The Role of Chromium in Animal Nutrition (1997)

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Chromium in Animal Nutrition Tnformation obtained on the nutritional essentiality and physiological function lof chromium (Cr) has been less definitive for animals than it has been for humans. The experimental approaches and criteria used to evaluate the influence of supplemental dietary chromium on cattle, sheep, swine, poultry, horses, rats, rabbits, and fish are discussed in this chapter. RUMINANTS Lactating Cows Assessment of performance or metabolic responses to chromium supplemen- tation in lactating dairy cattle has focused on the transition and early-lactation cow. Results of a study with periparturient and early-lactation dairy cows indi- cated that chromium supplementation influences the immune response in stressed cattle (Burton et al., 1993~. Twenty cows fed a basal diet containing an unspeci- fied quantity of chromium were compared with those fed 5.5 mg supplemental chromium per day as chelated chromium for 6 weeks before calving, and 9.98 mg/day for the first 16 weeks of lactation. Humoral immune responses were assessed by measuring antibodies either to ovalbumin or to human red blood cells. The chromium-supplemented cows had greater antiovalbumin antibody response profiles than did unsupplemented cows. Differences in antibody titers to human red blood cells were not evident. These results suggest that supplemen- tal chromium influences production of IgG, but not IgM antibodies. Cows fed supplemental chromium had greater mitogen-stimulated blastogenic responses 15

16 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION by peripheral blood mononuclear cells prepartum and at calving than did those of controls, also indicating a greater cell-mediated immune response because of chromium supplementation. Burton et al. (1996) conducted a subsequent study to determine whether chromium affected cytokine production by activated periph- eral blood mononuclear cells. They observed lower concentrations of interleukin- 2, interferon-y, and tumor necrosis factored in the culture supernatants of mito- gen-stimulated mononuclear cells from chromium-supplemented cows than from unsupplemented cows in one or more of four sample times during the trial. This observation suggests that chromium can affect blastogenic activity by altering cytokines production during the early stages of peripheral blood mononuclear cell activation. Results of experiments involving 74 Holstein cows suggested differences in response between primiparous and multiparous cows fed diets supplemented with 0.5 mg Cr/kg dry matter (DM) as amino acid chelated chromium (Yang et al., 1996~. Supplemental chromium increased milk yield of primiparous cows by 13.2 percent in Experiment 1 (P = 0.06) and 7 percent in Experiment 2 (P < 0.05) during the first 16 weeks of lactation. Supplemental chromium had no effect, however, on milk yield of multiparous cows when compared with that of cows consuming unsupplemented prepartal diets (containing 0.79 and 1.23 mg Cr/kg DM) and unsupplemented postpartal diets (containing 1.01 and 1.60 mg Cr/kg DM) in Experiments 1 and 2, respectively. Although primiparous cows tended to increase their dry matter intake (DMI) in the first 4 to 8 weeks of lactation when fed supplemental chromium, DMI was not influenced by treatment. In one of the experiments, supplemental chromium decreased mean serum nonesterified fatty acid (NEFA) and p-hydroxybutyric acid (~-HBA) in cows of three or more parities. Results of adrenocorticotrophic-releasing hormone and gonadotropin- releasing hormone (ACTH and GnRH) stimulation-response tests on primiparous cows suggest that chromium-supplemented cows experienced a greater stress with their increased milk production. Further studies with prepartum and early-lactation cows confirmed that supplementation with 5.5 mg Cr/day as chelated chromium during the dry period, and with 10 mg Cr/day during the initial six weeks of lactation, tends to improve milk yield in the initial stages of lactation for primiparous cows, but not for multiparous cows (Subiyatno et al., 1996~. Glucose tolerance tests conducted on cows two weeks prepartum and two weeks postpartum showed no effect of Cr supplementation on multiparous cow plasma glucose and insulin basal or peak levels, clearance rates, or insulin-to-glucose ratios; however, the prepartum plasma insulin-to-glucose ratio and triglyceride and insulin concentrations were decreased (P < 0.10) by supplemental chromium in primiparous cows. Insulin sensitivity of postpartum primiparous cows was reduced (P < 0.10) due to chromium supple- mentation. Subiyatno et al. (1996) also studied the effects of propionate loading on a second group of primiparous cows maintained on the same feeding program. Effects of chromium supplementation were most apparent at two weeks postpar

CHROMIUM AND NUTRITION 17 tum, causing reduced basal but higher peak serum propionate levels, elevated basal and peak glucose levels, reduced basal and peak insulin levels, and a lower insulin-to-glucose ratio (P < 0.10~. These responses were not evident when the test was repeated at six weeks postpartum; however, reduced basal serum NEFA levels were observed for supplemented cows following propionate infusion. Chromium supplementation has, relatively consistently, improved milk pro- duction of primiparous cows in the first four to six weeks of lactation when chromium supplementation was initiated in late gestation and continued into the first weeks of lactation. Primiparous cows can be more resistant to insulin than multiparous cows during late gestation (McClary et al., 1988~. Supplementation with chromium seems to improve insulin sensitivity for these primiparous ani- mals before calving, if one can assume that the ratio of insulin to glucose can be used as a crude indicator. After calving, however, supplemental chromium seems to reduce insulin sensitivity and increase plasma triglycerides. Subiyatno et al. (1996) suggested that this may relate to the ability of the liver to mobilize triglyc- erides as very low density lipoproteins during negative energy balance. Recently completed work by Besong et al. (1996) supports this theory. Decreased plasma p-HBA and liver triglyceride concentrations were observed 30 days after calving in cows supplemented with 0.8 mg Cr/day as chromium picolinate (CrPic). A summary of experimental results with lactating cows is presented in Table 3-1. Preweaned Calves Newborn calves offered 1.0 mg Cr/kg DM as CrPic in either the milk re- placer or calf starter for a 53-day study did not show a performance response (DePew et al., 1995~. Glucose responses to an intravenous glucose tolerance test and an intravenous propionate load test also were not affected by treatment. Prefeeding blood glucose and insulin levels were not affected by dietary treat- ment during the course of the trial, but there was some indication that prefeeding NEFA concentrations were decreased in calves given chromium-supplemented diets. Further work to determine the effects of dietary chromium on metabolic criteria during the first weeks of life have not been conducted. One-week-old bull calves, however, were used to determine the effect of either organic or inorganic chromium on cell-mediated and humoral immune response (Kegley et al., 1996~. Calves were fed a milk replacer containing 0.31 mg Cr/kg DM for an 87-day trial. Chromium was supplemented at 0.4 mg/kg DM as either chromium nicotinate (CrNic) or as chromic chloride (CrCl3~. Animal performance criteria were not affected by chromium supplementation. Immunoglobulin G. IgM, and total antibody titers to porcine red blood cells also were not affected by dietary treatment. Chromium supplementation did not influence blastogenic response of lymphocytes when either T-cell-stimulating or thymus-dependent B-cell mito- gens were used. Intradermal injection of phytohemagglutinin resulted in greater skinfold thickness responses for calves fed either supplemental chromium source.

18 THE ROLE OF CHROMIUM INANIMA:L NUTRITION TABLE 3-1. Influence of Supplemental Dietary Chromium on Lactating Dairy Cowsa Reference Dietary Cr Concentration per kg DM Unless Stated Otherwise (Source) Lactation Stage and Duration of Experiment Feed Intake Milk Yield Besong et al. (1996) Basal diet = unknown; Cr diet = basal + 0.8 mg (CrPic). 24 Holstein cows 30 d pre- to 60 d postpartum 1~ DMI. 1~ milk yield. Subiyatno et al. Basal prepartum diet = 12 Holstein cows - No effect. (1996) 0.79 ma; 4 PP and 8 MP 6 Cr prepartum diet = basal wk pre- to 2 wk + 5.5 mg/d; Basal postpartum diet 1.23 ma; Cr postpartum diet = basal + 10 mg/d (chelated Cr). No effect. postpartum

CHROMIUM AND NUTRITION 19 Hypoglycemic Body Change in Response or Milk Weight and Serum/Plasma Improved Insulin Components Change Constituents Efficiency Other No effect. ND No effect on plasma glucose, NEFA, and . , . serum 1nsulm. 1' plasma p-HBA and liver tri- glyceride on d 30 nost mortem. ND No effect. ~ plasma triglyce- rides, 1' peak insulin, 1' insulin: glucose ratio in prepartum PP cows, and 1~ plasma triglycerides and 1~ insulin:glucose ratio in postpartum PP cows during glucose tolerance test P<0.10.

20 TABLE 3-1. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Dietary Cr Concentration per kg DM Unless Lactation Stage Stated Otherwise and Duration of Reference (Source) Experiment Feed Intake Milk Yield Yang et al. (1996) Basal prepartum diet = 0.79 34 Holstein cows - No overall PP cows mg; 12 PP and 22 MP effect. Cr-fed 13.2% Cr prepartum diet = 1.29 ma; 6 wk pre- to 16 PP cows had increase Basal postpartum diet = 1.01 wk postpartum 15% greater P=0.06; MP ma; DMI in first 4 cows - no Cr postpartum diet = 1.51 mg wk of lactation. effect. (amino-acid-chelated Cr). Definitions of abbreviations used in table: AUC - area under curve p-HBA - beta hydroxybutyric acid con-A - concanavalin A Cr- chromium CrPic - chromium picolinate d- day DM - dry matter DMI - dry matter intake HRBC - human red blood cells LH - luteinizing hormone MP- multiparous ND - not determined NEFA - nonesterified fatty acids OVA- ovalbumin PP - primiparous wk- week

CHROMIUM AND NUTRITION 21 Body Milk Weight and Components Change No effect. PP cows lost more weight, P=0.07; MP cows - no effect. Change in Serum/Plasma Constituents No effect on NEFA or p-HBA. Hypoglycemic Response or Improved Insulin Efficiency Other No effect on reproduction.

22 THE ROLE OF CHROMIUM INANIMA:L NUTRITION Antibody titers to Pasteurella hemolytica were not affected by chromium supple- mentation. Serum cortisol concentrations were not influenced by treatment on day 7 of the trial; however, calves fed chromium-supplemented diets had lower cortisol concentrations and higher white blood cell counts 5 days after infection with infectious bovine rhinotracheitis virus (IBR). These differences disappeared by day 12 after infection. A summary of experimental results is presented in Table 3-2, which also presents results of studies on growing-finishing cattle. Growing-Finishing Cattle The first research results suggesting a beneficial effect of chromium supple- mentation to the diet of cattle were reported by Chang and Mowat (1992~. Aver- age daily gain (ADO) and ADG/DMI of 108 transportation-stressed, Charolais- cross calves, provided with 0.4 mg Cr/kg DM as high-chromium yeast, were improved by 30 and 27 percent, respectively, during the initial 28 days after arrival at the feedlot, compared with stressed calves not receiving chromium supplemented feed. This benefit of supplemental chromium was not observed when calves received a long-acting antibiotic within 48 hours of arrival at the feedlot. The measured chromium concentration in the control diets (corn silage with urea-corn or with soybean meal) of this study was 12.12 mg/kg DM. Selecting 96 steers from the above group of 108 calves, Chang et al. (1992) fed diets with O or 0.2 mg Cr/kg DM from high-chromium yeast during the growing period, followed by diets with O or 1.87 mg Cr/kg DM during the finishing period. Chromium supplementation had no effect on performance dur- ing either period. Chromium supplementation, however, decreased serum corti- sol in all calves and increased serum IgM and total immunoglobulin in calves fed corn silage diets supplemented with soybean meal. The immune response was not evident for calves fed a urea-corn grain supplement. Carcass characteristics were not altered as a result of chromium supplementation during the growing- finishing phase. Results of this series of experiments suggest that chromium might be essential to cattle during periods of stress. A second trial with transportation-stressed calves showed a 27 percent in- crease in ADG, but no feed efficiency response, when 0.2 or 1.0 mg Cr/kg DM as high-chromium yeast was added to a diet containing 0.16 mg Cr/kg DM (Moonsie-Shageer and Mowat, 1993~. No performance response was observed for calves fed an intermediate concentration (0.5 mg/kg DM) of chromium in this 30-day trial. Unlike the earlier trial, chromium supplementation reduced morbid- ity from days 2 to 30, and the 0.2 mg Cr/kg supplementation rate was most effective. Rectal temperatures were lower for the first 5 days after arriving in the feedlot, and elevated serum calcium and magnesium were evident in the first week but not thereafter. Increased hemagglutinating antibody titers to human red blood cells on day 14 suggested an improved humoral immune response in stressed feeder calves fed chromium supplements. Serum cortisol levels were

CHROMIUM AND NUTRITION 23 decreased in calves fed supplemental chromium by the end of the 28-day trial. Chromium supplementation had no effect on concentrations of potassium, cho- lesterol, globulin, glucose, protein, urea, and alkaline phosphatase in serum, and it had a transitory effect on serum albumin. Feedlot-adjusted steers offered a supplement of 1 mg Cr/kg DM in a chelated form did not show improved ADG, DMI, or feed efficiency but had decreased serum glucose and cortisol during a 56-day trial (Mowat et al., 1993~. Results of this study confirmed that the responses observed in previous studies conducted by Chang and Mowat (1992) were not caused by the yeast component of the chromium supplement, but rather were related to chromium. A second trial with newly arrived steer calves compared high-chromium yeast with chelated chro- mium and showed decreased morbidity for the chelated-chromium-treated calves relative to controls; calves receiving the high-chromium yeast had an intermedi- ate response. Chromium supplementation did not affect calf weight gain in the 35-day trial, but feed efficiency was improved with diets containing the high- chromium yeast and one of the two chelated forms of chromium. Serum cortisol was not affected by treatment in the study (Mowat et al., 1993~. The chromium concentration of the unsupplemented diet was 0.16 mg/kg in each trial. In another study, calves fed a lower level of supplemental chromium (0.14 mg added Cr/kg DM as chelated chromium) did not have improved ADG, DMI, or feed efficiencies in a 49-day trial using corn-silage-based diets (Wright et al., 1994~. Morbidity was not decreased in calves; however, the number of illness relapses for calves receiving the chromium supplement was smaller. The authors indicated that the failure to demonstrate improved calf performance with chro- mium supplementation in this trial could have been related to the fact that the calves were sick early in the trial, allowing little opportunity for chromium to exert its effect. Chromium concentrations in the unsupplemented diets were reported to be 0.32 mg/kg DM for the first 28 days and 1.05 mg/kg DM thereaf- ter. Serum total and free triiodothyronine and plasma cortisol were not influ- enced by chromium supplementation. Chromium-supplemented calves receiving vaccinations had greater plasma ascorbate concentrations than did calves receiv- ing vaccinations only, or no vaccinations and no supplemental chromium, sug- gesting a link between immune function and dietary chromium. To determine a link between immune function and dietary chromium, Burton et al. (1994) tested whether supplemental chromium affected antibody responses of stressed feedlot cattle to clinically relevant antigens (IBR and parainfluenza-3 [PI-31) contained in a commercial vaccine. Steers were offered 0.5 mg Cr/kg DM as chelated chromium in a corn-silage-based diet containing 0.16 mg Cr/kg DM. Chromium supplementation increased the magnitude of the peak antibody re- sponse to IBR but had no effect on anti-PI-3 antibody titers. Chang et al. (1994) supplemented the diet of transportation-stressed steers with 0.14 mg Cr/kg DM as chelated chromium for 49 days. They reported that blood lymphocytes obtained from morbid steers fed supplemental chromium

24 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION TABLE 3-2. Influence of Supplemental Dietary Chromium on Cattlea Reference PREWEANED CALVES Dietary Cr Concentration per kg DM (Source) DePew et al. Basal diet = unknown; (1995) Cr diet = basal + 1.0 mg (CrPic). K, ~, ~ ~ I, . . ~ ~ ~ egley eta. ~ ~ ~ nasal~a~et =~U.~l ma; : :: :: : :: :: :: : : : ~ : : ~ 199~63 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ CrNic~ = ~0.7~1 ~mg; ~ ~ ~ ~ ~ ~-~ :: : : ::: : : : : :: : : : : (:iCl3 diet =~0.7l~g. WEANED, STRESSED CALVES Chang and Mowat Basal diet = 12.12 ma; (1992) Cr diet = 12.52 mg (Cr yeast). Body Weight at Start and Duration of Experiment Improved Improved Feed Growth Rate Efficiency 42 newborn Holstein No effect. No effect. calves 39 kg 53-d trial :: : : : : : : :: l-`vk~ld~lstbit~bull ~ No effect. ~ ~ ~ ~No~effect. calves ~87 108 calves - 245 kg 28-d trial transportation stressed ~. . ~ Moonsie-Shageer~ ~ ~ Basal diet =~0.1~6 mg;~ ~84 steers~-~236~kg :: : : : :: :: : :: ::::: ::: ~30-d~hial lrans~?ortation; Stressed Mowat et al. (1993)  Bunting~et ~ 61. (~1~994) ~ ~ ~ Basal diet = unknown; Cr yeast diet = basal + 0.5 ma; chelated Cr diet = basal + 0.5 ma; chelated Cr. Zn, Cu + Mn for 1st 7 d = basal + 0.5 mugs Basal diet = 0.53 Dig, C:r Beth= O.90~mg~ CiPic).~: Bunting et al. Basal diet = 0.34 ma; (1994) Cr diet = 0.71 mg (CrPic). : : : ::::: :: :: : : ~ : ~ :: : ~ : : I: : : :: ~ :: 72 steers- 233 kg 35-d trial mixing/transportation stressed No overall No overall effect. 1~ in effect. 1~ in calves not calves not . . . . receiving receiving antibiotics. antibiotics. ~ ~ ~ . ~ ,~,- ~ ., o~e~ect. ~ ~ ~ ~ ~ : :~: :,::::. ::~ -. ~ :~::~::::::~:::~::~ I:: ~ ~ ::: ~ :: :: ::: :: ::: : :: :: :: ::: ::: :::: : : : : :: :: . ~ I: No effect. ~ for Cr yeast and one of chelated Cr diets. tein~s£eers - ~ ~ Juno Effect. ~ ~ ~ Juno effect. ~ ~98~kg ~ ~ ~ ~ ~ ~ ~ : :~ ;: 58-d :~ti;~ ~ ~:~ ~ ~ ~ :~ :: . ~ Not stressed ~ ~ . . ~:: : : ::: ~: : : . :: : I: : : : : : : : : : : : : :: : : ~ : :: : ::: ::: : :: :::: ::: ::: :~: : I: : ~ :::: :: :: : : :: I: : : :: ::: ::: :::: ~ I:: :~ I:: : : ::: . :: : :: : : ·~ ~ ~ ~ ~ 14 Holstein heifers - No effect. No effect 122 kg 56-d trial  not stressed : : :::: : I: :: :: :::: :::

CHROMIUM AND NUTRITION 25 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other ND ND No differences in blood glucose or insulin 1' NEFA until wk 2. No difference in re- sponse to i.v. glucose tolerance test or i.v. pionate load test. pro ND ND ND ND ND ND No effect. ND 1' morbidity. ND ND HI plasma cholesterol I rate of glucose No difference in GH in wk 6 only. No ef- clearance during i.v. or GHRH. No feet on plasma NEFA, glucose tolerance test. difference in N glucose, insulin, No difference for balance. creatinine, urea N. or insulin challenge test. total protein.

26 TABLE 3-2. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Dietary Cr Body Weight Concentration at Start and Improved per kg DM Duration of Improved Feed Reference (Source) Experiment Growth Rate Efficiency Chang et al. Basal diet= unknown; 24 steers - 258 kg ND ND (1994) Cr diet = basal + 0.14 mg 49-d trial (chelated Cr). mixing/transportation stressed Chang et al. Basal diet = 1.41 ma; 135 steers - 236 kg No effect. No effect. (1995) Cr yeast diet = 2.50 ma; 56-d trial CrC13 diet = 2.50 ma; mixing/transportation CrC13 and niacin diet = stressed 2.50 mg (Cr yeast or CrC13). Mathison and Engstrom (1995) Basal diet = unknown; Basal diet, no stress = unknown; mixing/transportation Cr diet, stress = basal + stressed 0.57 = 0.83; Cr diet, no stress = basal + 0.69 - 0.83 chelated Cr) 192 steers - 262 kg No effect. No effect. 28-d trial Chang et al. (1996) Basal diet (first 28 d) = 0.32 ma; Cr diet Cast 18 d) = 1.05 ma; Cr diet = basal + 0.14 mg (chelated Cr). 66 steers - 250 kg ND ND 49-d trial mixing/transportation stressed

CHROMIUM AND NUTRITION 27 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other ND ND 1~ blastogenic response in lymphocytes to con- A for morbid calves. ND ND Cr - yeast ~ serum Fe and total iron- binding capacity in healthy calves. No effect for serum cortisol, serum glucose, plasma ascorbate, plasma ~rr~t~.in nmmr~nin ND ND ND ND No effect on morbidity. ND ND ~d28titer for BVD. No response for IBR, PI-3, BRSV, P. hemolytica.

28 TABLE 3-2. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Reference Dietary Cr Concentration per kg DM (Source) Body Weight at Start and Duration of Experiment Improved Improved Feed Growth Rate Efficiency Kegley et al. (1997) GROWING-FINISHING CATTLE Basal diet= unknown; 48 steers - 263 kg Cr diet = basal + 0.4 mg 56-d trial (CrNic). transportation stressed (P<0. 10). No effect. Chang et al. Basal finishing diet = (1992) 1.87 ma; Cr finishing diet = 2.07 mg 96 steers - 377 kg 68-d finishing trial No effect. No effect. Mathison and Engstrom (1995) Basal diet = unknown; 190 steers - 294 kg Cr diet = basal + 0.5 mg feed lot adjusted, fed (chelated Cr). until finished No effect. No effect. Definitions of abbreviations used in table: BVD - bovine viral diarrhea BRSV - bovine respiratory syncytial virus con-A - concanavalin A Cr- chromium CrC13 - chromium chloride CrNic - chromium nicotinate CrPic - chromium picolinate d- day DM - dry matter GH - growth hormone GHRH - growth hormone releasing hormone IBR - infectious bovine rhinotracheitis virus i.d. - intradermal IgG - immunoglobulin G IgM - immunoglobulin M i.v. - intravenous ND - not determined NEFA - nonesterified fatty acids OVA- ovalbumin  PHA- phytohemagglutination PI-3 - parainfluenza-3 RBC - red blood cells SBM - soybean meal wk- week

CHROMIUM AND NUTRITION 29 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other ND ND 1' serum IgG after stress. No difference in rectal temperature or anti- body response after IBR challenge. No antibody response difference to porcine RBC. No effect. No effect. ND No effect. No effect. ~ liver Cr for cattle fed urea (not SBM) in growing phase. No effect on morbidity.

30 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION responded more to concanavalin A stimulation than did lymphocytes from steers not receiving added chromium. Added chromium did affect performance of the steers. Growing Holstein steer and heifer calves were offered either a control diet (containing 0.53 or 0.34 mg Cr/kg DM, respectively, for steers and heifers) or the control diet plus 0.37 mg Cr/kg DM as CrPic for heifers (Bunting et al., 1994~. Calf ADG, DMI, feed efficiency, and nitrogen balance were not influenced by dietary treatment. There were transient declines in plasma cholesterol for both heifers and steers; however, average plasma cholesterol concentrations were not affected by treatment. Plasma NEFA, glucose, insulin, creatinine, urea nitrogen, and total protein were not affected by treatment. Calves receiving supplemental chromium showed a 27 to 40 percent more rapid clearance of plasma glucose and a shorter plasma glucose half-life during an intravenous glucose tolerance test than did controls. Calves also were subjected to an intravenous insulin challenge test, and heifers, but not steers, had faster glucose clearance rates and shorter plasma glucose half-lives when fed supplemental chromium. These data suggest that chromium affects carbohydrate metabolism by enhancing the response of tissues to insulin. The potential interaction between supplemental inorganic chromium as CrCl3 and niacin was investigated by Chang et al. (1995~. Unsupplemented and chro- mium-yeast-supplemented diets were included as controls. Newly arrived steers were offered an alfalfa-silage-based diet containing 1.41 to 1.47 mg Cr/kg DM, with chromium supplemented at 0.75 mg/kg DM. There were tendencies toward increased weight gain and improved feed efficiency in the initial 28 days for calves fed the inorganic chromium-plus-niacin treatment, with no improvements for calves receiving chromium-yeast or inorganic chromium alone. Calf morbid- ity and response to antibiotic treatment were not influenced by dietary treatment. Serum iron and total iron-binding capacity were increased for healthy calves receiving the inorganic chromium-plus-niacin treatment. No consistent response to treatment was observed for serum cortisol or glucose concentrations nor for plasma ascorbate, protein, urea, and ammonia concentrations. Interest generated from the work conducted at the University of Guelph, Ontario, led to similar studies at other sites in North America. Three chromium supplements, CrCl3, high-chromium yeast, and CrNic, were compared with re- spect to growth, glucose metabolism, and immune responses of stressed feeder calves (Kegley and Spears, 1995~. Chromium was added at 0.4 mg/kg DM to a corn-silage-based diet containing 0.66 mg Cr/kg DM. Steer performance was not influenced by treatment during this 56-day study. Serum cortisol did not respond to treatment. Serum insulin concentrations were greater in cattle receiving the CrNic than in cattle receiving the other chromium supplements at 15 and 30 minutes after glucose infusion; and they were greater (at 30 minutes after infu- sion) than were those in animals fed control diets. Steers fed high-chromium yeast tended to have a greater inflammatory response to an intradermal injection

CHROMIUM AND NUTRITION 31 of phytohemagglutinin than did control steers. The inflammatory response of steers fed the other chromium supplements was significantly less than that of steers fed high-chromium yeast. Kegley and Spears (1995) concluded that the form of dietary chromium affects its function in cattle. Moreover, CrNic in- creased total serum IgG, and peripheral lymphocytes isolated from these steers had a greater blastogenic response to phytohemagglutinin than did lymphocytes from steers fed diets supplemented with CrCl3. Mathison and Engstrom (1995) evaluated the effect of chromium supple- mentation on rate and efficiency of weight gain and morbidity of steer calves during the initial 28 days in a feedlot under well-managed or stress-imposed conditions. Supplemental chelated chromium resulted in total chromium intake ranging from 0.69 to 0.83, and from 0.57 to 0.83 mg/kg DM, respectively, for unstressed and stressed calves during the 28-day trial. Chromium supplementa- tion did not affect any of the criteria measured in this trial. A subsequent grow- ing-finishing trial in which a diet of barley silage and barley grain was supple- mented with chromium at 0.5 mg/kg DM showed no improvement in DM intake, growth, feed efficiency, carcass characteristics, or morbidity in steers as a result of chromium supplementation. Acute-phase response, as indicated by serum haptoglobulin and total hemolytic complement activity, was measured in 72 steer calves that had been stressed by going through a sale barn marketing process (Wright et al., 1995~. Calves fed a corn-silage-based diet containing 0.32 to 1.05 mg Cr/kg DM supple- mented with 0.14 mg Cr/kg DM as chelated chromium had lower serum haptoglobulin concentrations 7 days after arriving at the feedlot than did the calves whose feed was not supplemented. Morbidity among the calves was greatest at that time. By day 14, however, no effects of chromium supplementa- tion on serum haptoglobulin were observed, and the increase in complement activity observed with the control calves was not evident with chromium-supple- mented calves. Chang et al. (1996) worked with the same calves to determine the effect of a diet supplemented with 0.14 mg Cr/kg diet on antibody response to vaccination for IBR, PI-3, bovine respiratory syncytial virus (BRSV), bovine viral diarrhea (BVD), and P. hemolytica on days O and 21 after arrival at the feedlot. Blood samples were taken on days 0, 28, and 35 for determination of antibody titers. Supplemental chromium increased the titer for BVD only on day 28 and caused no effect on antibody responses to other vaccines. Chang et al. (1996) conducted a second trial with another group of market- transport-stressed steer calves to determine the effect of 0.75 mg Cr/kg DM as chromium yeast on antibody responses to IBR, PI-3, BRSV, BVD, and P. hemolytica. The basal diet for this trial contained 75 percent alfalfa silage and 1.45 mg Cr/kg DM. No changes in antibody titers were caused by chromium supplementation. Calves also were injected with 2 mg ovalbumin at vaccination (days O and 21~. Supplemental chromium enhanced the primary response to this

32 THE ROLE OF CHROMIUM INANIMA:L NUTRITION protein antigen and tended to increase the secondary response as well. Results of these two experiments (Chang et al., 1996) and those obtained by Burton et al. (1994) illustrate the inconsistent immune responses observed in stressed calves fed chromium supplements. Kegley et al. (1997) observed that calves fed 0.4 mg Cr/kg DM as CrNic for 56 days before transportation, handling, fasting, and vaccination stress, tended to have improved ADG for the subsequent 80-day feeding period. Serum total IgG was decreased, but rectal temperature and antibody response to IBR or porcine red blood cells were not affected by chromium supplementation. Sheep Some of the earliest work to suggest a requirement for chromium by rumi- nants was related to the investigation of a stimulatory factor in cane molasses (Britton et al., 1968~. Lambs were fed a molasses-free control diet, a molasses supplement, a molasses ash supplement, or 0.037 mg Cr/day as CrCl3. Lambs fed the molasses ash and chromium-supplemented diet had increases in nitrogen utilization similar to those of lambs receiving the molasses supplement and greater than lambs fed the control diet. They did not exhibit improved ability to digest fiber, as had been observed for the molasses-supplemented group. Supplementation of 0.25 mg Cr/kg DM as CrPic did not influence ADG, DMI, or nitrogen utilization of female lambs (38 + 2.7 kg body weight) that were fed a basal diet containing less than 1 mg Cr/kg DM for 85 days (Kitchalong et al., l 995~. Prefeeding and three-hour postprandial blood samples collected from lambs on three occasions during the study showed similar plasma albumin, total protein, urea nitrogen, glucose, insulin, glucagon, T3, and T4 concentrations, irrespective of dietary chromium concentration. A 17 percent decrease in plasma total cholesterol was observed in the chromium-supple- mented lambs during the first week but not at weeks 7 or 11. Plasma NEFA concentrations were 21.7 percent less, and the extent of decline in NEFA after feeding was substantially less for lambs fed the chromium-supplemented diet. Carcass data indicated no treatment effects on carcass weight or on heart and kidney weights expressed as a percentage of body weight of the lambs. Chro- mium-supplemented lambs had lower lean carcass yields and lower liver weights, and they tended to have less fat over the tenth rib (P = 0.09) and decreased pelvic fat (P = 0.15) than did their control counterparts. Samsell and Spears (1989), using low- and high-fiber diets containing 0.175 and 0.295 mg Cr/kg diet DM, respectively, noted that supplemental chromium (0.01 mg/kg DM as CrCl3) did not influence plasma glucose or insulin concentrations and did not affect plasma glucose clearance when lambs were given a glucose tolerance test. A 48-hour fasting period resulted in reduced plasma glucose concentrations for lambs fed the low-fiber diet. A second study conducted by

CHROMIUM AND NUTRITION 33 Samsell and Spears (1989) showed that serum NEFA was decreased by chro- mium addition when lambs were fed the high-fiber diet. In contrast to results with female lambs (Kitchalong et al., 1995), Samsell and Spears (1989) observed that supplementing 0.01 mg Cr/kg DM as CrCl3 reduced fasting plasma glucose concentrations in male lambs fed a low-fiber diet. The 48-hour fasting period for the Samsell and Spears (1989) study was much longer than that imposed by Kitchalong et al. (1995~. Williams et al. (1994) evaluated the effects of supplementing 0 or 0.80 mg Cr/kg DM as CrPic to the diet of 29 kg lambs exposed to thermal stress. Lambs received a basal diet of corn silage and soybean meal (78 to 16 ratio, DM basis) at 2.7 percent body weight. Basal diet chromium levels were not determined. Chromium supplementation did not influence nitrogen, potassium, or zinc reten- tion by lambs. The authors reported a yeast culture by chromium interaction for serum glucose concentrations in lambs. DePew et al. (1996) fed diets with or without 0.5 mg supplemental Cr/kg DM as CrPic to lambs for 42 days. Chromium supplementation did not affect performance of the lambs, and no effects of chromium were observed for plasma glucose, NEFA, triglycerides, cholesterol, or insulin when lambs were fed or fasted. Sano et al. (1996) conducted an isotope study to determine the effect of supplemental chromium on whole-body kinetics of glucose, lactate, and propi- onate in rams fed a high-grain diet. This study showed an increase in the percent- age of glucose derived from propionate but no other differences in plasma con- centrations or turnover rates of glucose. Rams were given either a control diet with an undetermined amount of chromium or the control diet with 0.5 mg supplemental Cr/kg DM as chelated chromium. The work of Samsell and Spears (1989), Kitchalong et al. (1995), and Sano et al. (1996) suggested that chromium supplementation exerts subtle effects on carbohydrate and lipid metabolism in sheep. The significance of these effects, however, is not clear. The basal diet chromium concentrations were not always provided by the researchers, and the chromium supplementation concentrations used in the work conducted with sheep ranged from 0.01 to 0.80 mg/kg DM. The results of studies involving sheep are summarized in Table 3-3. Summary Availability of chromium contained in commercial diets fed to ruminant animals is not known. Further efforts need to be directed toward the determina- tion of chromium concentrations and chromium availability in foodstuffs and chromium supplements offered to ruminant animals. A range of chromium supplements including CrNic, CrCl3, CrPic, chelated chromium, and high-chro- mium yeast have been used in ruminant studies. Comparative studies have been limited and thus little is known about the relative availability of these sources for

34 THE ROLE OF CHROMIUM INANIMA:L NUTRITION TABLE 3-3. Influence of Supplemental Dietary Chromium on Sheepa Reference Dietary Cr Concentration per kg DM (Source) Body Weight at Start and Duration of Experiment Improved Growth Rate Improved Feed Efficiency Britton et al. (1968) Basal diet= unknown; Cr diet = 0.037 mg/d and basal (molasses ash or CrCl~). Growing lambs ND ND Samsell and Basal diet, low fiber Spears (1989) 0.175 ma; Basal diet, high fiber = 0.295 ma; Cr diet, low fiber = 0.185 ma; Cr diet, high fiber = 0.305 ma; (CrCl~). 16 lambs - 50 kg 28-d trial ND ND Kitchalong et al. Basal diet= <1 ma; (1995) Cr diet = basal + 0.25 mg (CrPic). 241ambs-38kg 85-d trial ND ND Sano et al. (1996) Basal diet= unknown; Cr diet = basal + 0.5 mg (chelated Cr). 6 rams Yes - no ND 28-d trial statistics provided. Definitions of abbreviations used in table: Cr- chromium CrC13 - chromium chloride CrPic - chromium picolinate d- day DM - dry matter FFA - free fatty acids h - hour HDL - high density lipoprotein i.v. - intravenous K- potassium N - nitrogen ND - not determined  NEFA - nonesterified fatty acids wk- week Zn - zinc

CHROMIUM AND NUTRITION 35 Increased Carcass Reduced Muscling Carcass Fat Change in Serum/Plasma Constituents Hypoglycemic Response or Improved Insulin Efficiency Other 1~ N balance. ND ND ND ND 1' serum FFA at 3 h post feeding. No effect on plasma glucose, or insulin. ND 1' pelvic fat in wethers, P = 0.15. No effect on total cholesterol, albumin, total protein, T3 and T4, glucose, urea N. 1' NEFA. No effect on glucose No effect on N clearance rates during tolerance tests. 1~ insulin and 1' glucose in wk 2 but not wk 10 of i.v. insulin challenge test. balance. ND ND

36 THE ROLE OF CHROMIUM INANIMA:L NUTRITION ruminants, although it should be noted that performance responses observed to date are limited to work conducted with CrPic, high-chromium yeast, and che- lated chromium. The literature does not support a general recommendation for chromium supplementation of commercial ruminant diets. Research efforts, however, have identified two situations in which chromium supplementation might have com- mercial application: newly arrived feedlot cattle and first-lactation dairy cattle during the transition period. Three of eight studies conducted with newly arrived feedlot cattle subjected to the stresses of transportation, mixing, and handling have demonstrated a posi- tive performance response in the initial weeks following arrival. Reduced mor- bidity and plasma cortisol levels due to chromium supplementation have been reported; however, the responses have not been consistent. Efforts to establish an enhanced immune response using response to vaccination or to foreign proteins have not shown consistent results, although blastogenesis in peripheral blood mononuclear cells cultured with T-lymphocyte mitogens was greater in cattle fed chromium-supplemented versus unsupplemented diets. The transition period, including parturition, early lactation, and late lacta- tion, is known to cause metabolic stress in dairy cows. There is evidence that chromium supplementation during this transition period can improve performance for first-lactation cows. This response was not observed for multiparous cows offered similar levels of supplemental chromium. Improved performance may be associated with a shift in ketone body metabolism, as reduced circulating ketone levels in cows consuming chromium supplemental diets have been observed in several studies. There is also evidence that chromium supplementation of first- lactation, postpartum cows will reduce sensitivity to insulin. As was observed for the receiving feedlot animals, chromium supplementation enhanced blastogenic responses of the peripheral blood mononuclear cells obtained of early-lactation cows. Controlled studies will be required to establish the specific role of chromium for cattle undergoing stress and to establish recommendations for rates of chro- mium supplementation where its use may be appropriate. NONRUMINANTS Growing-Finishing Swine Most of the recent research on dietary chromium supplementation for swine has focused on performance and carcass characteristics of growing swine. An early study by Steele et al. (1977), however, showed that a synthetic, chromium- containing glucose tolerance factor (GTF) potentiated the hypoglycemic response in swine after an intravenous insulin challenge, showing that the chromium- containing substance was biologically active, as had been described for humans

CHROMIUM AND NUTRITION 37 and rats by Mertz et al. (1974~. Subsequent research evaluated the effects on swine of supplemental dietary chromium, as inorganic CrCl3 or as organically bound CrPic. Page et al. (1990) fed diets containing 0, 30, or 60 mg Cr/kg as CrCl3 to growing swine for 43 days. They observed no influence of chromium on growth rate or carcass traits, including backfat thickness, loin eye area, or per- centage muscling. Concentrations of several constituents of blood serum also were unaffected. In a subsequent study, Page et al. (1992a,b) fed swine of two genotypes diets containing O or 200 ,ug Cr/kg as CrPic for 102 days. Average daily gain and feed intake were increased by dietary CrPic, whereas feed effi- ciency, carcass dressing percentage, and concentrations of several constituents of blood serum were unaffected. Feeding 200 ,ug of Cr/kg decreased tenth-rib fat and increased loin eye area and percentage of carcass muscling. Serum choles- terol concentration also was reduced by dietary CrPic. Page et al. (1992a) re- ported that the favorable effects of CrPic on swine carcass traits and serum cholesterol occurred irrespective of genotype. In a follow-up study, Page et al. (1992b) found that feeding CrPic did not affect pork quality in terms of tender- ness (shear force) or cooking loss. In 1993, Page et al. reported the results of three experiments in which chro- mium, supplied as CrPic, was evaluated at concentrations ranging from 25 to 800 ,ug/kg of feed. The basal diet contained 735,ug Cr/kg. Effects of CrPic on weight gain and feed efficiency were inconsistent among experiments. In two experi- ments, feeding 100 or 200 ,ug Cr/kg decreased tenth-rib backfat and increased loin eye area and percentage of muscling. A comparison of CrCl3 and CrPic as dietary sources of chromium in one experiment showed that CrPic was effective in improving carcass traits, whereas CrCl3 was not. Lien et al. (1993) fed diets containing 0, 200,400, or 800,ug Cr/kg as CrPic to swine and reported no effects on ADG or feed efficiency. Feeding 400 or 800 ,ug Cr/kg reduced backfat thickness and serum glucose concentration. Serum insulin concentrations were directly related to dietary CrPic, and serum lipid concentration was reduced by either 200 or 400 ,ug CrPic/kg of feed; serum cholesterol was not affected. Berrio et al. (1995) conducted in vitro studies on red blood cell ghosts and adipocytes obtained from swine fed O or 200 ,ug supplemental Cr/kg of diet as CrPic. They reported that insulin binding was increased with red blood cell ghosts obtained from swine fed 200,ug Cr/kg of diet in two experiments. Insulin binding to adipocytes from swine fed 200,ug Cr/kg of diet also was increased in one experiment. These observations indicated that chromium may play a role in . .. . . . Insulin action In swine. Evock-Clover et al. (1993) evaluated dietary CrPic for swine treated or not treated with pituitary-derived porcine somatotropin (pST). Feeding 300,ug Cr/kg of diet did not affect rate of growth, feed efficiency, or carcass composition of swine reared from 30 to 60 kg body weight, irrespective of pST treatment. Di- etary CrPic, however, decreased serum insulin and glucose concentrations and normalized the increase in glucose and insulin resulting from pST treatment. No

38 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION interactions between dietary CrPic and pST were observed. In a subsequent study, Evock-Clover and Steele (1994) fed 300,ug Cr to swine reared from 60 to 90 kg body weight. Dietary CrPic had no effect on growth rate, feed efficiency, or nutrient partitioning. Feeding CrPic increased the percentage of carcass pro- tein of swine treated with recombinant pST. The influence of 200,ug Cr/kg of diet, supplied as CrCl3, CrPic, or CrNic, on immune response of young swine was evaluated by Van Heugten and Spears (1994~. Dietary chromium increased weight gain and feed intake of swine not challenged by an injection of E. cold lipopolysaccharide (LPS). Chromium, however, did not prevent the adverse effects of LPS on swine performance, and no clear-cut effects of chromium supplementation on other measures of immune response were discernible. Boleman et al. (1995) conducted an experiment to evaluate the effects of feeding 200 ,ug Cr/kg of diet as CrPic on growth and carcass composition of swine and on sensory characteristics of pork. Feeding CrPic reduced weight gain and feed intake of swine reared from 30 to 103 kg. Carcass data showed that feeding CrPic during the late growing and finishing periods decreased body fat and increased the percentage of muscle of swine. Cooking loss, drip loss, shear force, and sensory characteristics of meat were not affected by chromium treat- ment. Smith et al. (1994) reported that 200 ,ug Cr/kg of diet as CrNic had no effect on weight gain and feed efficiency of swine but that backfat thickness was decreased by chromium treatment. Harper et al. (1995) reported that feeding 200 ,ug Cr/kg of diet as CrNic improved ADG of weaning pigs and of swine during the finishing period. Chro- mium supplementation also reduced backfat thickness but had no effect on long- issimus muscle cross-sectional area. Harris et al. (1995) conducted a similar study and found that feeding 200,ug Cr/kg of diet as CrPic did not affect perfor- mance or carcass characteristics of swine fed either adequate or low-protein diets. Amoikon et al. (1995) conducted two experiments to determine the effects of dietary chromium on performance, glucose tolerance, and plasma metabolites of swine. Feeding 200,ug Cr/kg of diet as CrPic to swine weighing 21 to 25 kg for 19 or 21 days had no effect on weight gain or feed efficiency. Concentrations of plasma cholesterol were increased, whereas plasma NEFA, urea nitrogen, and insulin concentrations were decreased in swine fed chromium. During a glucose tolerance test, the glucose disappearance rate increased and glucose half-life decreased in swine fed chromium; insulin kinetics were not affected. Dietary chromium did not affect plasma growth hormone concentrations. Mooney and Cromwell (1995) reported that when 200,ug Cr (CrPic)/kg was fed to swine throughout the growing-finishing periods, gain in body weight, gain in muscle mass, and accretion rate of muscle increased, whereas gain in fat and fat accretion rate decreased. The net effects of dietary chromium were swine carcasses that contained an increased percentage of muscle and a decreased per- centage of fat. Similar observations were reported by Lindemann et al. (1995b)

CHROMIUM AND NUTRITION 39 in that the inclusion of 200 ,ug Cr/kg of diet as CrPic increased muscling and decreased backfat of swine. In contrast, Ward et al. (1995) reported that feeding eight sources of chromium, including CrPic and CrCl3, at 200,ug Cr/kg of diet for 77 days had no effect on carcass traits and did not affect growth rate or feed efficiency. Determination of the concentrations of several serum constituents revealed only one inconsistent effect of dietary chromium: Reagent grade CrPic reduced serum NEFA, whereas a commercial source of CrPic had no effect on serum NEFA. Ward et al. (1994) measured the effect of dietary chromium on insulin binding by hepatic plasma membranes in vitro. Feeding swine 200,ug Cr/ kg of diet as CrPic for 42 or 64 days did not affect in vitro insulin binding by hepatic plasma membranes. Wenk et al. (1995) compared the effect of 500 ,ug of Cr/kg of diet given as CrCl3, CrPic, or high-Cr yeast on swine that weighed 27.4 kg each at the start of the experiment. The experiment was terminated when each pig weighed 106.5 kg. Supplementing the diet with 500,ug Cr/kg as CrCl3 improved rate of gain and feed efficiency, and there was a trend for improved performance by swine fed CrPic or high-Cr yeast. No effects of supplemental Cr were observed for carcass fatness. Several reports of studies on the effects of feeding supplemental chromium to growing swine were presented at the 1996 meeting of the American Society of Animal Science. Mooney and Cromwell (1996) fed 200,ug Cr/kg of diet as CrPic to barrows of medium-lean-gain and high-lean-gain genetic backgrounds. Dietary chromium did not affect performance when barrows were reared from 21 to 104 kg body weight. In addition, supplemental chromium did not improve carcass charac- teristics, chemical composition, or accretion rates of carcass protein or fat, regard- less of the genetic potential of the swine. LeMieux et al. (1996) report that supple- mental CrPic did not alter performance of weaning pigs whether fed with or without supplemental betaine or zinc. Harper and Kornegay (1996) also observed no effects of dietary CrPic supplementation on performance or carcass quality of swine grown from weaning to 102 kg body weight in the presence or absence of 5 percent dietary fish meal. In contrast, Lien et al. (1996b) reported improvements in rate of gain of swine fed 200,ug Cr/kg of diet as CrPic for 95 days. This effect was accompanied by reductions in serum triglycerides and low-density lipoprotein cho- lesterol and by increases in serum insulin, creatinine, and high-density lipoprotein cholesterol. Chromium picolinate did not affect backfat thickness. Dietary CrCl3 (200,ug Cr/kg) did not elicit the responses observed with CrPic. Yi et al. (1996) fed gilts averaging 73 kg each in body weight 105 ,ug Cr/head as CrPic during a 160- day growing period. Carcass data obtained when gilts weighed 114.5 kg showed that supplemental chromium increased loin eye area and percentage lean but did not affect other carcass traits, compared with carcasses of swine given no supplemental dietary chromium. Supplemental chromium had no effect on corpus luteum num- bers in the gilts. Min et al. (1997b) fed 20 kg swine diets supplemented with 0, 100, 200, or

40 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION 400 ,ug Cr/kg as CrPic for 103 days. They reported that rate of gain was not affected by dietary chromium, but feed efficiency was improved slightly by feeding 200 or 400,ug Cr/kg of diet, and carcass fat was reduced by 200,ug Cr/ kg of diet. In vitro tests also showed that lipolytic activity and protein synthe- sis of adipocytes obtained from swine fed 200,ug Cr/kg of diet were increased over that of the control groups. Serum triglycerides were decreased by feeding 200 or 400 ,ug Cr/kg of diet, but glucose, insulin, cholesterol and NEFA con- centrations in serum were unaffected. Min et al. (1997a) fed 60 kg swine a diet supplemented with 200 ,ug Cr/kg as CrPic in combination with daily injections of pST. Treatment with pST improved weight gain and feed effi- ciency during the 52-day experiment. Carcass fat also was decreased by pST. Chromium supplementation did not alter the effects of pST on performance or carcass traits, nor did chromium affect lipogenic or lipolytic activity of adi- pose tissue of the swine. In an experiment involving 1,000 pigs, Lindemann and Purser (1997) fed diets supplemented with 0 or 200 ,ug Cr/kg as CrPic. They reported that chro- mium supplementation improved weight gain and feed efficiency of barrows but not of gilts. Percentage lean in the carcass of barrows also was increased by supplemental chromium. Responses of barrows to chromium supplementation were evident whether 200 ,ug Cr/kg was fed throughout the growout period or whether 200,ug Cr/kg was fed in the first grower diet followed by 100,ug Cr/kg in the next three grower diets. Crow et al. (1997) did not observe any effects of feeding diets containing 100, 200, or 500 ,ug Cr/kg as CrPic on performance or carcass composition of swine reared from 45 to 109 kg body weight. They did, however, report that the blood insulin-to-glucose ratio increased linearly as dietary chromium increased. Crow and Newcomb (1997) also found that weight gain, feed efficiency, loin eye area, and backfat thickness were not influenced by supplementing diets with 200,ug Cr/kg as CrPic. In this experiment, no effect of supplemental chromium on blood insulin-to-glucose ratio was detected. Mooney and Cromwell (1997) compared the effects of dietary sources and concentrations of supplemental chromium (200 or 400,ug Cr/kg as CrPic versus 5 or 25 mg Cr/kg as CrCl3) on performance and carcass traits of swine. They reported that 200,ug Cr/kg as CrPic increased ADG and feed intake and increased lipid accretion rate in the carcass compared with feeding no supplemental chro- mium. Other carcass traits, however, were not affected by chromium source or concentration. Results of a second experiment showed that inclusion of either 200 ,ug Cr/kg as CrPic or 5 mg Cr/kg as CrCl3 had no effect on performance or backfat thickness of swine but both chromium sources increased loin eye area. Percentage of muscle tissue in carcasses and accretion rate of muscle tissue were increased and percentage fat was decreased in swine fed supplemental chromium, with CrPic being more effective than CrCl3. No changes in blood metabolites occurred as a result of supplemental chromium in either experiment. Kornegay et al. (1997) conducted four experiments to determine the influ

CHROMIUM AND NUTRITION 41 once of supplemental CrPic on nutrient digestibility and retention in swine. Ani mats weighed 26.3, 13.2, and 30.9 kg at the start of Experiments 1, 2, and 3, respectively. Data obtained from the three experiments, each using 12 barrows, were pooled for statistical evaluation. Supplementation of diets with 200,ug Cr/ kg of diet as CrPic improved dry matter digestibility and absorption of nitrogen as compared with those of swine fed no supplemental chromium. Carcass analy- sis also showed that supplemental chromium increased the area of the longissi- mus muscle. In Experiment 4, 200 ,ug Cr/kg of diet again improved dry matter digestibility and nitrogen absorption during the finishing period. Although there were no effects of supplemental chromium on nitrogen retention in the four experiments, the data showed that dietary nutrient availability was improved when chromium was added to the diet of swine. The reasons for the inconsistencies in responses of growing swine to dietary chromium supplementation observed by several researchers are not known. Anderson (1994) and Mowat (1994) suggested, on the basis of studies with humans and cattle, that animals respond most favorably to supplemental chro- mium when exposed to stress, and that the inconsistent responses to chromium reported in the literature were related to poorly controlled stress conditions. Re- sults of recent research with swine in which limited floor space was used to cause stress, however, have not supported their suggestion. Siberio et al. (1996a) used 21-day-old pigs in a 35-day feeding study to determine the influence of dietary chromium, supplied by a high-chromium yeast, on immune status when swine . were provided with up to 59 percent less than the recommended amount of floor space. They reported that feeding 500 ,ug Cr/kg of diet, with or without supple- mental copper, had no effect on performance or immune response of young swine reared in a crowded environment. Siberio et al. (1996b) also conducted a 15-day trial with 21-day-old pigs in which the animals were fed diets supplemented with 0,500, or 1,000,ug Cr/kg of diet to determine the effect on retention of copper or zinc, which has been shown to increase during stress. Stress was imposed by providing only 0.15 m2 of floor space per pig. Supplemental chromium, provided as high-chromium yeast, had no effect on retention of copper or zinc by the stressed baby pigs. Ward et al. (1997) used a complete factorial arrangement of two supplemen- tal chromium concentrations (O and 400 ,ug/kg of diet as CrPic), two dietary lysine (protein) concentrations (80 and 120 percent of levels recommended by National Research Council, 1988) and two floor space allowances (0.025 and 0.035 m2/kg body weight067) in a study with growing swine. Supplemental chromium improved weight gain and feed efficiency of swine fed the 80 percent lysine diet but slightly impaired performance of swine fed the 120 percent lysine diet. There were no indications that supplemental chromium alleviated the ad- verse effects of the stress of inadequate floor space on pig performance. An interaction between supplemental chromium and floor space, however, was ob

42 THE ROLE OF CHROMIUM INANIMA:L NUTRITION served in that thickness of tenth-rib fat of swine reared in inadequate floor space (0.025 m2/kg body weight067) was increased by chromium supplementation. Sows As a follow-up to feeding 200 ,ug Cr/kg of diet as CrPic to growing swine, Lindemann (1995b) continued feeding 200,ug Cr/kg to gilts through two parities. They found that sows fed chromium had more total pigs born, more live pigs born, and more pigs at 21 days than did sows fed an unsupplemented diet. They also observed that changes in serum insulin concentrations and insulin-to-glucose ratios in the sows indicated an improvement in the efficiency of insulin action as a result of chromium supplementation. In another study, Lindemann et al. (1995a) used gilts that had received no previous dietary chromium supplementation. The treatments evaluated were no supplemental chromium, 200,ug Cr/kg of diet as CrPic during nonlactation only, 200 ,ug Cr/kg during lactation only, or 200 ,ug Cr/kg during both periods. No effects of supplemental CrPic were observed on total pigs born, live pigs born, or pigs alive at 21 days. The authors surmised that these results, which did not support those of Lindemann et al. (1995b), provided evidence of the need for chromium supplementation early in the gilt' s life (from 18 kg body weight). No information describing chromium toxicity for swine was found in the literature. Summary Reported responses of growing-finishing swine to supplemental dietary chro- mium have been inconsistent (Table 3-4~. Statistically significant (P < 0.10) improvements in growth rate as a result of supplementing diets with 200 to 500 ,ug Cr/kg as CrPic were reported in 11 of 31 studies. Similarly, feed efficiency was improved by adding CrPic to diets in 8 of 31 studies. In addition to reports of improved performance of swine, favorable effects of added chromium on selected carcass traits have been observed. When supplemental dietary CrCl3 or CrPic was used, increases in carcass leanness (muscling) were reported in 9 of 24 experiments and decreases in carcass fat were reported for 11 of 26 experiments. Research to determine the influence of added dietary CrPic on reproductive per- formance has been meager and the results were inconsistent: Litter size was increased in one experiment but reproductive traits were unaffected in a second trial. There also is limited information on metabolic changes that could be caused by supplementing swine diets with CrPic. Results of several experiments have shown that supplemental CrPic induced a hypoglycemic response or improved insulin efficiency in swine and, in some instances, in vitro lipogenic and lipolytic activities of adipocytes and livers were altered when swine were fed supplemen- tal dietary chromium. Although responses of swine to supplemental chromium

CHROMIUM AND NUTRITION 43 have been inconsistent, there is an increasing amount of evidence indicating that chromium may favorably alter metabolism of swine under some circumstances, with resultant improvements in growth rate, carcass traits, and reproductive performance. The needfor chromium supplementation of practical swine diets, however, depends on the chromium status of the animals, the amount of biouvailable chromium in the feedstuffs, and exposure of the animals to certain environmental stresses. Thus, a decision to use supplemental chromium in prac- tical swine diets must be based on the potential benefits in individual circum- stances versus the cost of supplementation. Poultry Interest in chromium as a dietary supplement for poultry dates to Hill and Matrone's 1970 study reporting that adding chromium to chick diets decreased the toxicity of dietary vanadium. They found that the growth-depressing effects of 20 mg vanadium/kg of diet decreased as dietary chromium (as CrCl3) concen- trations were increased from 500 to 2,000 mg Cr/kg of diet, although total recov- ery of growth rate was not achieved. They also found that rates of oxidative phosphorylation (P:O ratio) of chick liver tissue were reduced by vanadium and that the in vitro effects of vanadium on the P:O ratio could be prevented by including chromium in the medium at a 10:1 ratio to vanadium. Hafez and Kratzer (1976) confirmed the observations of Hill and Matrone (1970) that inclu sion of 1,000 mg Cr (as CrCl3) alleviates most of the slower growth and mortality of chicks fed diets containing 50 or 100 mg of vanadium/kg of diet. Interest in dietary chromium for laying hens was stimulated when Jensen et al. (1978a,b) reported a favorable effect of chromium (as CrCl3) on albumen quality (Haugh unit score) of eggs. Jensen et al. (1978b) suggested that dietary chromium may be necessary for maintaining the normal physical state of the egg albumen. This suggestion was supported by data showing that 5 mg Cr/kg of diet as CrCl3 prevented the adverse effect on Haugh unit score of 10 mg vanadium/kg of diet. In subsequent research, Jensen and Maurice (1980) were unable to show that chromium fed in the absence of dietary vanadium was essential for optimal albumen quality of eggs, although they confirmed that chromium alleviated the deleterious effects of vanadium on albumen quality. They also reported that dietary chromium had no effect on glucose tolerance of White Leghorn hens, contrary to what has been reported for humans and rats (Mertz, 1992~. Not all research done with laying hens has shown that dietary chromium overcomes the adverse effects of vanadium on albumen quality. Sauveur and Thapon (1983) evaluated CrCl3 and a natural source of chromium and reported that neither source influenced the adverse effects of 5 or 30 mg vanadium/kg of diet on albumen quality. Similarly, Benabdeljelil and Jensen (1989, 1990) were unable to show that 5 to 50 mg Cr/kg of diet improved albumen quality of eggs produced by hens fed 10 or 30 mg vanadium/kg. The reasons for the disparate

44 THE ROLE OF CHROMIUM INANIMA:L NUTRITION TABLE 3-4. Influence of Supplemental Dietary Chromium on Swinea Reference Concentration of Supplemental Dietary Cr per kg (Source) Body Weight at Start and End and Duration Improved of Experiment Growth Rateb Improved Feed Efficiency GROWING PIGS Steele et al. (1977) Tested a Cr-containing GTF 6, 30 kg pigs/trt for NDb in viva study. Adipose tissue samples from 8 pigs for in vitro study. ND Page et al. 200 ,ug (1992a,b) (CrPic) 50 pigs; Yes No Start = 21.9 kg; 102-d trial. Lien et al. (1993) 200, 400, 800 ,ug (CrPic) 32 pigs; Start = ~50 kg; End = ~100 to 110 kg; 75-d trial. No No Evock-Clover and 300 ,ug Steele (1994) (CrPic) 50 barrows; Start = 20 kg; End = 90 kg. No No Van Heugten and 200 ,ug Spears (1994) (CrC13, CrPic, CrNic) 96, 21-d-old pigs; 31-d trial. Yes Yes

CHROMIUM AND NUTRITION 45 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other ND ND ND Yes Yes Yes Reduced ND cholesterol, increased triglycerides. Cooking qualities of pork not affected. ND Yes Reduced glucose ND and lipid, increased insulin. No No ND ND ND ND ND ND No consistent benefit for immune response.

46 TABLE 3-4. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Reference Concentration of Supplemental Dietary Cr per kg (Source) O and 200 ,ug (CrPic) Isolated adipocytes from subcutaneous adipose tissue and red blood cell ghosts. Body Weight at Start and End and Duration Improved of Experiment Growth Rateb Improved Feed Efficiency Berrio et al. (1995) Not stated. ND ND Harper et al. 200 ,ug (1995) (CrPic) 144 pigs in 3 Yes Yes experiments; Start = 7.3 kg; 35-d trial. Lindemann et al. 200 ,ug (1995a,b) (CrPic) Two experiments; No Yes Start = 40.9 and 14.5 kg; End = 98 and 98 kg. Ward et al. (1995) 200 ,ug 128 crossbred pigs; No No (CrPic plus seven other Cr Start = 18.1 kg; sources) 77-d trial. Harper and 200 ,ug Kornegay (1996) (CrPic) 96 pigs; Start = 5.9 kg; End = 10.2 kg. No No

CHROMIUM AND NUTRITION 47 Increased Carcass Reduced Muscling Carcass Fat Change in Serum/Plasma Constituents Hypoglycemic Response or Improved Insulin Efficiency Other ND ND ND ND Cr increased in vitro binding of insulin to RBC ghosts in two experiments and to adipocytes in one experiment. No Yes ND ND Yes Yes ND ND No No Slight, inconsistent, ND e.g., reagent CrPic reduced NEFA but commercial sources did not affect NEFA. No No ND ND Factorial with 5% fish meal.

48 TABLE 3-4. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Concentration of Body Weight Supplemental at Start and Improved Dietary Cr per kg End and Duration Improved Feed Reference (Source) of Experiment Growth Rateb Efficiency Lien et al. 200 ,ug 36 pigs; Yes with CrPic, No (1996a,b) (CrPic or CrC13) Start = ~36 kg; not CrC13. 95-d trial. Siberio et al. 500 ,ug Start = 21 d, 7.3 kg; No No (1996a) (high-Cr yeast) 35-d trial. Siberio et al. 500 and 1,000 ,ug; 21 barrows; No No (1996b) Basal diet = 1,440 ,ug Start = 6 kg; (high-Cr yeast). 15-d trial. Crow and O and 200 ,ug (CrPic) 224 pigs; No No Newcomb (1997) (96 barrows, 128 gilts); Start = 25 kg; End = 109 kg. Kornegay et al. 200 ,ug (1997) (CrPic) 200 ,ug (CrPic) Three experiments, No ND 12 barrows in each experiment; Start = 26.3, 13.2, and 30.9 kg in Experiments 1, 2, & 3, respectively. 12 barrows in ND ND switch-back design; Start = 82 kg.

CHROMIUM AND NUTRITION 49 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other ND No CrPic reduced triglycerides and LDL cholesterol; increased insulin, creatinine, HDL cholesterol. ND ND ND ND ND No effect on immune response. ND ND ND ND No effect on retention of copper or zinc by stressed pigs. No No No ND No effect of Cr on blood insulin-to glucose ratio. Increased No ND ND Improved DM longissimus digestion and muscle area. absorption of nitrogen. No significant effect on nitrogen retention. ND ND ND ND Improved DM digestion and increased nitrogen absorption (P < 0.06). No effect on nitrogen retention.

so TABLE 3-4. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Reference Concentration of Supplemental Dietary Cr per kg (Source) Body Weight at Start and End and Duration of Experiment Improved Improved Feed Growth Rateb Efficiency Min et al. (1997a) 0, 4 mg pST/head daily, and 4 mg pST plus 200 ,ug Cr (No Cr treatment alone). 72 pigs; Start = 60 kg; End = 105 kg; 52-d trial. Yes, with pST, Yes, with pST but Cr had no but Cr had no additional effect. additional effect. Mooney and Cromwell (1997) Exper. 1 0, 200, or 400 ,ug (CrPic) or 5 or 25 mg (CrC13). 35 pigs, individually Yes, by 200 ,ug No penned; from CrPic Start = 19.6 kg; (P<0.07). End = 43.2 kg; 35-d trial. Ward et al. (1997) 400 ,ug (CrPic) SOWS 64 barrows, 64 gilts; Yes, with 80% Yes, with Start = 27.2 kg; lys diet, not 80% lys diet, Dietary lys = 80 or with 120% lys not with 120% of NRC; diet. 120% lys diet. Floor space = 0.035 m2/kg BWO.67 versus 0.025 m2/kg BWO.67

CHROMIUM AND NUTRITION 5 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other No effect of Yes, with pST ND ND No effect of Cr on Cr. but Cr had no lipogenic or additional lipolytic activity in effect. adipose tissues. No No ND ND 200 ,ug from CrPic increased (P<0.07) lipid accretion in carcass. No Cr increased Minimal effects on ND tenth-rib fat of metabolites and pigs reared hormones. with inadequate floor space (P<0.07). Cr did not overcome effect of stress.

52 TABLE 3-4. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Reference Concentration of Supplemental Dietary Cr per kg (Source) 200 ,ug Body Weight at Start and End and Duration of Experiment Improved Improved Feed Growth Rateb Efficiency Lindemann et al. ( 1 995b) or 500/1,000 ,ug (CrPic) During growing Increased litter Indicated period and two size. P<O.10 parities. During growing period only. 39 gilts used. Definitions of abbreviations used in table: BW - body weight Cr- chromium CrNic - chromium nicotinate CrPic - chromium picolinate DM - dry matter d- day GTF - glucose tolerance factor HDL - high density lipoprotein LDL - low density lipoprotein lys - lysine ND - Not determined NEFA - nonesterified fatty acids pST - porcine somatotropin RBC - red blood cells wk- week bOnly statistically significant responses (P < 0.05) are indicated unless stated otherwise.

CHROMIUM AND NUTRITION 53 Hypoglycemic Increased Change in Response or Carcass Reduced Serum/Plasma Improved Insulin Muscling Carcass Fat Constituents Efficiency Other Yes Yes Reduced serum ND insulin and insulin: glucose ratio.

54 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION results obtained by these studies as compared with those of Jensen and Maurice (1980) and Jensen et al. (1978a,b) are not evident. The influence of chromium supplementation on rate of egg production by laying hens also has varied. When CrCl3~6H2O was used as the supplemental source of chromium, no effects on egg production were observed (Benabdeljelil and Jensen, 1989, 1990; Jensen and Maurice, 1980; Jensen et al., 1978b). How- ever, Southern and Page (1994) reported that including 100 or 200 ,ug Cr/kg of diet as CrPic enhanced egg production in one of three experiments with White Leghorn laying hens. Lien et al. (1996a) fed diets containing 0, 200, 400, or 800 ,ug Cr/kg as CrPic and observed no chromium effect on egg production. A1- though weights of eggs and egg yolks were not affected by dietary CrPic in the Lien et al. (1996a) study, cholesterol concentration in egg yolks and in serum of hens was related inversely to dietary CrPic concentration. Kim et al. (1997) fed diets containing 0, 200, 400, or 800,ug Cr/kg as CrPic to laying hens for 6 weeks. Supplemental chromium did not affect rate of egg production, but average egg weight was increased and serum cholesterol was decreased for hens fed 400,ug Cr/kg of diet. Dry matter and protein digestibilities were improved when diets were supplemented with 200, 400, or 800 ,ug Cr. Working with chicks, Cupo and Donaldson (1987) determined the effects of chromium and vanadium on glucose metabolism and lipid synthesis. They re- ported that chromium (as CrCl3) and vanadium increased the rate of glucose utilization of liver tissue 16 percent and 33 percent, respectively. Feeding 20 mg Cr/kg or 20 mg vanadium/kg, alone or in combination, enhanced incorporation of i4C glucose into liver fatty acids. They concluded that chromium and vanadium do not always act antagonistically in biologic processes. Results of research with growing poultry designed to determine the influence of dietary chromium on performance have varied. Baker and Molitoris (1975) found that omission of chromium from a purified, crystalline amino acid diet did not affect growth or feed efficiency of chicks. Steele and Rosebrough (1979, 1981), however, reported that inclusion of 20 mg Cr/kg of diet as CrCl3 increased growth rates of poults fed corn-soybean-meal diets from 7 to 21 or from 1 to 21 days of age, respectively. Steele and Rosebrough (1981) also reported that added dietary chromium accelerated the rate of glucose uptake by chick liver incubated in vitro. Rosebrough and Steele (1981) observed a stimulation of poult growth when 20 mg of chromium were included in a 23 percent crude protein starter diet but not when the diet contained 30 percent crude protein. Supplemental chro- mium also increased the activity of hepatic glycogen synthetase of poults. Because of the interest in modifying chromium content of foods, Anderson et al. (1989) fed diets containing 25, 100, or 200 mg Cr/kg of diet as CrCl3 to turkeys for five weeks. Chromium content of tissues increased as dietary chro- mium increased, with liver tissue accumulating the greatest concentrations. Because research to determine the influence of dietary CrPic on young poul- try was not done until the l990s, few data are available. Liarn et al. (1993) fed

CHROMIUM AND NUTRITION 55 diets containing 0, 100, 200, 300, or 400 ,ug Cr/kg as CrPic to broiler chickens from 1 to 56 days of age. The basal starter, grower, and finisher diets used in this study contained 2,600, 1,400, and 71 ,ug Cr/kg, respectively, as determined by laboratory analysis. Supplemental chromium had no effect on rate of gain, feed efficiency, or carcass composition. In vitro activity of hepatic ATP citrate lyase was increased when 100 or 200,ug Cr/kg of diet were fed but activity of hepatic fructose-1,6-diphosphatase was not. Ward et al. (1993) reported that diets con- taining 200 or 400 ,ug Cr/kg of diet as CrPic did not affect weight gain, feed intake, feed efficiency, or protein, fat, or ash content of muscle of 5- to 19-day- old broiler chicks. Feeding 200,ug Cr/kg, however, tended to increase the protein concentration in whole bodies of chicks. Ward and Southern (1995a) determined growth, organ weight, and selected plasma metabolites of broilers as influenced by dietary CrPic in concentrations of 0, 400, 1,600, and 16,000,ug Cr/kg. In one experiment, CrPic did not affect weight gain, feed intake, feed efficiency, plasma insulin, glucagon, NEFA, or liver, heart, or abdominal fat pad weights. Gener- ally, weights of most body parts were not affected consistently by dietary CrPic. Rate of gain and feed efficiency of chicks improved during the last 7 days of a second experiment of 49 days duration. In a subsequent study, Ward and South- ern (1995b) found that feeding a diet containing 200,ug of Cr/kg as CrPic did not influence weight gain or feed efficiency of broilers from 4 to 8 weeks of age. Also, plasma glucagon and NEFA and glucose clearance rates in 11-week-old broilers were not affected by dietary CrPic. Kim et al. (1996a) fed diets supplemented with 0,100, 200,400,600, or 800 ,ug Cr/kg as CrPic to broiler chickens from 1 to 42 days of age. Supplemental chromium did not affect growth rate or feed efficiency. Carcass fat was de- creased by feeding 100 or 200 ,ug Cr/kg of diet. Two hundred ,ug Cr/kg of diet also decreased serum cholesterol, and HDL cholesterol of serum increased as dietary chromium increased. Percentage mortality of the chickens was inversely related to dietary chromium supplementation. Information is meager on chromium toxicity for poultry. Dietary concentra- tions of chromium ranging from 3 to 1,000 CrCl3 mg/kg caused no adverse effectsongrowingchicks(HillandMatrone,1970;BakerandMolitoris,19751. Similarly, 30 or 100 mg Cr/kg as Na2CrO4 and 100 mg Cr/kg as K2CrO4 had no deleterious effects on growth and health of chicks to 21 days old (Romoser et al., 1961; Mertz and Roginski, 1975~. Feeding a diet containing 2,000 mg Cr/kg as CrCl3 decreased growth in chicks (Hill and Matrone, 1970~. Kim et al. (1996a) fed diets supplemented with 0,800,1,600, or 2,400,ug Cr/ kg as CrPic to broiler chickens from 1 to 42 days of age. No signs of chromium toxicity were observed. Percentage mortality decreased as dietary chromium increased, and serum glucose and cholesterol decreased in chickens fed 800 or 1,600 ,ug Cr/kg of diet. Results of research with young poultry are summarized in Table 3-5 and results obtained with laying hens are summarized in Table 3-6.

56 THE ROLE OF CHROMIUM INANIMA:L NUTRITION TABLE 3-5. Influence of Supplemental Dietary Chromium on Young Poultrya Dietary Cr Concentration per kg Reference (Source) Age/Duration of Increased Experiment Growth Rateb Baker and 3 mg (CrC13) Molitoris (1975) 1- to 27-d-old No response with purified chicks. crystalline amino acid diet. Steele and 20, 40, 80 mg (CrC13) Rosebrough (1979) 7- to 21-d-old turkeys. Yes, with 20 mg/kg in absence of added nicotinic acid. Cupo and 20 mg (CrC13) Donaldson (1987) Four chicks per No treatment; 1- to 21-d-old chicks. Ward et al. (1993) 200, 400 ,ug (CrPic) 60, 5-d-old chicks; No 14-d trial. Ward and 400, 1,600, 16,000 ,ug Southern (1995b) (CrPic); 400, 1,600 ,ug (CrPic). Exp. 1 - 60 chicks; No Start- 5 d; Duration - 28 d. Exp. 2 - 60 chicks; Start- 3 d; Duration 49 d; 5- to 54-day old chicks. Slight from 47 to 54 days of age.

CHROMIUM AND NUTRITION 57 Improved Feed Decreased Efficiency Carcass Fat Change in Serum/Plasma Hypoglycem Constituents Response Other 1C No ND ND ND Yes ND ND ND No No No effect on ND Increased rate of serum fatty acids glucose utilization or cholesterol. by liver in viva and in vitro. No No ND ND No effect on N balance. No No No effect on ND ND plasma glucagon, insulin, urea N. NEFA. Yes Yes, slightly. Reduced plasma ND ND glucose and NEFA. Increased insulin. No effect on glucagon.

58 TABLE 3-5. Continued THE ROLE OF CHROMIUM IN ANIMAL NUTRITION Dietary Cr Concentration per kg (Source) Age/Duration of Increased Experiment Growth Rateb Kim et al. (1996b) 0, 100, 200, 400, 600, or 800 ,ug (CrPic) 288, 1-d-old broiler No chicks; 42-d trial. Definitions of abbreviations used in table: ATP - adenosine triphosphate Cr- chromium CrC13 - chromium chloride CrPic - chromium picolinate d- day HDL - high density lipoprotein N -nitrogen ND - Not determined NEFA - nonesterified fatty acids S- sulfur V - vanadium wk- week bOnly statistically significant responses (P < 0.05) are indicated unless stated otherwise.

CHROMIUM AND NUTRITION 59 Improved Feed Decreased Efficiency Carcass Fat Change in Serum/Plasma Hypoglycem Constituents Response Other 1C No Yes, with 100 Decreased and 200 ,ug. cholesterol with greatest decrease from 200 ,ug Cr. Increased percentage HDL cholesterol as dietary Cr increased. ND Notable decrease in mortality as dietary Cr increased.

60 THE ROLE OF CHROMIUM INANIMA:L NUTRITION TABLE 3-6. Influence of Supplemental Dietary Chromium on Laying Hensa b Reference Dietary Cr Concentration per kg (Source) Age/Duration Jensen et al. (1978b) 5 mg (CrC13) 60-wk-old hens; 6- or 16-wk trial. Sauveur and Thapon (1983) mg (CrC13) 40-wk-old 6- to 8-wk trials. Southern and Page (1994) 100, 200 ,ug (CrPic) 32-, 28-, and 48-wk-old hens; 4- to 8-wk trials. Kim et al. (1997) O. 200, 400, 800 ,ug (CrPic). Complete factorial arrangements with 14 and 16% protein. 960, 37-wk-old hens; 6-wk trial. Definitions of abbreviations used in table: Cr- chromium CrC13 - chromium chloride CrPic - chromium picolinate d- day DM - dry matter ND - Not determined wk- week bOnly statistically significant responses (P < 0.05) are indicated unless stated otherwise.

CHROMIUM AND NUTRITION 6 Effect on Egg Production Effect on Quality of Egg Albumen None Increased albumen quality in presence or absence of dietary vanadium. None No effect on albumen quality in presence or absence of dietary vanadium. Increased in one of three experiments. ND None. Egg weight increased with 400 ,ug. Improved DM and protein digestibility with 200, 400, and 800 ,ug. Decreased serum cholesterol with 400 ,ug. ND

62 Summary THE ROLE OF CHROMIUM INANIMA:L NUTRITION Research with poultry has shown that supplemental dietary chromium can be used to alleviate some of the toxic effects of vanadium in growing chicks and laying hens. Evidence also has been obtained that supplemental chromium at 20 mg/kg of diet as CrCl3 increases the rate of glucose utilization by livers of chicks and poults in vivo and in vitro. The effects of added dietary chromium on growth rate and feed efficiency of growing poultry have differed, with improvements reported in four of eight experiments. An improvement in performance of young poultry was observed when CrCl3 was used to supply 20 mg Cr/kg of diet. Supplementing diets with 200 ,ug to 1,600 ,ug Cr/kg as CrPic increased growth rate of chicks in only two of seven studies. Recent research with broiler chickens, however, has shown that supplemental chromium decreased mortality during periods of rapid growth. The evidence available today, although meager, indicates that supplemental dietary chromium can affect metabolism and well-being of poultry. Additional information is needed, however, to describe the circumstances in which supple- mental dietary chromium can be used to greatest advantage. Horses A limited amount of work has been done to determine the effects of chromium supplementation on performance and metabolic responses of horses. Daily supplementation of 5 mg chromium as chromium yeast to a diet contain- ing an undetermined concentration of chromium for a 14-day period did not affect fasting (8 hours) concentrations of plasma glucose, insulin, cortisol, or triacylglycerols (Pagan et al., 1995~. Trained thoroughbred horses fed chro- mium supplements tended to have lower plasma insulin concentrations 1 hour after feeding, but other plasma metabolites were not affected by chromium. Plasma glucose tended to decrease during exercise, and post-exercise concen- trations of triacylglycerols were increased in horses receiving chromium supplements. Peak plasma lactate concentrations tended to be lower, and those of cortisol were lower just prior to and in the early stages of the exercise program. Recently completed research at Louisiana State University (D. Thompson, personal communication) showed that feeding 5 mg chromium per head daily as CrPic did not affect metabolic or hormonal responses when adult mares were subjected to a glucose tolerance test, insulin challenge, epinephrine challenge, feeding challenge, or exercise challenge. Pokeweed mitogen tended to stimulate greater proliferation of lymphocytes in chromium-supplemented mares than it did in control mares. The meager amount of research conducted with horses, described here, has not shown any definitive benefits of dietary chromium supple- mentation.

CHROMIUM AND NUTRITION 63 Rats Because supplemental trivalent chromium (Cr+3) has been reported to en- hance insulin and glucose metabolism, it has been suggested that chromium is essential for the rat and that its function is to aid in the utilization of glucose. The work of Schwarz and Mertz (1959), Schroeder et al. (1963), Schroeder (1966), Roginski and Mertz (1967), Mertz et al. (1965), Mertz and Roginski (1969), and Roginski and Mertz (1969), using highly restrictive environmental conditions, often is cited as evidence for the essentiality of chromium for the rat. Many of the chromium deficiency symptoms are typical of those associated with aging. Be- cause of the larger number of responses associated with this process, life span should be positively affected by chromium supplementation. This effect of chro- mium was assessed by Evans and Meyer (1992, 1994) in rats. They found an increase in life span of greater than 25 percent due to supplementation of the diet with 1,000 ,ug Cr/kg as CrPic. Whether chromium contributes to a beneficial physiological function is uncertain. Specificity is questioned because other heavy metals seem to initiate similar effects (Fagin et al., 1987; Pederson et al., 1989~. Other studies have failed to show positive effects of chromium on glucose tolerance or glucose utilization in tissues of rats (Flats et al., 1989; Holdsworth and Neville, 1990; Woolliscroft and Barbosa, 1977~. Woolliscroft and Barbosa (1977) fed 6-week-old Sprague-Dawley rats 30 percent Torula yeast diets con- taining low chromium concentrations (30 to 100 ,ug/kg, as CrCl3 estimated) or diets that contained 5,000 ,ug Cr/kg. After 6 weeks, there was no significant difference in intravenous glucose tolerance between the two groups. Flatt et al. (1989) found no significant difference in food intake, body weight gain, glycosylated hemoglobin, plasma glucose, plasma insulin, glucose tolerance, or insulin sensitivity between two groups of weaning Wistar rats fed either 30 or 1,000,ug Cr/kg of diet as CrCl3 for 32 days. Differences in chromium concentra- tions in tissues between the two groups varied from no change in skeletal muscle to a 44 percent reduction in the pancreas. Holdsworth and Neville (1990) found that supplementing the diet with chro- mium acetate had no effect on glucose metabolism in rats. They fed weanling Wistar rats Torula yeast diets similar to those designed by Schwarz (1951) but supplemented with ~-cystine, ~-methionine, and ~-histidine. These supplemented diets supported more rapid growth than did the original diet and contained 100 (low-chromium diet) or 1,000 ,ug Cr/kg (high-chromium diet). A control group was fed a commercial natural-ingredient diet. After five weeks, the rats fed the Torula yeast diets gained 30 percent less weight than did the control rats, regard- less of whether supplemental chromium was used. Those fed chromium-supple- mented yeast diets did not grow at a significantly greater rate than did those without supplemental chromium. The incorporation of glucose carbon into liver glycogen in the rats fed the low-chromium diet was only one-fifth that of the control rats, but was not different from that of rats given the chromium-supple

64 THE ROLE OF CHROMIUM IN ANIMAL NUTRITION mented yeast diet. Yeast was grown in media with or without chromium. Ex- tracts from this yeast enhanced glucose incorporation into glycogen of hepato- cytes isolated from rats fed low- or high-chromium diets regardless of whether chromium was present in the extract. Others reported lower sperm counts in semen of rats fed low-chromium diets (<100,ug/kg) for eight months, compared with semen of rats fed high-chromium diets (2,000,ug/kg) (Anderson and Polansky,1981~. Effects of chromium supple- mentation on weight gain of rats are achieved only with restrictive environmental conditions (Schroeder et al., 1963~. Under similar conditions, supplementation of the diet with other heavy metals, such as cadmium and lead, also enhanced initial weight gain, suggesting a nonspecific pharmacological response rather than a nutritional response (Schroeder et al., 1963~. Anderson et al. (1996b) measured the incorporation of chromium into the tissues of rats fed nine different forms of chromium. They postulated that insu- lin-chromium-nicotinic acid complexes stabilized with amino acids would have the greatest incorporation into tissues because these complexes would be similar to the naturally occurring, biologically active complexes that have been shown to be better utilized. Chromium absorption in conjunction with tissue chromium concentrations was determined in order to ascertain whether tissue chromium stores, and presumably status, are regulated at the level of absorption. Chromium compounds tested were chromium chloride, chromium acetate, chromium potas- sium sulfate, chromium trihistidine, chromium triglycine, chromium trinicotinic acid, chromium dinicotinic acid dihistidine, chromium tripicolinic acid, and chro- mium dinicotinic acid diglycine cysteine glutamic acid. Complexes were fed to weanling rats for three weeks at 5,000 ,ug Cr/kg of diet. The basal control diet was a cornstarch-based diet containing 30 ,ug Cr/kg. Chromium incorporation into the kidney was greatest for chromium dinicotinic acid diglycine cysteine glutamic acid (850 ng/g dry weight) followed by chromium potassium sulfate (407 ng/g), chromium acetate (397 ng/g), chromium dinicotinic acid dihistidine (394 ng/g), CrPic (368 ng/g), chromium glycine (343 ng/g), CrNic (166 ng/g), chromium chloride (74 ng/g), chromium trihistidine (49 mg/g), and control (23 ng/g). Chromium concentration of the liver was greatest for the chromium picolinate compound (50 ng/g) followed by chromium dinicotinic acid diglycine cysteine glutamic acid and chromium acetate. Liver chromium concentrations of rats fed the remaining complexes were not significantly different from those of control animals that received no added chromium. Chromium concentrations were significantly greater in the kidney than in the liver, spleen, heart, lungs, and gastrocnemius muscle. Differences in absorption of radioactive forms of chro- mium did not explain the differences in tissue chromium concentrations. Chro- mium absorption after 4 hours and retention after 24 hours were not significantly different for the forms of chromium tested. These data demonstrate that chro- mium concentrations are greatest in the kidney and that the form of dietary chromium significantly affects tissue chromium concentrations. Absorption of

CHROMIUM AND NUTRITION 65 chromium does not correlate with tissue chromium concentrations, and blood chromium is not in equilibrium with tissue chromium stores. Although the early studies seemed to indicate that dietary chromium supple- ments enhanced glucose metabolism, more recent studies do not. Perhaps the duration of the experiments and the environmental conditions in the later studies were insufficient to allow chromium stores to be depleted and deficiency signs to be expressed. Signs of chromium deprivation might have been more evident if longer feeding periods or multiple-generation studies had been used. Trivalent chromium salts, chromic oxide, and metallic chromium are not very toxic. Because of their oxidizing and protein-precipitating properties, chro- mium trioxide, chromates, and bichromates are potent poisons. The LD50 (the dose that is lethal to 50 percent of test animals) for a Cr+3-nicotinic acid complex injected intravenously was 60 mg/kg of body weight in rats. The lethal single oral dose for Cr+6 in young rats was 130 mg/kg, whereas as much as 650 mg Cr+3/ kg produced no overt toxicosis (National Research Council, 1980~. Summary Chromium has been shown to be essential for glucose metabolism in rats only under highly controlled experimental conditions in which body stores of chromium were depleted. Mainly on this basis, chromium has been listed in the National Research Council (1995) publication Nutrient Requirements of Labora- tory Animals as a potentially beneficial dietary constituent for rats, but a dietary requirement per se was not given. Rabbits Chromium is present in very low concentrations in the tissues and body fluids of rabbits. Plus (1988) reported mean chromium concentrations of 0.65, 1.00, 4.90, and 0.80 ppm in rabbit liver, kidney, serum, and brain, respectively. Chromium deficiency in rabbits has not been demonstrated. However, posi- tive effects of chromium on cholesterol metabolism and sucrose utilization have been reported. For 135 days, Abraham et al. (1991) treated 33 Loewenstein (Yoknian) male rabbits with daily injections of distilled water; of 20 ,ug potas- sium chromate; or of 1,5,10, or 20,ug CrCl3. The rabbits were fed a cholesterol- enriched (1 percent cholesterol) diet ad libitum. The mean serum chromium concentrations were increased by 2- to 10-fold for treated rabbits within 1 week of treatment. It also was observed that the cholesterol content in aorta, aortic weight, and aortic minimal surfaces covered by plaque were decreased by ap- proximately 50 percent for rabbits treated with either form of chromium. Similar decreases in cholesterol-induced plaque in rabbits injected daily with potassium chromate also have been previously reported (Abraham et al., 1980, 1982a). Moersen and Borgman (1984) studied changes in zinc and chromium me

66 THE ROLE OF CHROMIUM INANIMA:L NUTRITION tabolism in rabbits fed purified diets with either corn starch or sucrose as the sole dietary source of carbohydrate. Sixteen mature New Zealand White rabbits were assigned to two dietary treatments for 14 weeks. Hair samples obtained at the beginning and end of the experiment were analyzed for zinc and chromium content. The chromium concentration in hair of rabbits fed the sucrose diet decreased by about 30 percent at the end of week 14. Similar studies with rats and humans have reported a negative chromium balance on diets high in glucose or sucrose (Roginski and Mertz, 1969; Mertz et al., 1974~. The minimal toxic concentration of chromium for rabbits is unknown. Chro- mium in high doses can be toxic to rabbit liver and kidney. Tandon et al. (1978) treated male albino rabbits with daily injections of either normal saline, 2 mg Cr/kg of body weight as chromium nitrate (trivalent form), or potassium bichromate (hexavalent form) for a period of six weeks. Serum urea concentration of chro- mium-treated rabbits increased threefold within three weeks of treatment. This change was coupled with cellular damage to liver and kidney. Serum chromium concentrations were about five times greater for rabbits injected with the hexa- valent form than they were in the rabbits treated with trivalent chromium. Aerosol exposure of rabbits to Cr+3 or Cr+6 (hexavalent) at a rate of 0.9 mg/m3 for six weeks (five days/week and six hours/day) resulted in nodular accumulations of the alveo- lar macrophages, with most macrophages showing enlarged lysosomes. However, gross appearance of lung tissue was normal (Johansson et al., 1986~. Overall, the hexavalent form induced greater toxic effects than did the trivalent form. Summary Research with rabbits indicates that cholesterol and plaque content of the vascular system were decreased by supplemental dietary chromium and that the dietary carbohydrate source can modify chromium metabolism. There is, how- ever, insufficient information on which to base conclusions or recommendations concerning rabbits' need for dietary chromium. Fish Investigations of the influence of chromium in fish nutrition have been lim- ited, although more attention has been focused on this topic in recent years. Tacon and Beveridge (1982) fed rainbow trout (Oncorhynchus mykiss) chemi- cally defined diets (1.56 mg Cr/kg provided by the ingredients) supplemented with 0,1,3, or 6 mg Cr/kg as CrCl3 and observed no differences in weight gain or changes in tissue chromium distribution. In a more recent study, supplementa- tion of practical diets with chelated chromium at 0.5 mg/kg of diet increased blood glucose clearance in rainbow trout but did not affect weight gain or protein and energy retention (Bureau et al., 1995~. Hertz et al. (1989) reported that chromium supplementation of a fishmeal-soybean-meal-based diet improved glu

CHROMIUM AND NUTRITION 67 cose utilization in common carp (Cyprinus carpio). Increased intestinal absorp- tion of glucose was reported in snakehead (Channa punctatus) at concentrations as low as 1 mM (Sastry and Sunita, 1982~. Significant effects of dietary chromium on growth and carbohydrate utiliza- tion of hybrid tilapia (Oreochromis niloticus x O. aureus) have been reported. In a study by Shiau and Lin (1993), hybrid tilapia were fed fishmeal-based diets containing either cornstarch or glucose at 40 percent dry weight with or without supplemental chromium (2 mg Cr/kg) as CrCl3. The unsupplemented diets were analyzed to contain 0.4 mg Cr/kg. Weight gain and retention of dietary protein and energy in fish fed the diets containing cornstarch were not affected by chro- mium supplementation but were significantly greater than in fish fed diets con- taining glucose. Chromium supplementation of the diet containing glucose did, however, significantly increase weight gain, energy deposition, and liver glyco- gen of fish, and it delayed the postprandial peak in plasma glucose from two to three hours. Thus, chromium supplementation enhanced the utilization of dietary glucose but not cornstarch by hybrid tilapia. In a related study, Shiau and Chen (1993) fed hybrid tilapia fishmeal-based diets similar to those described previ- ously, containing either cornstarch or glucose with or without supplemental chro- mium (2 mg Cr/kg) as CrCl3, Na2CrO4.4H2O, or Cr2O3. Fish fed the diets with cornstarch, regardless of chromium supplementation or source, had significantly greater weight gain, feed intake, protein and energy retention, and body lipid compared with those fed diets with glucose. The fish fed the glucose diet without supplemental chromium showed significantly less weight gain as well as more rapid plasma glucose peak time and elevated glucose-6-phosphate activity, com- pared with fish fed glucose diets with supplemental chromium, regardless of source. Fish fed the glucose diet supplemented with Cr2O3 had weight gain, feed intake, protein and energy retention, body lipid, and phosphofructokinase activity similar to that of fish fed cornstarch diets and much greater than found in fish fed the other glucose diets. Thus, supplementation of Cr2O3 to a diet containing glucose improved glucose utilization by hybrid tilapia and was much more effec- tive than were the other sources of chromium. Specific mechanisms by which chromium enhances the utilization of dietary glucose in hybrid tilapia, however, were not elucidated. In a subsequent study, Shiau and Liang (1995) observed enhanced weight gain and feed utilization of hybrid tilapia fed diets containing cornstarch relative to fish fed diets containing glucose. Also, fish fed diets containing glucose supplemented with Cr2O3 at 0.5 percent exhibited increased weight gain, feed efficiency, protein efficiency ratio, protein deposition, and phosphofructokinase activity, as well as lower tissue chromium, than did fish fed a similar diet supple- mented with 2 percent Cr2O3. The Indian major carp (Labeo rohita) also has been reported to exhibit growth enhancement when fed 10 mg Cr/kg of diet, but growth reduction when fed 20 and 40 mg Cr/kg; and carcass chromium was increased by chromium intake (Jain et al., 1994~.

68 THE ROLE OF CHROMIUM INANIMA:L NUTRITION The effects of waterborne chromium also have been studied in some fish species. Chronic exposure to waterborne Cr+6 has been reported to inhibit Na/K- ATPase activity but not Mg-ATPase activity in several tissues of rainbow trout (Kuhnert et al., 1976~. It has also been reported to increase the frequency of micronuclei induction in erythrocytes of Prussian carp (Carassius auratus gibelio) (Al-Sabti et al., 1994), and to alter several enzyme activities in snakehead (Sastry and Sunita, 1983), rainbow trout, and seabass (Dicentrarchus labrax) (Boge et al., 1988). Summary Investigations concerning the influences of dietary chromium on fish have been limited. Some studies reported no effect on growth or tissue chromium distribution. Others showed that chromium supplementation of diets, especially those containing glucose, caused significant increases in weight gain, energy deposition, and liver glycogen, and altered postprandial plasma glucose concen- trations. Some differences in response to different sources of dietary chromium also have been reported. Specific mechanisms by which chromium influences dietary carbohydrate utilization of fish have not been elucidated. CONCLUSIONS Based on careful analysis of the available scientific literature, as presented in this chapter, the following conclusions can be made. (1) It is not possible to make specific recommendations as to dietary form and concentration of chromium supplementation for cattle, poultry, and swine because · there are insufficient comparative data for the determination of relative biouvailabilities of chromium from supplemental sources; · only meager data are available from titration studies designed to deter- mine supplemental chromium concentrations that are most effective for cattle, poultry, and swine; and · there have been no studies designed or conducted to determine dietary chromium requirements of cattle, poultry, or swine. (2) Supplementing practical diets with trivalent sources of chromium might be beneficialfor health and well-being of cattle during times of stress; however, the factors that affect the efficacy of supplemental chromium and the dietary chromium concentrations required have not been determined. (3) Chromium supplementation of swine diets, beginning at an early age and continuing through thefinishing period, could improve carcass leanness and subsequent reproductive efficiency, but these responses to chromium are likely to be inconsistent until the factors that affect the efficacy of dietary chromium inclusion are more clearly defined.

CHROMIUM AND NUTRITION 69 (4) Information in the scientific literature on the need for supplemental chromium in practical diets offish, horses, sheep, rabbits, and rats is too sparse to allow conclusions. (5) Although most research on potential toxicity of trivalentforms of dietary chromium has been conducted with rats and chickens, the results show that the concentrations of trivalent chromium typically added to diets offood-producing animals are safe and nontoxic (National Research Council, 19804. (6) Additional research is needed to determine the biouvailability of chro- mium contained in feed ingredients and to obtain more definitive data on the comparative biouvailability of chromium from dietary supplements. (7) Research should be designed to create reproducible signs of chromium deficiency in animals, which would facilitate the establishment of dietary chro- mium requirements by way of appropriate titration studies.