The discovery of a novel series of glucokinase activators based on a pyrazolopyrimidine scaffold.

Glucokinase is a key enzyme in glucose homeostasis since it phosphorylates glucose to give glucose-6-phosphate, which is the first step in glycolysis. GK activators have been proven to lower blood-glucose, and therefore have potential as treatments for type 2 diabetes. Here the discovery of pyrazolopyrimidine GKAs is reported. An original singleton hit from a high-throughput screen with micromolar levels of potency was optimised to give compounds with nanomolar activities. Key steps in this success were the introduction of an extra side-chain, which increased potency, and changing the linking functionality from a thioether to an ether, which led to improved potency and lipophilic ligand efficiency. This also led to more stable compounds with improved profiles in biological assays.

Peroxisomal trans-2-enoyl-CoA reductase is involved in phytol degradation.

Phytol is a naturally occurring precursor of phytanic acid. The last step in the conversion of phytol to phytanoyl-CoA is the reduction of phytenoyl-CoA mediated by an, as yet, unidentified enzyme. A candidate for this reaction is a previously described peroxisomal trans-2-enoyl-CoA reductase (TER). To investigate this, human TER was expressed in E. coli as an MBP-fusion protein. The purified recombinant protein was shown to have high reductase activity towards trans-phytenoyl-CoA, but not towards the peroxisomal beta-oxidation intermediates C24:1-CoA and pristenoyl-CoA. In conclusion, our results show that human TER is responsible for the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes.

From behavior to molecules: an integrated approach to the study of neuropeptides.

Despite extensive information on many aspects of peptide neurobiology, the links between the behavioral effects of neuropeptides and their actions at the cellular and molecular levels are not fully understood. A pair of insect neuropeptides, the cardioacceleratory peptides (CAPs) of the tobacco hawkmoth Manduca sexta, provide an opportunity to elucidate these links. The CAPs are involved in the modulation of four distinct types of behavior during the life cycle of this moth. Functional differences at these four developmental periods can be explained by stage-specific changes in target sensitivity and the distribution of the CAP-containing neurons, including a set of peptidergic neurons that alter their transmitter phenotype postembryonically. Studies show that inositol 1, 4, 5-trisphosphate (IP3), linked to intracellular Ca2+, mediates the response of the cells to the CAPs. This preparation thus provides additional insights into the mechanisms underlying the action of multifunctional neuropeptides.

Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations.

In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a 'proteostasis network' and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge - the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.

Amphetamine challenge test, response to treatment, and lateral ventricle size in schizophrenia.

The hypothesis of two independent pathologies in schizophrenia proposed by Crow (1980) were tested. Two dimensions of the dopamine variable, namely, the behavioral response during the Amphetamine Challenge Test (ACT) and the response to neuroleptic treatment, were studied in a cohort of 19 subjects with a research diagnosis of schizophrenia (n = 18) or schizoaffective disorder (n = 1) in an acute inpatient setting. The size of the lateral ventricle was assessed by mesauring the ventricle-brain ratio (VBR) on the computerized tomographic brain scan. Patients who had greater symptom reduction with the neuroleptic treatment worsened more in their positive psychotic symptoms during the ACT. Those with larger VBRs showed less treatment responsiveness and no worsening during the ACT. The findings are supportive of Crow's hypothesis. The ACT has the potential to be an index of both Type I and Type II pathologies.

How "blind" are double-blind placebo-controlled trials of benzodiazepine hypnotics?

This study examined the accuracy of insomnia patients and their treating physicians in rating whether an active hypnotic drug or a placebo was given in treatment. Forty older adults with primary insomnia were randomly assigned to either an active (temazepam) or a placebo condition using a double-blind strategy. Ratings of treatment conditions were obtained at 1 week (early treatment), 4 weeks (midtreatment), and 8 weeks (late treatment). Patients were able to accurately discriminate (beyond chance levels) between the active and placebo medications at the early (76.9% accuracy) and late treatment assessment timepoints (78.1% accuracy), but not at midtreatment (51.5% accuracy). Therapists, however, were able to make accurate discriminations at the late treatment assessment timepoint only (80% accuracy); early (69.2% accuracy) and midtreatment (47.2% accuracy) ratings did not exceed chance levels. Patients who had used hypnotic drugs prior to this trial were more accurate in their judgments of treatment conditions than those without prior exposure. The findings raise an important issue about the internal validity of the double-blind strategy, which may in fact be only a single-blind procedure.

Case of lipoblastoma with two derivative chromosomes 8 containing homogeneously staining-like regions and a review of the literature: lipoblastoma and chromosome 8.

We report a case of a lipoblastoma in a 10-month-old girl in which the cytogenetic aberration showed a homogeneously staining-like region (hsr) within two derivative chromosomes 8. There was a loss of one normal copy of chromosome 8 and gain of two identical derivative chromosomes 8 with the karyotype designation 47,XX,psu idic(8)(pter-->q12 approximately 13::hsr::q12 approximately 13-->pter),+psu idic (8)(pter-->q12 approximately 13::hsr::q12 approximately 13-->pter). This is the first report of a chromosomal aberration of this type seen in lipoblastoma.

Locally correlated activity in the goldfish tectum in the absence of optic innervation.

Despite a substantial literature on the role of correlated presynaptic activity in the patterning of nerve connections during synaptogenesis in the central nervous system, few studies have focuses on postsynaptic activity during this process. To address the possibility that the target exhibits correlated activity independently of presynaptic activity, extracellular activity was recorded from the main optic innervation layer stratum fibrosum et griseum superficiale (SFGS) in goldfish in which the optic nerve was crushed or the eye removed. At about 2 weeks after denervation, multiunit recordings revealed phasic temporally correlated discharge between different tectal units. Auto-correlation analysis of these trains showed a broad peak 75-100 ms wide confirming temporal correlation. Using cross-correlation analysis of two simultaneous recordings at different distances across tectum, this correlation was shown to be local. Strong positive correlations were detected over about 200 micron and decrease with greater distances disappearing beyond about 400 micron. These correlograms showed a broad symmetrical peak about 75-100 ms wide. This pattern of activity persisted from the day following nerve crush into the period of activity dependent reinnervation at 1 month. When the eye was removed, the pattern could be demonstrated for up to 3 months of denervation indicating the circuitry responsible for the correlated activity was quite stable in the absence of optic innervation. We conclude that tectal elements are capable of locally correlated discharge independently of optic innervation. We propose that locally correlated discharge represents cooperative groups of tectal cells and that these groups, rather than single cells, are the target of the activity dependent synaptic rearrangement such as ocular dominance columns which occurs during synaptogenesis.

The effect of flurbiprofen on steady-state plasma lithium levels.

STUDY OBJECTIVE: To evaluate the effects of flurbiprofen therapy on the pharmacokinetics of lithium. DESIGN: Placebo-controlled, single-blind, crossover study. SETTING: University-affiliated hospital. PATIENTS: Eleven healthy women with bipolar disorder. INTERVENTIONS: The subjects received therapeutic doses of lithium administered as an immediate-release capsule every 12 hours. In addition, they received one placebo tablet every 12 hours during phase I and flurbiprofen 100 mg every 12 hours during phase II of the study. MEASUREMENTS AND MAIN RESULTS: Steady-state pharmacokinetic parameters were measured for each phase. Lithium trough plasma concentration (Cmin) and area under the curve were statistically significantly increased (p < 0.05) when patients received flurbiprofen. Flurbiprofen also caused decreases in lithium clearance and 24-hour lithium urine excretion, although the changes did not reach statistical significance. Clinically significant increases in Cmin appeared to be associated with a greater than 1000-microgram/24 hour decrease in urinary excretion of prostaglandin E2. CONCLUSION: Patients with clinically normal renal function may experience an increase in lithium levels with the initiation of flurbiprofen therapy.

Leukemic phase of mantle cell lymphoma presenting as anemia: diagnosis by combining flow cytometry and cytomorphology.

We report a case of mantle cell lymphoma in leukemic phase, which was diagnosed by a bone marrow biopsy performed as part of a workup for chronic anemia in a patient without lymphadenopathy. The patient, a 79-year-old man with diabetes mellitus, hypertension, chronic renal failure, congestive heart failure, and atherosclerosis, presented with claudication. On admission, he also had an 8-month history of anemia, during which time he experienced a 18-kg weight loss. On presentation, the patient had normal vital signs, anemia, leukocytosis (as well as an absolute lymphocytosis), and splenomegaly; as mentioned, lymphadenopathy was absent. A bone marrow biopsy showed an increase in small to intermediate-sized, slightly irregular lymphocytes in interstitial nodules. Flow cytometric immunophenotyping of the bone marrow identified a monoclonal population of cells, representing 25% of cells within the bone marrow, with expression of CD19, CD20, immunoglobulin M/D, lambda light chain, HLA-DR, and CD5; reactions for CD10 and CD23 were absent. Based on morphologic and immunophenotypic analysis of the bone marrow, as well as morphologic review of the peripheral blood smear, a diagnosis of mantle cell lymphoma involving the bone marrow and in leukemic phase was made. Subsequent polymerase chain reaction analysis of DNA from peripheral blood identified a population of cells with the bcl-1 rearrangement. This case is unique in that the diagnosis of mantle cell lymphoma was made without lymph node or spleen analysis and the patient, although exhibiting bone marrow and peripheral blood involvement by mantle cell lymphoma at presentation, did not have lymphadenopathy.

Site and extent of starch and neutral detergent fiber digestion as affected by source of calcium and level of corn.

Five steers (385 kg) fitted with permanent abomasal cannulae were used to compare Ca source (limestone or dicalcium phosphate) and corn level on site and extent of digestion. Diets contained 50, 70 or 90% corn, with corn silage and supplement to provide .70% Ca from either limestone or dicalcium phosphate. Limestone did not affect ruminal digestion, but postruminal starch and neutral detergent fiber (NDF) digestion were higher (P less than .05) for limestone compared with dicalcium phosphate, which suggests that starch utilization may be increased postruminally by a compound with buffering capacity. As a percentage of total starch intake, total tract and ruminal starch digestion increased (linear, P less than .01) while postruminal starch digestion decreased (linear, P less than .01) with corn level. Neutral detergent fiber digested in the rumen decreased (linear, P less than .01) and postruminal NDF digestion increased (linear, P less than .01) when level of corn in the diet increased. Effects of corn level were not different when organic matter and starch intake were included as covariates.

Procedural and mathematical considerations in urea dilution estimation of body composition in lambs.

Two experiments (n = 46 and 56, respectively) were conducted to evaluate urea dilution as an estimator of body composition in lambs and to address certain procedural and mathematical considerations in this technique. In Exp. 1, 14 blood samples were taken over 240 min after urea infusion. The equation describing the urea clearance curve was: delta PUN = 9.7e-.1727(min) + 10.4e-.0021(min), pools 1 and 2, respectively (r2 = .99, P less than .001; individual lamb effects removed). In the combined experiments, urea space (US) was related to percentage of empty body water (PEBH2O) by the equation 31.7 + .471 US (empty body weight basis; r2 = .56, P less than .001). The regression equation indicates that the US-PEBH2O relationship in lambs is different from that reported in cattle, even though urea clearance kinetics are similar. Although the prediction equations appeared to be biologically valid, considerable error was associated with the composition estimates. The PEBH2O was predicted as well by live weight (r2 = .69; SEy.x = 3.0) as by US in these experiments. The two-sample method (T12 minus T0) to determine the change in marker concentration was shown to be related more closely (r2 = .56) to PEBH2O than the standard multisample extrapolation to T0 method (r2 = .0 and .38 for pools 1 and 2, respectively). An equilibration time of 9 to 12 min provided the best estimate of body composition in lambs.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of growth rate and compensatory growth on body composition in lambs.

Fifty lambs were used in a comparative slaughter experiment to determine the effects of growth rates and compensatory growth on body composition. The study consisted of a growing and a finishing phase. During the growing phase, lambs (20 to 30 kg) were fed three different concentrate levels (30, 50 or 70%) to gain at three different rates (slow, medium and rapid). The finishing phase was evaluated in two periods (early, 30 to 38 kg; late, 38 to 45 kg). All lambs received 70% concentrate diets during the finishing phase. Groups of five lambs were sacrificed at 20, 30, 38 and 45 kg fleece-free weights for whole-body chemical analysis. At 30 kg live weight, lambs on a rapid growth diet were the fattest (P less than .01) and contained the least protein (P less than .05) in their empty bodies. The slower the lambs gained during the growing phase, the greater (P less than .05) was the response in rate of gain and feed efficiency during both periods of the finishing phase. Compensatory growth occurred in two stages; a greater proportion of protein gain was made early while a greater proportion of the fat gain was made during the late period of the finishing phase.

Feeding combinations of dry corn and wheat to finishing lambs and cattle.

Two finishing trials, one with lambs and one with cattle, were conducted to determine the effect of combinations of dry corn (whole or rolled) with dry rolled wheat on animal performance. The cattle (mixed crossbred yearlings) trial also evaluated methods of getting cattle to full feed by stepping-up using either grain combinations or dry rolled corn and then feeding the appropriate grain combination. In the lamb trial, as the level of dry whole corn increased in the diet of 80 Rambouillet X Suffolk lambs (29 kg), feed intake (linear, P less than .01), gain (linear, P less than .01; quadratic, P less than .15) and feed efficiency (linear, P less than .01; quadratic, P less than .15) were improved. The major improvement occurred in the first 30 d of feeding when feed efficiency was improved (linear, P less than .01; quadratic, P less than .01) by the inclusion of dry whole corn. In the cattle trial (272 mixed crossbred yearling cattle, avg = 358 kg), as the level of dry rolled corn increased, feed intake (linear, P less than .01), gain (linear, P less than .01; quadratic, P less than .01) and feed efficiency (linear, P less than .05; quadratic, P less than .05) were improved. Cattle fed 67 or 33% dry rolled corn with 33 or 67% dry rolled wheat gained 4% faster and 4.4% more efficiently than the average performance of cattle fed 100% corn or wheat. Cattle stepped-up on dry rolled corn and then switched to 100% wheat, tended to gain faster (.1 kg/d) than cattle stepped-up on 100% wheat.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol-17 beta effects on lipid metabolism of adipose tissue in nutritionally induced lean and obese ovariectomized ewes.

Nutritionally manipulated lean (68 kg) and obese (87 kg) ovariectomized ewes were administered estradiol-17 beta (E2) or sham implants. Ewes individually had ad libitum access to corn silage. Rates of de novo lipogenesis, palmitate esterification, and glycerol and fatty acid release were determined with slices of subcutaneous adipose tissue at 0, 5, and 30 d after implantation. Condition and E2 interacted over time (linear, P less than .12; quadratic, P less than .05) to affect intake. Lean ewes implanted with E2 decreased intake initially after implantation, whereas obese ewes implanted with E2 decreased intake later after implantation. The linear effect of time x condition x E2 interacted (P less than .02) for lipogenesis. Lipogenesis was inhibited in both the lean and obese ewes implanted with E2. Lean compared with obese ewes without E2 had increased lipogenesis at a faster rate over time. Esterification increased (linear, P less than .01) in the lean ewes and decreased (quadratic, P less than .01) in the obese ewes over time. A time x E2 interaction occurred for esterification (P less than .02). Glycerol and fatty acid release were variable over time within condition and E2. A 48-h adipose tissue culture determined the effect of E2 on lipid metabolism. Estradiol-17 beta decreased (P less than .05) lipogenesis, decreased (P less than .08) esterification, and maintained fatty acid and glycerol release. Data in vivo and in culture indicated that E2 acted to decrease de novo lipogenesis and palmitate esterification and had little or no effect on lipolysis.

Impact of weighing procedures and variation in protein degradation rate on measured performance of growing lambs and cattle.

A lamb growth trial was conducted to evaluate soybean meal and blood meal and to compare two experimental designs (a completely randomized design and a switchback design). A larger amount of variation was observed with the switchback than with the randomized design. A larger number of animals, longer treatment period or increased number of weighings may be needed to overcome end point weighing errors and decrease the variation in the switchback design. In both designs, there was a significantly greater conversion of protein to gain for blood meal (BM)-supplemented lambs than for soybean meal (SBM)-supplemented lambs. The relative value of BM compared with SBM was 251% (completely randomized design) or 296% (switchback design). Weighing steers 2 or 3 d at the beginning and end of the trial reduced variation and increased the probability of detecting differences in daily gain, gain/feed and protein efficiency when compared with a standard weighing method (once at the beginning and once at the end of the trial). Regressing 11 weekly weights on an average of one, two or three initial and final weights also reduced variation and increased the probability of detecting treatment differences. Regressions of weekly weights were more useful as the number of initial and final weights was decreased. Daily gain and gain/feed increased linearly (P less than .05) with protein level when BM or SBM was fed. A nonlinear model was used to predict protein requirement, maximum gain and relative protein values. There was a range in relative values for BM of 255 to 348% depending on the method of analysis used.

Severity of liver abscesses and efficiency of feed utilization of feedlot cattle.

Relationships of gain, intake, feed efficiency and severity of liver abscesses were evaluated in 12 experiments involving 566 head of individually fed cattle. Concentrate level in the diets ranged from 64 to 95%. In all experiments, livers were scored as unabscessed (0), one or two small abscesses (A-), two to four small active abscesses (A) or one or more large, active abscesses (A+). Based on homogeneity of variances, nine of the experiments were divided into two groups. In one group (four experiments) the incidence of liver abscesses was 32.1% and no significant (P greater than .25) effects of liver abscess severity score on feedlot performance variables were found. In the second group (five experiments), the incidence of liver abscesses was 77.7%. In the second group, liver abscess severity score affected final live weight (P less than .10), hot carcass weight (P less than .0001), dry matter intake (P less than .10), daily gain based on live weight recorded 24 h prior to slaughter (P less than .10), daily gain based on live weight estimated from hot carcass weight with a 62% dressing percentage (P less than .0001), feed efficiency using final live weight estimated from hot carcass weight (P less than .0001) and dressing percentage (P less than .01). In all cases, performance means for cattle with A+ liver scores were the only ones that differed significantly from those of non-abscessed cattle.

Effects of inorganic and organic copper supplemented to first-calf cows on cow reproduction and calf health and performance.

Two experiments were conducted to determine whether the supplementation of Cu in the organic or inorganic form to 2-yr-old cows, before and after calving, affects reproduction rate, calf health and performance, passive transfer of immunoglobulin, or liver and serum Cu concentrations compared with unsupplemented controls. Cows (n = 75 in 1997; n = 120 in 1998) were randomly assigned by estimated calving date and body condition score to one of three treatments: 1) Control, control; 2) Inorganic, inorganic Cu supplement (200 mg Cu from CuSO4); 3) Organic, organic Cu supplement (100 mg Cu from AvailaCu). In 1998, a fourth treatment was added; 4) CU-ZN, organic Cu and Zn (400 mg Zn from AvailaZn in the Organic diet). Cows were fed a hay-based diet and individually fed supplements for approximately 45 d before and 60 d after calving (approximately January 15 to May 15 each year). Liver biopsies were obtained from cows before supplementation began, and from cows and calves at 10 and 30 d after calving. Blood samples were obtained from both cows and calves at calving, and colostrum samples were collected for IgG and mineral content. Cow liver Cu concentrations before supplementation began were 58 mg/kg in 1997 and 40 mg/kg (DM basis) in 1998. By 10 d after calving, liver Cu concentrations of Control cows had decreased (P < 0.05) to 24 mg/kg (Cu deficient) in both years, whereas liver Cu concentrations of Cu-supplemented cows increased (P < 0.05) in both years. Calf liver Cu concentrations at 10 d of age were similar (P > 0.10) for all treatment groups. No differences (P > 0.10) were found in colostrum Cu concentrations, or in calf health among treatments. No differences (P > 0.10) were found in cow BW change, calf serum Cu concentrations, calf weaning weights, or in cow 60-d pregnancy rates among treatments in either year. In 1998, cows in the Organic group had higher (P < 0.05) 30-d pregnancy rate than Control cows. Neither serum samples nor placental tissue were reliable indicators of Cu status in cows. Feeding supplemental Cu (either inorganic, organic, or organic with extra Zn) to cows with liver Cu concentrations of approximately 50 mg/kg before calving did not improve cow 60-d pregnancy rates or the health and performance of their calves when compared with unsupplemented cows.

Phosphorus requirement of finishing feedlot calves.

Dietary P supplied to feedlot cattle is important because an inadequate supply will compromise performance, whereas excess P may harm the environment. However, P requirements of feedlot cattle are not well documented. Therefore, 45 steer calves (265.2+/-16.6 kg) were individually fed to determine the P required for gain and bone integrity over a 204-d finishing period. The basal diet consisted of 33.5% high-moisture corn, 30% brewers grits, 20% corn bran, 7.5% cottonseed hulls, 3% tallow, and 6% supplement. Treatments consisted of 0.16 (no supplemental inorganic P), 0.22, 0.28, 0.34, and 0.40% P (DM basis). Supplemental P was provided by monosodium phosphate top-dressed to the daily feed allotment. Blood was sampled every 56 d to assess P status. At slaughter, phalanx and metacarpal bones were collected from the front leg to determine bone ash and assess P resorption from bone. Dry matter intake and ADG did not change linearly (P > 0.86) or quadratically (P > 0.28) due to P treatment. Feed efficiency was not influenced (P > 0.30) by P treatment and averaged 0.169. Plasma inorganic P averaged across d 56 to 204 responded quadratically, with calves fed 0.16% P having the lowest concentration of plasma inorganic P. However, plasma inorganic P concentration (5.7 mg/dL) for steers fed 0.16% P is generally considered adequate. Total bone ash weight was not influenced by dietary P for phalanx (P = 0.19) or metacarpal bones (P = 0.37). Total P intake ranged from 14.2 to 35.5 g/d. The NRC (1996) recommendation for these calves was 18.7 g/d, assuming 68% absorption. Based on performance results, P requirements for finishing calves is < 0.16% of diet DM or 14.2 g/d. Based on these observations, we suggest that typical grain-based feedlot cattle diets do not require supplementation of inorganic mineral P to meet P requirements.

Effects of supplementation of organic and inorganic combinations of copper, cobalt, manganese, and zinc above nutrient requirement levels on postpartum two-year-old cows.

The objective of this study was to determine whether a combination of Cu, Co, Mn, and Zn in an organic or inorganic form fed at higher than nutrient recommendations for 2-yr-old cows from calving to breeding would affect pregnancy rate, calving date, calf performance, and cow liver and serum mineral concentrations. Crossbred 2-yr-old cows were used after calving in 1994 (n = 127) and 1995 (n = 109). Cows were blocked by calving date to one of three treatments: 1) no supplemental minerals (CTL), 2) organic minerals (ORG), or 3) inorganic minerals (ING). Minerals were fed for the same daily intake for both organic and inorganic treatments: Cu (125 mg), Co (25 mg), Mn (200 mg), and Zn (360 mg). Cows were individually fed a mineral-protein supplement with grass hay from calving (February-March) to before breeding (May 15). Hay intakes were calculated using chromium oxide boluses to determine fecal output. Fecal excretion of minerals was calculated following trace element analysis of feces. Liver biopsies were obtained before calving, after calving (start of supplementation), at the end of supplementation, and in midsummer. Over 2 yr, more cows did not become pregnant (P < .01) in ORG (11/78) and ING (11/78) treatments than in CTL (0/80) treatments. A treatment x year interaction was found for day of conception. Cows in the ORG group conceived later (P < .01) than cows in the ING or CTL groups in 1994. In 1995, there was no difference (P > .10) in day of conception among groups. Liver Zn and Mn concentrations were not different (P > .10) and Cu concentrations increased (P < .01) for the ORG and ING groups. Cows in the ORG and ING groups had higher (P < .01) concentrations of Cu, Mn, and Zn in the feces than the CTL cows. Trace elements in the feces did not differ for ORG and ING groups. Results indicate that combinations of Cu, Co, Mn, and Zn fed at higher levels than are required reduced reproductive performance.