Growth performance and hematology characteristics in pigs treated with iron at weaning as influenced by nursery diets supplemented with copper

Copper is an essential trace mineral used for the synthesis of hemoglobin and several oxidative enzymes critical for normal metabolism. Although the dietary copper requirement for weaned pigs is 5 to 6 ppm,¹ diets supplemented with levels of copper in excess of requirements (100 to 250 ppm) enhance growth during the nursery phase of production.^2-5 Dietary copper at levels deficient or in excess of nutritional requirements, however, have negative effects on iron absorption from the gastrointestinal tract.^6,7 Recent research has demonstrated that fast-growing pigs of modern genotypes are often iron deficient or anemic at weaning, despite having received intramuscular (IM) iron during the first week of life.^8-10 Clinically, pigs are considered anemic if blood concentrations of hemoglobin are less than 9.0 g/dL, and iron deficient if hemoglobin levels are above 9.0 g/dL but less than 11.0 g/dL.^8,11 An additional iron treatment at weaning could be important, particularly for nursery pigs consuming diets supplemented with pharmacological levels of copper to enhance growth performance. Thus, the experiment reported herein was conducted to determine the effects of an additional 100 mg iron treatment at weaning on growth performance and hematology characteristics in nursery pigs fed a diet supplemented with 250 ppm copper.

Materials and methods

The protocol for this experiment was reviewed and approved by the Institutional Animal Care and Use Committee at Virginia Tech (Blacksburg, Virginia).

Study animals and housing

Yorkshire × Landrace sows (n = 18) farrowed 169 Duroc-sired piglets, of which 144 high-health pigs (n = 76 males and n = 68 females) were employed. Piglets (n = 25) were excluded from the experiment because of unusually heavy or light body weights, signs of being unthrifty, hernias, or leg problems. Within 24 hours after birth, piglets were ear notched for identification, weighed, needle teeth were resected, and tails docked. All pigs received an IM injection of 100 mg iron hydrogenated dextran (Iron-100; Durvet, Inc) in the neck muscle behind the ear using a 20-gauge, 1.27 cm-long needle and a disposable 3 cc syringe (Becton, Dickinson and Company). To simulate commercial procedures, a standard amount of iron was administered shortly after birth rather than amounts based on body weight.¹ Similar to our previous work,¹⁰ the dosage of iron was chosen because 1) lower doses are less likely to be toxic and cause oxidative stress; 2) greater doses of iron increase liver hepcidin secretion, which perturbs systemic iron metabolism; and 3) the 100 mg dose soon after birth would likely increase the number of anemic pigs at weaning, allowing for the evaluation of how these pigs respond to dietary copper supplementation. Boar piglets were castrated at seven days of age using a sterile scalpel. All piglets had access to sow feeders but no access to creep feed during the suckling period. At weaning, pigs were moved to an environmentally controlled nursery facility. Each nursery pen measured 0.91 × 1.22 m² over galvanized steel bar slats and contained a nipple drinker and a stainless-steel feeder with four feeding spaces.

Study design

At 21.8 (0.5) days of age (mean [SE]), pigs were weaned, vaccinated against porcine circovirus type 2 and Mycoplasma hyopneumoniae (Circumvent PCV-M G2; Merck Animal Health), weighed, and divided into equal groups of the largest and smallest pigs (8.72 [0.40] and 5.97 [0.40] kg, respectively). Six blocks of eight pens each were created by placing a total of 12 pigs of each size category in pens of three pigs each. Each pen had at least one barrow and one gilt and pigs from at least two different litters. The eight pens within a block were randomly allocated to a 2 × 2 × 2 factorial arrangement of treatments. The factors were: 1) size of pig (large or small); 2) number of 100 mg IM iron doses (one dose administered within 24 hours after birth or two doses [one administered within 24 hours after birth and the other at weaning]); and 3) level of dietary copper (14.2 [control] or 250 ppm). There were six replicate pens per treatment combination (total of 48 pens).

Experimental diets

Pigs were allowed ad libitum access to a three-phase feeding regimen with all diets meeting the requirements for the various nutrients¹ and copper adjusted to concentrations previously indicated. For each phase, a basal diet was first prepared, containing most of the corn and all the common ingredients for each experimental diet. Copper sulfate (Pestell Minerals and Ingredients) or an equal amount of ground corn was added to the basal diet to create the copper or control diets, respectively (Table 1).

Data and sample collection

Pigs were weighed at weaning (day 0) and at days 7, 21, and 49 post weaning. Average daily gain (ADG) was determined for day 0 to 7, day 8 to 21, day 22 to 49, and day 0 to 49. Feed additions were recorded so that for each period, average daily feed intake (ADFI) and the gain to feed ratio (G:F) could be calculated. Feed remaining in feeders was removed with a vacuum (Shop-vac) and weighed.

A blood sample from the barrow weighing closest to the mean weight of pigs in each pen was collected at weaning (before the second dose of iron was administered to the appropriate pigs), and at days 7 and 49 post weaning. The same pig was used for each collection. For sampling, barrows were placed supine on a v-board and approximately 7 mL of blood was collected via jugular venipuncture (20-gauge, 2.54 cm-long needle) into a Vacutainer tube (Becton, Dickinson and Company) containing EDTA. Hematology analyses were conducted using a Coulter Multisizer 3 cell counter (Beckman Coulter, Inc) by Animal Laboratory Services of the Virginia-Maryland College of Veterinary Medicine (Blacksburg, Virginia). The following hematological determinations were made: number of red blood cells, reticulocytes, white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and platelets, percentage of reticulocytes, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, and mean platelet volume.

Statistical analysis

Data were subjected to ANOVA using the mixed models procedure of SAS (SAS Institute Inc). Body weights, ADG, ADFI, and G:F were analyzed using a model that included pig size, number of iron doses, diet, and all two- and three-way interactions as possible sources of variation. Block was included as a random variable and pen served as the experimental unit. A repeated measures model was used for analyzing hematological characteristics and included pig size, number of iron treatments, diet, day, and all two-, three-, and four-way interactions as possible sources of variation. Block was included as a random variable and individual pig was the experimental unit. Individual means were compared using the LSMEANS option of PROC MIXED and were adjusted using the Tukey-Kramer procedure. Differences in means were considered statistically significant at P < .05.

Results

There were no pig deaths or removals during the experiment.

Hematology characteristics

There were no three- or four-way interactions of main effects on hematology characteristics. The number and percentage of reticulocytes were affected (P = .05) by an interaction of pig size and the number of iron doses (Figure 1). In large pigs only, reticulocyte number and percentage were greater (P = .05) for animals receiving two versus one iron dose.

Mean corpuscular volume (P = .01) and reticulocyte percentage (P = .001) and number (P = .001) were affected by an interaction between number of iron doses and day post weaning (Figure 2). On day 7, but not on days 0 or 49, mean corpuscular volume (P = .06) and reticulocyte percentage (P < .001) and number (P < .001) were greater for pigs receiving two versus one iron dose.

There was an interaction of number of iron doses and diet for mean corpuscular hemoglobin (P = .04). Compared to pigs receiving iron only at birth, pigs receiving an additional iron dose at weaning had slightly greater mean corpuscular hemoglobin if fed the copper diet, but slightly decreased values if fed the control diet (Figure 3). Basophil concentration (P = .05; Figure 3) was also affected by an interaction of number of iron doses and diet. For pigs receiving only one dose of iron, basophil concentrations were less (P = .04) in animals fed copper. This effect of diet did not exist (P = .98) for pigs receiving iron at both birth and at weaning.

The interaction of pig size and day affected hemoglobin concentrations (P < .001), hematocrit (P < .001), mean corpuscular volume (P < .001), mean corpuscular hemoglobin (P = .008), red blood cell distribution width (P = .001), and reticulocyte percentage (P = .02) and number (P = .02; Figure 4). Hemoglobin concentrations and mean corpuscular volume on days 0 (P < .001 and P < .001, respectively) and 7 (P = .02 and P < .001, respectively), hematocrit on day 0 (P = .002), and mean corpuscular hemoglobin concentration on day 7 (P = .008) were less in large versus small pigs; there were no differences detected on day 49. In contrast, red blood cell distribution width was greater in the large pigs on both day 0 (P = .001) and 7 (P = .002). Reticulocyte percentage (P = .09) and number (P = .04) were greater for large versus small pigs on day 7, but not on the other days.

Table 2 contains hematology characteristics in nursery pigs as affected by the main effects of pig size, number of iron doses, diet, and day post weaning. Concentration of eosinophils (P = .03) were greater in the large versus small pigs. Hemoglobin (P = .05) and hematocrit (P = .04) were greater, and the number of platelets was less (P = .05) in pigs receiving iron doses at birth and at weaning compared to at birth only. Hemoglobin (P = .03), hematocrit (P = .03), and mean corpuscular volume (P = .04) were greater in pigs fed the control versus copper-supplemented diet. Red (P < .001) and white (P = .006) blood cell numbers, and mean platelet volume (P < .001) increased from day 0 to day 7, and then remained similar until day 49. Concentrations of lymphocytes (P < .001), monocytes (P < .001), basophils (P < .001), and platelets (P < .001) decreased from day 0 to day 7, and further decreased from day 7 to day 49.

Growth performance

There were no three-way interactions among pig size, number of iron doses, and diet for body weights at weaning or days 7, 21, or 49 post weaning. Day 49 body weights were affected by interactions of size of pig and number of iron doses (P = .05; Figure 5), and number of iron doses and diet (P = .04; Figure 6). In large pigs only, body weight was greater (P = .05) for individuals receiving two versus one dose of iron (Figure 5). Body weight was greater (P = .04) for copper-fed pigs that received two versus one dose of iron, however, body weights were not affected (P = .99) by the number of iron doses in control-fed pigs (Figure 6).

The interaction between number of iron doses and diet affected ADG and G:F from day 0 to 7 (P = .04 and P = .05, respectively) and day 8 to 21 (P = .009 and P = .01, respectively), ADFI from day 22 to 49 (P = .03), and ADG (P = .02) and ADFI (P = .04) for day 0 to 49 (Figures 7, 8, 9, and 10). In all cases, performance measures were greater in copper-fed pigs receiving two versus one dose of iron. In contrast, growth was unaffected by the number of iron doses in animals fed the control diet.

Table 3 summarizes body weights and growth performance in nursery pigs as affected by the main effects of size of pig, number of iron doses, and diet. Large pigs weighed more than small pigs on days 0, 7, and 21 of the experiment (P < .001). From day 0 (weaning) to 7, size of pig did not affect ADG (P = .50), ADFI (P = .18), or G:F (P = .18). For days 8 to 21 and 22 to 49, ADG and ADFI were greater in the large versus small pigs (P < .001 and P = .02, and P < .001 and P < .001, respectively). The G:F was also greater for large pigs from day 8 to 21 (P < .001) but not from day 22 to 49 (P = .41). For the overall trial (day 0 to 49 post weaning), ADG and ADFI were greater (P < .001) in large pigs, and G:F was similar (P = .34) for the different sized animals. Body weights were greater at day 21 (P < .001) in pigs receiving two versus one dose of iron.

Discussion

Hemoglobin is a protein molecule that allows red blood cells to carry oxygen from the lungs to bodily tissues and return carbon dioxide from tissues back to the lungs. Iron is a critical constituent of hemoglobin and iron deficiency anemia occurs if iron levels in the body are inadequate to maintain normal concentrations of hemoglobin in the blood. Clinically, pigs are considered anemic if blood concentrations of hemoglobin are less than 9.0 g/dL, and iron deficient if hemoglobin levels are above 9.0 g/dL but less than 11.0 g/dL.^8,11 To prevent iron deficiency anemia, pigs reared in confinement operations typically receive IM treatment with iron, usually in the form of iron dextran, within a few days after birth. The exact timing, dosage, and number of injections of iron dextran, however, varies widely among commercial pig farms.¹² Nevertheless, several research groups reported that a significant number of pigs, particularly the fastest growing animals within a litter, were iron deficient or anemic at weaning, despite receiving treatment with iron early in life,^8-10 and pigs that are anemic at weaning display poorer growth in the nursery compared with non-anemic pigs.¹³ The results of the current experiment, when 100 mg iron was administered at birth, are consistent with those previous studies that demonstrated an increased risk of anemia at weaning in larger pigs. Indeed, small pigs weaned in the present study had greater hemoglobin concentrations, hematocrit, and mean corpuscular volume compared to large pigs. Mean corpuscular hemoglobin was also greater in small pigs on day 7 post weaning. In contrast, red blood cell distribution width, a measure of variability in the size of cells that increases in anemic individuals, was greater at weaning in the large versus small pigs. By the end of the 49-day trial there were no differences between size groups for hemoglobin, hematocrit, mean corpuscular volume, or red blood cell distribution width. In the current experiment, a standard dose of iron was employed and our finding that 100 mg iron was sufficient to prevent anemia in smaller but not larger pigs, suggests that body weight should be considered when iron is administered to newborns.

Others have reported that eosinophil concentrations were less in anemic versus non-anemic pigs.¹⁴ Interestingly, in the current study, eosinophil concentrations were greater in the pigs classified as large at weaning, despite the display of hematological data consistent with iron deficiency. Cases of concurrent iron deficiency anemia and eosinophilia have been reported in humans diagnosed with internal parasites,¹⁵ and oral inoculation of pigs with infective ascaris eggs resulted in eosinophilia in the peripheral blood and a serum antibody response.¹⁶ It is doubtful, however, that pigs in the current study had high numbers of internal parasites. The experiment was conducted in an intensively managed and highly sanitary university facility and sows were treated with 1.8% fenbendazole as per label (Safe-Guard; Merck Animal Health) before farrowing. 

Hemoglobin concentrations, hematocrit, and mean corpuscular volume were greater in control pigs versus pigs fed a diet supplemented with copper. These findings are consistent with the hypothesis that pharmacological levels of dietary copper decrease iron absorption in pigs.^6,7 Interestingly, basophil concentrations in pigs receiving iron at birth only were less in copper-fed individuals compared to controls. The biological significance of this finding, however, is unclear.

In the current experiment, hemoglobin concentrations, hematocrit, and reticulocyte number and percentage were affected by day of sampling and pig size. Hemoglobin and hematocrit were greater, and platelet counts were less, in pigs receiving an additional iron injection at weaning. These changes reflect a positive effect of iron therapy in individuals that may be anemic or iron deficient, and are consistent with previous reports in the literature.^17,18 For example, a second injection of 200 mg iron dextran at 20 days of age increased hemoglobin concentrations in pigs weaned and blood sampled at 34 days of age.¹⁸ Our finding that platelet counts were less in pigs receiving an additional dose of iron at weaning are consistent with a previous study in which humans with iron deficiency anemia had greater platelet counts than those with adequate iron stores; oral iron supplementation decreased platelet counts in anemic individuals but not in those with normal iron levels.¹⁹

For the study reported here, pigs classified as large at weaning weighed approximately 2.8 kg more than pigs classified as small. The difference between size groups increased during the study and was approximately 7.6 kg at day 49 post weaning. That larger pigs at weaning maintain or expand a size advantage over small pigs at weaning has been previously reported.^10,20-23 Feed conversion efficiency is a function of body weight, and as a pig grows, it may become less efficient at converting feed into body weight gain,²⁴ which could explain our finding that small pigs displayed greater G:F from day 8 to 21 in the nursery than large pigs. In the current experiment, however, final body weight was impacted by an interaction between pig size and the number of iron doses. As mentioned above, hemoglobin levels were less in large versus small pigs at weaning. Perhaps an iron deficiency was mitigated by iron treatment at weaning, allowing the large pigs to achieve maximum size. Moreover, we cannot discount potential effects of dietary iron on improved growth responses.

Increased growth in nursery pigs provided pharmacological concentrations of dietary copper have been well-documented^2-5 and, consistent with previous reports, pigs fed the copper-supplemented diet in the current study exhibited greater weight gain, feed intake, and feed conversion efficiency than control pigs during the first week post weaning. More importantly, many aspects of growth performance were influenced by an interaction between the number of iron treatments and diet. Indeed, ADG (days 0 to 7, 8 to 21, and 0 to 49), ADFI (days 22 to 49 and 0 to 49) and G:F (days 0 to 7 and 8 to 21) were enhanced by dietary copper only if an additional 100 mg iron dose was administered at weaning. Based on these results, it appears that an adequate level of iron in the body is requisite for dietary copper to enhance growth performance in nursery pigs. As reported, iron injected at weaning did not enhance growth performance in pigs fed the control diet.

Studies in which additional iron was given by either increasing the dosage administered at birth or by administering an additional dose during the suckling period or at weaning, have yielded equivocal growth responses. Consistent with our results for the pigs receiving copper, pigs receiving injections of 200 mg iron at birth and 200 mg iron at 7 to 14 days prior to weaning had increased ADG compared to pigs receiving 200 mg iron at birth only.^18,25 In contrast, growth performance was not affected or was only slightly influenced by increasing the dosage of iron given at birth from 200 to 300 mg,^26,27 or by injecting 200 mg at birth and 100 to 200 mg at 17 days of age or at weaning.^17,27

In summary, hematological analyses conducted in this study reflect an increased risk of anemia at weaning in larger pigs. That 100 mg of iron given at birth was sufficient to prevent anemia in smaller but not larger pigs suggests that body weight should be considered when iron is administered to newborns. Based on hematological evidence, high levels of dietary copper appear to decrease iron absorption, and it appears that an adequate level of iron in the body is requisite for dietary copper to enhance growth performance in nursery pigs. These findings illustrate the complex relationship among trace minerals in swine and the need for further research in this area of nutrition.

Implications

Under the conditions of this study:

• Iron treatment at weaning increased hemoglobin levels.
• Copper enhanced nursery growth only if pigs received iron at weaning.
• Hemoglobin levels were less in copper-fed pigs compared to controls.

Acknowledgments

Funding for this work was provided in part, by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, US Department of Agriculture, and grants from the Virginia Agricultural Council, Virginia Pork Council, Inc, and the National Pork Board (Project NPB #17-052).

Conflict of interest

None reported.

Disclaimer

Scientific manuscripts published in the Journal of Swine Health and Production are peer reviewed. However, information on medications, feed, and management techniques may be specific to the research or commercial situation presented in the manuscript. It is the responsibility of the reader to use information responsibly and in accordance with the rules and regulations governing research or the practice of veterinary medicine in their country or region.

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