Load Cricket and Insect Food (Energy Savers Unlimited Inc., Carson, CA); treat ment 5 Jurassi'Diet Gutload for crickets and other prey (JurassiPet, Stone Mountain, GA) and treatment 6 TRex Calcium Plus Food for Crickets (TREX Products Inc., Chula Vista, CA). The ingredient statements (reproduced exactly as they appear on the label) and, when available, the lot codes for the four commercial products are shown in the appendix. The products were chosen because they were featured at our local pet stores and are presumably used by many reptile hobbyists. All food sources were purchased at retail. Particle Size Distribution Three 25 g samples were placed on a Tyler Screen series and shaken thoroughly. The amount of material retained on each of the screens was weighed and particle size distribution was determined. Feeding Experiments All experiments were carried out at a temperature of C (78 83 F) with a photope riod o f 12:12 (light:dark). Fifty to sixty adult crickets (individual weight mg; mean + SEM) or 200 to 250 cricket nymphs (individual weight mg) were placed in each of twelve (one container/treatment) plastic rectangular containers (26.5 x 15.9 x 17.1 cm [length x width x height]) with ventilated plastic tops (K ritter Keeper, Lee s Pet Products, San Marcos, CA). Cardboard egg cartons were stacked in the middle of each container to increase surface area and to provide cover. Food and dis tilled water were provided in small dishes (6.1 x 1.2 cm [diameter x height]). Small pieces o f paper towel were placed in the water dishes to prevent the crickets from drowning. For both food and water, two dishes were placed in the comer of each container to help minimize competition for food and water. Dishes were cleaned and food and water replaced at 20 and 40 hr. All crickets were fed the diets for 48 hr after which they were frozen. A time Journal of Herpetological Medicine and Surgery period of 48 hr was used as it has been previously shown 1 to be sufficient time to maximize calcium content of crick 1 ets when they are fed a calciumfortified diet (Allen & I Oftedal, 1989, Anderson 2000, Hunt et al, 2001). Chemical Analysis Diets and insects were kept frozen 1 until analyzed for m oisture, calcium and phosphorus. ^ M oisture was determ ined by adapting AOAC method I with oven drying at 80 C (176 F) for several days i followed by a minimum o f six hours in a vacuum oven 1 (AOAC, 1990). For calcium and phosphorus analysis, dry I samples (approximately cricket nymphs or 9 12 H I adult crickets) were digested in concentrated nitric acid I (trace metal grade) using a Microwave Sample Preparation 1 System (MDS2000, CEM Corporation, Matthews, NC). I Digested samples were diluted and analyzed for calcium I (at nm with background correction) and phospho1 rus (at nm ) on a L eem an L abs PS1000 I In d u ctiv ely C o u p led P lasm a A tom ic Em ission 3 Spectrom eter using a m odification o f AOAC method (AOAC 1990). Statistical Analysis Insect moisture, calcium and phos^h phorus concentrations were analyzed using an ANOVA and. between treatm ent differences detected with Tukey s 1 Studentized Range Test. Statistical analysis was performed I u sin g S tatg rap h ics P lus for W indow s 5.0 softw are I (Statistical Graphics Corp, Rockville, MD). R esults The particle size distribution of the six diets is shown in 1 table 1. Treatments 1 and 2 were both a fine, uniform pow 1 der and consisted mostly of particles less than mm in I diameter. Treatments 5 and 6 were also uniform but con1 sisted o f somewhat larger particles. For treatment 5 most a particles were between and 1.00 mm in diameter a while treatment 6 consisted o f small extruded nuggets 1 greater than 1.70 mm in diameter. In contrast treatments 3 1 and 4 were very heterogeneous with a small amount of I fairly large particles (greater than 3.35 mm in diameter)* that appeared to be course particles of grains and/or parti 1 cles of dried molasses. The remainder o f treatments 3 and 4 were fairly evenly distributed through the remaining par tide size classifications. The calcium and phosphorus content of the food sources used in the six treatments is shown in table 1. The experi1 mental calcium fortified diet contained 65.4 g Ca/kg diet I while the unfortified diet contained 10.0 g Ca/kg diet. The j four dry commercial gut loading diets contained 10.9, I 13.2, 17.1 and g Ca/kg diet for treatments 3 through 1 6 respectively. Three o f the commercial products had a I minimum guarantee for calcium on their label but only two I met their minimum guarantees. The low calcium content (10.9 g Ca/kg) o f treatment 3 relative to the guarantee on 1 the label (minimum 80.0 g Ca/kg diet) appears to be typi 1 cal for this product. This experiment was done in M ay and samples with the identical label obtained both, earlier (December 2002 and December 2003), and later 1 (July 2004) contained only 9.6, 18.4 and 14.4 g Ca/kg diet 1 respectively. Both o f the commercial products that had a i

3 minimum guarantee for phosphorus on their label exceed ed their guarantee. The effect o f the six treatments on cricket moisture, cal cium and phosphorus content is shown in table 2. Cricket moisture content was affected by cricket size (F = l67.23; po.oool) but not by diet (F=0.88; p=0.5052). There were significant diet (F=94.91; po.oool) and size (cricket nymphs versus adult crickets) (F=52.26; po.oool) effects on cricket calcium content. In addition for cricket calcium content there was also a significant (F=5.84; p=0.0010) interaction between cricket size and diet. For both cricket nymphs and adult crickets, those fed the calciumfortified dry diet (treatment 1 positive control) and treatment 6 (TRex C alcium P lus Food fo r C rick ets) co n tained significantly more calcium (nymphs p < 0.001; adults p < 0.005) than crickets fed the other four treatments. There were no significant differences (nymphs and adults p > 0.10) between the calcium content of the crickets on treat ment 2 (negative control) and those fed the other three commercial diets (treatments 3, 4 and 5). Cricket phospho rus content was affected by crick et size (F=40.85, po.oool) but not by diet (F = l.01; p=0.4268). Only crick et nymphs on treatments 1 and 6 had calcium:phosphorus ratios greater than 1:1. Adult crickets on treatments 1 and 6 likely contained sufficient calcium to meet the require ments for most reptiles but had calcium:phosphorus ratios less than 1:1. Both adult crickets and cricket nymphs on treatments 2 through 5 had calcium:phosphorus ratios less than 0.40:1. D is c u s s io n A palatable finely ground dry diet containing g Ca/kg diet has been shown to be suitable for gut loading crickets (Allen and Oftedal, 1989, Hunt, et al, 2001, Finke, 2003, Finke, et al, 2004). In this experiment feeding a finely ground dry diet containing 65.4 g Ca/kg diet and a commercial extruded dry diet containing g Ca/kg diet resulted in crickets that would meet the estimated min imum dietary calcium requirement (6.1 to 8.5 g Ca/kg dry matter basis) of leopard geckos (Allen, 1997). The calcium and phosphorus content of crickets from both treatments 1 and 2 are consistent with those o f two previous studies although in this study the calcium content of adult crickets on treatment 1 was slightly lower than that reported previ ously (Finke, 2003, Finke, et al, 2004). Three o f the commercial gut loading products tested resulted in crickets containing no more calcium than those fed an unfortified diet (treatment 2). As a food source, insects are likely to be lacking in sev eral nutrients with calcium being among the most critical (Pennino, et al, 1991, Ferguson, et al, 1996, Barker, et al, 1998, Finke, 2002, Finke, 2003). For commercial gut load ing diets calcium appears to be an important marketing message as three of the four products specifically imply their value as a calcium source with claims such as HighC alcium (treatm ent 3 F lu k er s HighCalcium Cricket Feed ), provides feeder crickets, meal worms and king mealies with a supercharge of carbohydrates, cal cium and beneficial calories to make them an ideal food source for reptiles (treatment 4 ESU Reptile Gut Table 1. Particle size and calcium and phosphorus content (g/kg) of the diets used in the experiment. All data reported on an as is basis. For particle size measurements values are means + SEM of three tests. For chemical assays values are means + SEM and num ber of replicates are shown in parenthesis. No guarantee. Food + 15% CaC 0 3 Treatment % retained on 3.35 mm screen Food Fluker's HighCalcium Cricket Feed iiil 111 retained on 1.70 mm screen ESU Reptile Gut Load Cricket and Insect Food ills 3.0 ± 0.6% 3.0 ± 0.4% 13.8 ± 1.1% 19.5 ±0.6% Jurassi Diet Gutload for TRex Calcium crickets and Plus Food for Crickets other prey ±4.2% % 61.5 ±4.0% % retained on 1.00 mm screen 2.0 ± 0.2% 2.2 ±0.1% 27.2 ± 0.9% 28.0 ±1.1% 3.6 ± 1.1% 0.8 ± 0.2% % retained on mm screen 12.1 ±0.1% 15.2 ± 1.2% 21.3 ±0.8% 16.2 ±1.2% 85.9 ±1.3% 0.1 ±0.1% % retained on mm screen 19.7 ±0.4% 22.5 ±0.1% 14.1 ±0.6% 9.5 ± 1.6% 8.1 ±0.2% % through the mm screen 66.2 ±0.6% 60.1 ±1.2% 20.6 ± 1.0% 23.8 ± 1.4% 2.4 ± 0.8% Actual 65.4 ± 2.0 (3) 10.0 ± 0.2 (4) 10.9 ± 0.3 (3) 13.2 ± 0.8 (4) 17.1 ± 0.2 (3) ±0.3 (4) Guaranteed (miri) Actual 6.7 ± 0.2 (3) 7.2 ± 0.3 (4) 7.2 ± 0.2 (3) 5.3 ± 0.2 (4) 11.6 ± 0.3 (3) 7.7 ± 0.2 (4) Guaranteed (min) Calcium (g/kg) Phosphorus (g/kg) Journal of Herpetological Medicine and Surgery 9

4 Load Cricket and Insect Food), and high calcium cricket formula (treatment 6 TRex Calcium Plus Food for Crickets). Despite these claims, only treatment 6 contained sufficient calcium to properly gut load crickets. When diet ingredients and diet particle size are kept constant, a significant linear relationship between dietary calcium content and cricket calcium content has been observed (Allen and Oftedal, 1989, Finke, 2003). In contrast, when crickets were fed a variety of commercial products that differed with respect to ingredients, product form and product texture (high calcium supplements, pellets, alfalfa or calciumfortified dry diets) there was no clear relationship between diet calcium content and cricket calcium content (Anderson, 2000, Winn, et al, 2003). Anderson also observed differences in food consumption between crickets fed the various treatments which may help explain the lack of a relationship between diet calcium and cricket calcium in her study (Anderson, 2000). Even though the diets in the present experiment differed with respect to ingredients, processing and particle size, a statistically significant relationship was observed between diet calcium content and cricket calcium content for both cricket nymphs (cricket Ca = [ x Diet Ca], F=50.53, p=0.0021) and adult crickets (cricket Ca = [ x Diet Ca], F=208.8, p=0.0001). Of note is that small cricket nymphs and adult crickets on treatment 6 contained only 8 and 30% more calcium than those on treatment 1 even though the diet for treatment 6 contained 58% more calcium than treatment 1. This suggests that the larger harder particles of diet 6 may have had a slight negative effect on food intake relative to the crickets fed a finely ground diet (treatment 1). Not surprisingly this appears to have had a more pronounced impact on food intake of the smaller cricket nymphs than on adult crickets. While the large food particles of diet 6 may have had a slight negative impact on food consumption and hence cricket calcium content, it still provided sufficient calcium to properly gut load crickets. For all six treatments the smaller cricket nymphs contained significantly more calcium (g Ca/kg dry matter) than adult crickets fed the same diet. Hunt and coworkers reported a similar pattern for both crickets and mealworms, Tenebrio molitor (Hunt, et al, 2001). In our experiment this appears to be a result of the higher moisture content of the small nymphs (relative to adult crickets) because when calcium is expressed on an as is basis no consistent pattern was evident (data not shown). The higher moisture of cricket nymphs versus adult crickets is consistent with previous studies (Barker, et al, 1998, Finke, 2002, Finke, 2003). Likewise the higher phosphorus content (dry matter basis) of cricket nymphs versus adult crickets has also been observed previously (Barker, et al, 1998, Hunt, et al, 2001, Finke, 2002, Finke, 2003). There is currently a variety o f published research available to aid in formulating appropriate gut loading diets for Table 2. Moisture, calcium and phosphorus content of the crickets after fed the various food sources for 48 hr. Moisture data is reported on an as is basis. Calcium and phosphorus is reported on a dry weight basis. Values are means + SEM and number of replicates are shown in parenthesis. Within each row, treatments with different superscripts denote significant differences p < Food + 15% CaC03 Food Fluker's HighCalcium Cricket Feed ESU Reptile Gut Load Cricket and Insect Food Jurassi*Diet Gutload for crickets and other prey TRex Calcium Plus Food for Crickets Treatment Moisture (g/kg) Small (3) (2) (3) 763 ± 5 (3) 781 ± 4 (3 ) 786 ± 3 (3) Large (5) (6) (5) 681 ± 6 (6 ) 682 ± 7 (5) 679 ± 15(5) Calcium (g/kg) Small 11.0 ± 0.8 (3) 2.1 ± 0.0 b (3) b (3) 4.1 ± 0.1b (3) 3.7 ± 0.6b (3) 11.9 ± 0.7a 3) Large a (4) 1.7±0.1b (3) b (4) 1.8 ±0.0 b (3) 2.9 ± 0.1b (3) 8.2 ± 1,0a (3) Phosphorus (g/kg) Small 9.8 ±0.8 (3) (3) (3) 10.5 ±0.1 (3) 10.0 ± 0.6 (3) 9.4 ± 0.7 (3) Large (4) (3) (4) 9.1 ±0.1 (3). 8.6 ±0.1 (3) 9.2 ± 0.0 (3) Ca:P Small 1.21: : : : : :1.0 Large 0.75: : : : : : Journal of Herpetological Medicine and Surgery

5 crickets (Allen and Oftedal, 1989, Ferguson, et al, 1996, Anderson, 2000, Hunt, et al, 2001, Finke, 2003). In addi tion, products can easily be tested for efficacy simply by analyzing the gut loaded cricket for calcium. Despite this, it is surprising that only one of the four commercial gut loading diets tested resulted in crickets containing ade quate calcium for insectivorous reptiles and amphibians. Three o f the four products had label claims suggesting their use as a suitable calcium source for gut loading crick ets however only one of the commercial products tested (TRex Calcium Plus Food for Crickets) was effective in increasing the calcium content of crickets. Even though most products containing 60 to 100 g Ca/kg diet should provide adequate calcium to gut load crickets, these data clearly demonstrate that selecting a product based solely on the label guarantee for calcium may not be appropriate. Based on the information on the label, Fluker s HighCalcium Cricket Feed with a minimum label guarantee of 80 g Ca/kg diet would appear to be an appropriate diet for gut loading crickets. However, four different samples col lected over a 20 month period contained an average of 13.3 g Ca/kg diet or only 16.7% of the label guarantee. For this reason we recommend hobbyists and institutions either make their own gut loading diets (see methods treatment 1) or use TRex Calcium Plus Food for Crickets in order to load crickets with sufficient calcium for insectivo rous reptiles and amphibians. Finke MD Complete nutrient composition of selected invertebrates commonly fed to insectivores. Zoo Biol, 21: Finke MD Gut loading to enhance the nutrient content of insects as food for reptiles: A mathematical approach. Zoo Biol, 22: Finke MD, Dunham SU, Cole JS Evaluation of various calcium fortified high moisture commercial products for improving the calcium content of crickets, Acheta domesticus. JHMS, 14:69. Hunt AS, Ward AW, Ferguson GW Effects of a high calci um diet on gut loading in varying ages of crickets ( Acheta domestica) and mealworms ( Tenebrio molitor). Proc 4th Conf on Zoo and Wildlife Nut, Pennino M, Dierenfeld ES, Behler JL Retinol, Atocopherol and proximate nutrient composition of invertebrates used as feed. Int Zoo Yearb, 30: Trusk AM, Crissey S Comparison of calcium and phos phorus levels in crickets fed a high calcium diet versus those dusted with supplement. In Meehan TP, and Allen ME (eds): Proc 7th Dr. Scholl Conf on the Nutrition o f Captive Wild Animals, 6:9399. Winn D, Dunham S, Mikulski S Food for insects and insects as food: Viable strategies for achieving adequate calci um. J Wildlife Rehab, 26:413. A ppendix 1 A cknow ledgem ents We would like to thank Todd Goodman of Industries for kindly providing the crickets used in this study. Thanks also to Diane Winn, Gary Ferguson and three unknow n review ers w hose com m ents g reatly improved this paper. Funding for this research was sup ported by the Howard Hughes Medical Institute and the Gerald and Myra Dorros Chair in Life Sciences at Colby College. R eferences Allen ME, Oftedal OT Dietary manipulation of the calci um content of feed crickets. J Zoo Wildl Med, 20:2633. Allen ME From blackbirds and thrushes... to the gut loaded cricket: a new approach to zoo animal nutrition. Brit J Nutr 78:S Anderson SJ Increasing calcium levels in cultured insects. Zoo Biol, 19:19. AO AC Official methods of analysis of the Association of Official Analytical Chemists, Washington, D.C. Assoc of Official Analytical Chemists. Barker D, Fitzpatrick MP, Dierenfeld ES Nutrient compo sition of selected whole invertebrates. Zoo Biol, 17: Ferguson GW, Jones JR, Gehrmann WH, Hammack SH, Talent LG, Hudson RD, Dierenfeld ES, Fitzpatrick MP, Frye FL, Holick MF, Chen TC, Lu Z, Gross TS, Vogel JJ Indoor husbandry of the panther chameleon, Chamaeleo [Furcifer] pardalis: effects of dietary vitamins A and D and ultraviolet irradiation on pathology and lifehistory traits. Zoo Biol, 15: Treatment 3 Fluker s HighCalcium Cricket Feed Ingredients Grain products, plant protein products, processed grain byproducts, animal protein products, for age products, cane molasses, calcium, calcium carbonate, dicalcium phosphate, salt, DLmethionine, vitamin A acetate, Dactivated animal sterol (source of vitamin D3), vitamin E supplement, vitamin B12 supplement, riboflavin supplement, niacin supplement, calcium pantothenate, choline chloride, menadione sodium bisulfite complex, folic acid, pyridoxine hydrochloride, thiamine mononi trate, sodium selenite, biotin, manganese sulfate, zinc sulfate, ferrous sulfate, copper sulfate, calcium iodate, cobalt sulfate. Treatment 4 ESU Reptile Gut Load Cricket and Insect Food Ingredients Grain products, plant protein products, processed grain byproducts, forage products, animal protein products, ground limestone, deflourinated phosphate, salt, vitamin A acetate, Dactivated animal steral (source o f vitamin D3), vitamin B12 supplement, chlorine chloride, riboflavin supplement, calcium pan tothenate, niacin, vitamin E supplement. Treatment 5 Jurassi*Diet Gutload for crickets and other prey (lot code 18290) Ingredients Krill meal, fish meal, alfalfa, amino acids, algae meal, fish oil, beta carotene, spirulina, vitamin A acetate, Dactivated animal sterol (D3), vitamin B 12, riboflavin, niacin, folic acid, cal cium pantothenate, pyridoxine hydrochloride, thiamine, biotin, DL alphatocopherol (E), Lascorbyl2polyphosphate (C), choline chloride, cobalt sulfate, copper sulfate, Journal of Herpetological Medicine and Surgery 11

6 ferrous sulfate, manganese sulfate, ethylenediamine dihydroiodide. Added mineral not more than 2.50% Treatment 6 TRex Calcium Plus Food for Crickets (lot code ; exp 10/10) Ingredients Ground com, ground wheat, calcium carbonate, soybean meal, soy hulls, wheat middlings, dicalcium phosphate, fish meal, animal fat, coconut fat, natural and artificial flavors, Llysine HCL, choline chloride, salt, DL methionine, ascorbic acid, vitamin A, biotin, hydroxocobalamin (source of vitamin B12) cholecalciferol (source of vitamin D3), menadione (source of vitamin K), niacin, riboflavin, thiamin HCL, calcium pantothenate, pyridoxine hydrochloride, folic acid, sodium selenite, cobalt carbonate, magnesium oxide, ferric sulfate, copper sulfate, calcium iodate, manganous oxide, zinc sulfate, zinc oxide. 12 Journal of Herpetological Medicine and Surgery