Effects of hydroxychloride and sulfate form of zinc and manganese supplementation on superoxide dismutase activity and immune responses post lipopolysaccharide challenge in poultry fed marginally lower doses of zinc and manganese.

Effects of hydroxychloride (OHCl) and sulfate form of zinc and manganese supplementation on immune responses of birds fed marginally lower levels of zinc and manganese during an experimental lipopolysaccharide (LPS) injection were studied. In experiment I, 30-week-old layer birds were fed 50 mg/kg Zn+45 mg/kg Mn or 100 mg/kg Zn+90 mg/kg Mn in sulfate or OHCl form and injected with 0 or 500 µg/kg LPS in a 2 (50 mg Zn+45 mg Mn and 100 mg Zn+90 mg Mn) X 2 (sulfate and OHCl) X 2 (0 and 500 µg LPS) factorial setup of treatments for 10 weeks. Among LPS-injected birds, those receiving 50 mg ZnOHCl+45 mg MnOHCl had comparable heterophil and monocyte superoxide dismutase (SOD) activity compared to the birds fed 100 mg Zn+90 mg Mn. Compared to the birds injected with PBS, LPS injection upregulated cathelicidin and IL-1 relative mRNA amounts in monocytes from birds fed 100 mg Zn+90 mg Mn, both in sulfate and OHCl form, and in birds fed 50 mg ZnOHCl+45 mg MnOHCl, but not in the birds fed 50 mg ZnSO4+45 mg MnSO4. In experiment II, one-day-old broiler birds were fed 50 mg ZnOHCl+45 mg MnOHCl, 50 mg ZnOHCl+90 mg MnOHCl, 100 mg ZnOHCL+45 mg MnOHCl, 100 mg ZnOHCl+90 mg MnOHCl, 50 mg ZnSO4+45 mg MnSO4, or 100 mg ZnSO4+90 mg MnSO4 for 21 and 42 days. Birds fed 100 mg ZnOHCl+45 mg MnOHCl form had a comparable heterophil and monocyte SOD activity and monocyte cathelicidin mRNA amounts compared to the group fed 100 mg Zn+90 mg Mn. Increasing the ZnOHCl content from 50 mg to 100 mg/kg Zn reversed (P > 0.05) the decrease in SOD activity and monocyte cathelicidin mRNA levels of the 50 mg ZnOHCl+45 mg MnOHCL fed group, and increasing the MnOHCl content from 45 mg to 90 mg/kg in the 100 mg ZnOHCl+45 mg MnOHCl group further increased SOD activity. In conclusion, birds fed diets with lower amounts of zinc and manganese in sulfate form decreased SOD activity and IL-1 and cathelicidin amounts during inflammation, and either increasing the dietary zinc and manganese content or feeding zinc and manganese in OHCl form synergistically increased the SOD activity and IL-1 and cathelicidin mRNA amounts in immune cells.

Effects of dietary copper and amino acid density on growth performance, apparent metabolizable energy, and nutrient digestibility in Eimeria acervulina-challenged broilers.

The objective of this experiment was to evaluate the influence of copper supplementation in diets varying in amino acid (AA) density on growth performance, apparent metabolizable energy (AMEn), apparent ileal nutrient digestibility (AID), and plasma carotenoids in broilers infected with Eimeria acervulina. Ross 308 male broilers (480 total) were housed in battery cages and allotted to 8 experimental treatments in a factorial arrangement of 2 dietary AA densities [1.00% (LAA) or 1.20% (HAA) digestible Lys], 2 supplemental copper concentrations (zero or 116 mg/kg), and 2 E. acervulina infection states (uninfected or infected). Essential AA ratios relative to digestible Lys were similar in both the LAA and HAA diets, and copper was provided by 200 mg/kg of tribasic copper chloride (58% copper). Chicks received experimental diets from 2 to 21 d post hatch and 6 replicate cages of 10 birds per cage were assigned to each treatment. Broilers were inoculated with zero or 6.3 × 105 sporulated E. acervulina oocysts at 15 d and blood and ileal digesta were collected at 21 days. From 2 to 15 d, body weight gain and G:F of broilers were improved (P < 0.05) with increasing AA density, and an AA density × copper interaction was observed (P < 0.05) for feed intake. Eimeria infection reduced (P < 0.05) plasma carotenoids, growth performance, dietary AMEn, and AID of organic matter, nitrogen, and total AA. There were no interactive effects of dietary treatments with E. acervulina infection on broiler growth performance or dietary AMEn. An AA density × copper supplementation interaction was observed (P < 0.05) for AID of total AA, whereby copper supplementation increased AID of total AA for birds fed the LAA diet and decreased AID of total AA for birds fed the HAA diet. In summary, E. acervulina-induced reductions in nutrient digestibility were dependent on dietary copper and AA status, but changes in digestibility had minimal impact on growth performance of broilers during the E. acervulina infection period.

Isoleucine requirements and ratios in starting (7 to 11 kg) pigs.

Two experiments were conducted to refine the Ile needs in 7- to 11-kg pigs. In Exp. 1, 1,680 pigs were fed a 1.25% digestible Lys diet containing 7.5% spray-dried blood cells (as-fed basis) with supplemental crystalline Ile (0.06% increments) to generate seven levels of apparent digestible Ile (0.47 to 0.83%). There were 12 replicates of each treatment with 20 pigs per pen, and treatments were imposed at an initial BW of 7 kg and continued for 16 d. Responses in ADG, ADFI, G:F, and plasma urea nitrogen (PUN) were quadratic (P < 0.01) over the 16-d period. Data were fitted to both a single-slope broken line and a quadratic fit, and when the quadratic response curve was superimposed on the broken line, the points at which the quadratic curve first intersected the plateau of the broken line occurred at 0.70, 0.73, 0.66, and 0.65% digestible Ile for ADG, ADFI, G:F, and PUN, respectively. Using the ADG and ADFI obtained at this intersection point resulted in an estimate of 9.1 mg of digestible Ile per gram of weight gain. In Exp. 2, 1,840 pigs were fed similarly composed diets, except that digestible Lys was lowered in six diets to 1.10% by decreasing soybean meal. Crystalline Ile was supplemented at 0.09% increments to generate six levels of digestible Ile (0.37 to 0.83%). A seventh diet contained 1.25% digestible Lys by supplementing the 0.83% digestible Ile diet with 0.19% L-Lys HCl to verify that 1.10% digestible Lys was deficient for these pigs. There were 12 replicates of each treatment with 22 pigs per pen, and treatments imposed at an initial BW of 7 kg and continued for 16 d. Supplementation of Lys to the 0.83% digestible Ile diet (1.10 vs. 1.25% digestible Lys) did not affect ADG (260 vs. 264 g/d, P = 0.60) and ADFI (359 vs. 343 g/d, P = 0.20), whereas G:F (725 vs. 774 g/kg, P < 0.01) was improved by increasing dietary Lys. Responses in ADG, ADFI, and G:F to the first six diets were quadratic (P < 0.01) over the 16-d period. The points at which the quadratic curve first intersected the plateau of the broken line occurred at 0.686, 0.638, and 0.684% digestible Ile for ADG, ADFI, and G:F, respectively. Using the ADG and ADFI obtained at this intersection point results in an estimate of 9.9 mg of digestible Ile per gram of weight gain. These results suggest that although the percent digestible Ile requirement and digestible Ile:Lys ratio for starter (7 to 11 kg) pigs may be higher than 1998 NRC recommendations, the requirement may be lower than current recommendations when taking gain and feed intake into account.

Utilization of spray-dried blood cells and crystalline isoleucine in nursery pig diets.

Three experiments were conducted to evaluate spray-dried blood cells (SDBC) and crystalline isoleucine in nursery pigs. In Exp. 1, 120 pigs were used to evaluate 0, 2, 4, and 6% SDBC (as-fed basis) in a sorghum-based diet. There were six replicates of each treatment and five pigs per pen, with treatments imposed at an initial BW of 9.3 kg and continued for 16 d. Increasing SDBC from 0 to 4% had no effect on ADG, ADFI, and G:F. Pigs fed the 6% SDBC diet had decreased ADG (P < 0.01) and G:F (P = 0.06) compared with pigs fed diets containing 0, 2, or 4% SDBC. In Exp. 2, 936 pigs were used to test diets containing 2.5 or 5% SDBC (as-fed basis) vs. two control diets. There were six replicates of each treatment at industry (20 pigs per pen) and university (six pigs per pen) locations. Treatments were imposed at an initial BW of 5.9 and 8.1 kg at the industry and the university locations, respectively, and continued for 16 d. Little effect on pig performance was noted by supplementing 2.5% SDBC, with or without crystalline Ile, in nursery diets. Pigs fed the 5% SDBC diet without crystalline Ile had decreased ADG (P < 0.01), ADFI (P < or = 0.10), and G:F (P < 0.05) compared with pigs fed the control diets. Supplementation of Ile restored ADG, ADFI, and G:F to levels that were not different from that of pigs fed the control diets. In Exp. 3, 1,050 pigs were used to test diets containing 5, 7.5, or 9% SDBC (as-fed basis) vs. a control diet. There were six replicates of each treatment at the industry (20 pigs per pen) location and five replicates at the university (six pigs per pen) locations. Treatments were imposed at an initial BW of 6.3 and 7.0 kg at the industry and university locations, respectively, and continued for 16 d. Supplementation of 5% SDBC without crystalline Ile decreased ADG and G:F (P < 0.01) compared with pigs fed the control diet, but addition of Ile increased ADG (P < 0.01) to a level not different from that of pigs fed the control diet. The decreased ADG, ADFI, and G:F noted in pigs fed the 7.5% SDBC diet was improved by addition of Ile (P < 0.01), such that ADG and ADFI did not differ from those of pigs fed the control diet. Pigs fed diets containing 9.5% SDBC exhibited decreased ADG, ADFI, and G:F (P < 0.01), all of which were improved by Ile addition (P < 0.01); however, ADG (P < 0.05) and G:F (P = 0.09) remained lower than for pigs fed the control diet. These data indicate that SDBC can be supplemented at relatively high levels to nursery diets, provided that Ile requirements are met.

Isoleucine requirement for late-finishing (87 to 100 kg) pigs.

Three pig trials were carried out to determine the true digestible Ile requirement for maximal weight gain and minimal plasma urea nitrogen (PUN) of late-finishing (87 to 105 kg) pigs. In Exp. 1, an Ile-deficient basal diet was developed and confirmed to be markedly deficient in Ile, yet fully efficacious when fortified with surfeit Ile. This diet contained corn and dried red blood cells (RBC) as Ile sources, and was analyzed to contain 10.5% CP, 0.25% Ile, and 0.63% lysine; ME was calculated to be 3,475 kcal/kg. True digestibility of Ile in the basal diet was 88% based on previous digestibility trials in ileal-cannulated pigs and cecectomized roosters. Experiment 2 was a growth trial that involved five graded levels of crystalline Ile supplementation (0.02%) to generate five dose levels of true digestible Ile (0.25 to 0.33%), Diets 1 through 5, respectively. Gain and feed efficiency showed a linear response to incremental doses of Ile (P = 0.003 and 0.036, respectively), with an apparent plateau at 0.31% true digestible Ile. In Exp. 3, a replicated 5 x 5 Latin square, five barrows (Square 1) and five gilts (Square 2) were used in five 4-d feeding periods, with five levels of true digestible Ile (0.22 to 0.30%). Using feed intake as a covariate, a linear decrease in PUN occurred in gilts (8.9, 8.6, 8.0, 7.0, and 5.5; P = 0.004) and in gilts and barrows combined (9.5, 9.2, 9.2, 8.5, and 7.6; P = 0.006) as Ile increased incrementally. The PUN results for barrows (10.5, 10.0, 10.2, 9.9, and 9.7) were not affected by dietary Ile (P = 0.417). The results of these experiments suggest that the factorial requirement estimate of 0.30% true digestible Ile for high-lean, late-finishing pigs suggested by the NRC Subcommittee on Swine Nutrition is accurate.

Isoleucine requirement of growing (25 to 45 kg) pigs.

Three pig bioassays and two digestibility trials were conducted to determine the true digestible Ile requirement for maximal weight gain and minimal plasma urea nitrogen (PUN) of growing (25 to 45 kg) pigs. In Exp. 1, an Ile-deficient basal diet was developed and confirmed to be markedly deficient in Ile, yet fully efficacious when fortified with surfeit Ile. This diet contained corn, red blood cells, and soybean meal as Ile sources, and was analyzed to contain 15.5% crude protein, 0.34% Ile, and 0.95% lysine; metabolizable energy was calculated to be 3,430 kcal/kg. True digestibility of Ile in the basal diet was 89% based on digestibility trials in ileal-cannulated pigs and cecectomized roosters. The first growth trial (Exp. 2) involved six dose levels of true digestible Ile (0.38 to 0.58%), which resulted in a quadratic (P < 0.02) response in weight gain by growing pigs over a 21-d period. The weight-gain data were fitted to both a single-slope broken line and a quadratic fit, and when the quadratic response curve was superimposed on the broken line, the point at which the quadratic curve first intersected the plateau of the broken line occurred at 0.50% true digestible Ile. This (objective) requirement estimate was similar to that determined by taking 90% of the upper asymptote of the quadratic fitted line. In Exp. 3, a replicated 5 x 5 Latin square, five barrows (square 1) and five gilts (square 2) together with five 4-d feeding periods and five levels of true digestible Ile were utilized. A linear (P < 0.01) decrease in PUN occurred as Ile was incremented, with an apparent plateau occurring at 0.50% true digestible Ile. The results of these experiments suggest that the true digestible Ile requirement of grower (25 to 45 kg) pigs is 0.50% of the diet, or 1.46 g/Mcal of metabolizable energy, somewhat higher than the 1.38 g/Mcal metabolizable energy estimated by the 1998 National Research Council Subcommittee on Swine Nutrition.

Ideal ratio (relative to lysine) of tryptophan, threonine, isoleucine, and valine for chicks during the second and third weeks posthatch.

Six bioassays were conducted to determine the ideal ratios of several amino acids relative to Lys. Young male crossbred chicks were fed diets based on corn gluten meal and synthetic amino acids that could be made singly deficient in Lys, Trp, Thr, Ile, or Val. Diets for all assays contained 3,400 kcal ME/kg, and L-glutamic acid was used to make all diets (within and among assays) equal in crude protein at 22.5% of the diet. True digestibility assessment of corn gluten meal in cecectomized roosters facilitated dose-titration studies so that least squares fitted one-slope broken-lines and quadratic regression equations could be calculated to establish inflection points for weight gain and gain:feed. Four battery pens of four chicks were fed one of six amino acid levels from 8 to 21 or 22 d posthatching. Weight gain and gain:feed responded quadratically (P < 0.01) to increasing doses of digestible Lys (0.68 to 1.28%), Trp (0.09 to 0.24%), Thr (0.41 to 0.81%), Ile (0.45 to 0.95%), and Val (0.51 to 1.06%). Broken-line least squares analysis predicted breakpoints for gain and gain:feed, respectively, of: Lys (0.85, 0.96%), Trp (0.16, 0.16%), Thr (0.53, 0.53%), Ile (0.59, 0.58%), and Val (0.74, 0.74%). The intercept of the quadratic regression curve and the plateau of the broken line predicted digestible Lys requirements for gain and gain:feed, respectively, of 0.95 and 1.03%. Similar calculations predicted digestible Trp requirements of 0.18% for gain and gain:feed, digestible Thr requirements of 0.59% for gain and 0.60% for gain:feed, digestible Ile requirements of 0.68% for gain and gain:feed, and digestible Val requirements of 0.81% for gain and 0.82% for gain:feed. Regardless of curve-fitting method, gain:feed requirements for Lys were much higher than weight gain requirements. Using the higher of the broken-line requirement estimates for gain and gain:feed, ideal ratios (% of Lys) were as follows: Lys (100), Trp (16.6), Thr (55.7), Ile (61.4), and Val (77.5).

Valine requirement of nursery pigs.

Six experiments were conducted to determine the true digestible valine requirement of 5- to 20-kg pigs. In Exp. 1, a valine-deficient diet for 5- to 10-kg pigs was developed and validated in terms of growth performance in response to supplemental L-valine. A different basal diet was validated for 10- to 20-kg pigs in Exp. 2. Both diets were demonstrated to be deficient in valine and to support performance equivalent to typical nursery diets when fortified with L-valine. In Exp. 3, true ileal digestibility of valine in the two basal diets was determined in eight pigs fitted with a simple T-cannula at the terminal ileum. Another four pigs received an enzymatically hydrolyzed casein-based diet to determine endogenous contributions to collected ileal digesta. The two diets were found to have true valine digestibilities of 82% (5- to 10-kg pigs) and 86% (10- to 20-kg pigs). In Exp. 4, 80 weaned pigs (5.8 kg) were offered the basal diet fortified with five incremental doses (0.08%) of L-valine. Weight gain increased quadratically (P < 0.05) with increasing levels of valine. Broken-line analysis revealed a true digestible valine requirement of 0.86 +/- 0.03%. In Exp. 5, the true digestible valine requirement of 10- to 20-kg pigs was estimated with 120 pigs (10.9 kg) using the second basal diet fortified with six incremental doses (0.05%) of L-valine. The data suggested a digestible valine requirement level of about 0.775%, which was reevaluated in Exp. 6, wherein pigs did not respond to levels of digestible valine higher than 0.775%. In conclusion, requirement estimates were 2.50 and 2.22 g of true digestible valine per megacalorie of ME for 5- to 10- and 10- to 20-kg pigs, respectively. These empirical estimates are in close agreement with recent estimates of the National Research Council Subcommittee on Swine Nutrition of 2.48 and 2.11 g of true digestible valine per megacalorie of ME, respectively.

True ileal digestibility of amino acids in sow's milk for 17-day-old pigs.

The digestibility of amino acids in sow's milk consumed by young pigs is currently unknown because of difficulties associated with collecting an adequate quantity of milk, and also problems in cannulating suckling pigs. A total of 14 kg of sow's milk was collected, two soluble indigestible markers (Co-EDTA and YbC13) were added, and the milk was fed to four pigs at 17 d of age that were fitted with a simple T-cannula at the terminal ileum. Another four cannulated pigs were offered a similar amount of a 20% DM liquid diet based on enzymatically hydrolyzed casein and lactose to assess endogenous amino acid losses. All pigs were fed about 875 g of each diet per day in 10 hourly meals from 0700 to 1700. Following 2 d of adaptation, ileal digesta were collected from 0800 to 1800 for 2 d. Diets and digesta were analyzed for amino acids using appropriate hydrolysis and preoxidation procedures. Average nitrogen true digestibility was 88%, whereas amino acid true digestibilities ranged from 84% (cystine and threonine) to 100% (methionine, histidine, and glutamic acid); the average for all amino acids was 92 +/- 4%. Based on average values, true digestibility of essential amino acids was not different from that of nonessential amino acids (P > 0.10). In whole milk, amino acids found in abundance in whey proteins (i.e., cystine, glycine, and threonine) were less (P < 0.05) digestible than amino acids predominating in casein proteins (i.e., glutamic acid, proline, and methionine). When true ileal digestible amino acid concentrations in sow's milk were expressed as ratios to digestible lysine, it appeared that threonine, tryptophan, and arginine were lower than what might be considered optimal. In conclusion, amino acids in sow's milk were highly digestible, but most of the amino acids had true ileal digestibility values significantly less than 100%.

Zinc bioavailability in tetrabasic zinc chloride and the dietary zinc requirement of young chicks fed a soy concentrate diet.

Three chick assays were conducted to determine a bioavailable Zn requirement and to establish a Zn relative bioavailability (RBV) value for tetrabasic Zn chloride [TBZC, i.e., Zn5Cl2(OH)8]. In Assay 1, 8-d-old chicks were fed a Zn-deficient soy concentrate diet (8.8 mg/kg bioavailable Zn) supplemented with 0, 5.81, 10.81, 15.10, and 20.25 mg Zn/kg from ZnSO4.7H2O for 14 d to establish a linear response range and estimate a bioavailable Zn requirement. Weight gain of chicks increased linearly (P < 0.01; r2 = 0.99) when the first three levels of Zn were fed. Broken-line analysis of the weight gain data indicated a bioavailable Zn requirement of 22.4 mg/kg. In Assay 2, RBV of Zn in TBZC was determined by multiple-linear regression slope-ratio methodology. The Zn-deficient basal diet was supplemented with 0, 5.81, and 10.81 mg Zn/kg from ZnSO4.7H2O to produce a linear growth response as determined in Assay 1. The same basal diet was supplemented with 5.38 and 10.81 mg Zn/kg from TBZC. The RBV of Zn in TBZC was found to be 102%, which was not different (P > 0.10) from 100%. A third assay was conducted as described for Assay 2, and the RBV of Zn in TBZC was 111%, which was not different from 100%. In summary, the RBV of Zn in TBZC was not different from that of ZnSO4.7H2O, and the minimal bioavailable Zn requirement for chicks 1 to 3 wk of age fed a soy concentrate diet was estimated at 22.4 mg/kg.

Growth-promoting efficacy in young pigs of two sources of zinc oxide having either a high or a low bioavailability of zinc.

Commercial sources of zinc oxide (ZnO) differ widely in Zn relative bioavailability (RBV), but it is unknown whether growth-promoting efficacy in young pigs is influenced by RBV of the ZnO sources used. We compared a low-RBV (39%) ZnO manufactured by the Waelz process (W) to a high-RBV (93%) ZnO manufactured by the hydrosulfide process (HS). Antibacterial agents were included in the diet in only one of the four trials (Exp. 4). In Exp. 1, pigs (n = 36, 6.5 kg, 28 d of age) were randomly assigned in three replicates to receive 0, 1,500, or 3,000 mg Zn/kg from HS Zn in a 21-d growth assay. Growth rates and feed intake responded linearly (P < 0.01) to incremental doses of Zn. In Exp. 2, pigs (n = 60, 6.1 kg, 28 d of age) were randomly assigned in five replicates to receive either 0 or 1,500 mg W or HS Zn/kg during a 21-d feeding period. Growth performance was improved (P < 0.01) by the addition of ZnO. During wk 1, however, pigs receiving HS Zn grew faster (P < 0.03) than those receiving W Zn, but the difference diminished to a trend (P < 0.08) during wk 2. Morphology of duodenal, jejunal, and ileal intestinal sections was examined at d 21 of the assay, but neither source of ZnO had an effect on crypt depth or on villus height or width. In Exp. 3, weaned pigs (n = 48, 5.4 kg, 21 d of age) were randomly assigned in four replicates to the same dietary treatments as in Exp. 2 for a 17-d growth assay. Growth performance was improved (P < 0.05) by the addition of ZnO, but no difference was detected between the two sources. In Exp. 4, pigs (n = 60, 6.2 kg, 28 d of age) were randomly assigned in five replicates to receive either 0 or 1,500 mg/kg W or HS Zn in an 11-d growth assay wherein antimicrobial agents were included in the basal diet. Growth rates during the first 6-d were improved (P < 0.06) by the addition of ZnO, with a trend (P < 0.10) for greater weight gain in pigs receiving HS than in those fed W Zn. During the entire 11-d, however, there was no difference in growth rates between pigs fed the two sources of ZnO. In conclusion, RBV of Zn in ZnO did not substantially affect the growth-promoting efficacy of ZnO in young pigs fed diets with or without antimicrobial agents.

Single versus multiple deficiencies of methionine, zinc, riboflavin, vitamin B-6 and choline elicit surprising growth responses in young chicks.

A soy-protein isolate diet that was deficient in methionine (Met), zinc (Zn), riboflavin, vitamin B-6 and choline for chick growth (Assay 1) was used to study individual or multiple deficiencies of several of these nutrients. In all cases, adding all three deficient nutrients together resulted in growth responses that were superior to those resulting from supplementation with any pairs of deficient nutrients. In Assay 2, single addition of Zn but not of methionine or riboflavin produced a growth response, but the combination of either Zn and Met or Zn and riboflavin resulted in growth responses that were greater than the response elicited by Zn alone. Assay 3 involved individual or multiple deficiencies of choline, riboflavin and vitamin B-6, and individual additions suggested that choline was first limiting. Choline + riboflavin supplementation, however, produced marked growth and gain:food responses that were far greater than those resulting from supplemental choline or riboflavin alone. Moreover, the growth response to a combination of choline + pyridoxine (PN) was also greater than that obtained from any of the three nutrients fed alone; even PN + riboflavin (in the absence of choline) produced responses greater than those observed with the unsupplemented negative-control diet. In Assay 4, chicks responded to individual additions of riboflavin, PN or Met, and in Assay 5, to either riboflavin or PN; all two-way combinations resulted in growth rates that were far greater than those occurring with any single addition. The data from these experiments show that unlike the situation with three deficient amino acids, the expected responses to first-, second- and third-limiting B-vitamins or deficient vitamins combined with deficient levels of Zn or Met do not follow the expected pattern of response to first-, further response to first- and second- and an even further response to first-, second- and third-limiting nutrients.