Impact of Trace Minerals on Wound Healing of Footpad Dermatitis in Broilers

Footpad dermatitis (FPD) is used in the poultry industry as an animal welfare criterion to determine stocking density. Trace minerals (TM) play a role in skin integrity and wound healing. This study evaluated the impact of TM on FPD and consisted of 3 treatments supplemented with 0 (NTM), low (LTM) and high (HTM) TM levels in the same basal diet. On d21, 71% birds in all treatments developed mild FPD and pens were top-dressed with dry litter to promote FPD healing. Compared to NTM, LTM reduced area under the curve (AUC) of FPD lesion scores during d21–42, HTM reduced the AUC of FPD lesion scores during d7–21 and d21–42. LTM improved growth performance on d14, HTM improved growth performance on d14 and d28. LTM and/or HTM increased gene expression of VEGF, TIMP3, TIMP4, MMP13, ITGA2, ITGA3 and CD40, which promoted collagen synthesis, deposition and organization; cell migration, matrix remodeling, and angiogenesis. LTM and/or HTM increased inflammation by upregulating TNFα and IL-1β during the early wound healing phase and reduced inflammation by downregulating IL-1β during the late wound healing phase. Our findings showed that TM not only improved growth performance but also reduced FPD development by promoting FPD wound healing.

normal management practices, until a temperature of 23 °C was achieved. Hours of light (L) and dark (D) were provided as follows: d 1 to 3, 23L:1D; d 4 to 12, 18L:6 D; d 13 to 30, 16L:8D. Birds were fed crumbled starter diets from d 0 to 14; switched to pelleted grower diets from d15 to 28, and then to pelleted finisher diets from d29 to 43 (Table 1). Birds had free access to feed and water at all times. Experimental diets were formulated to meet or exceed nutritional requirements of broiler chickens with the exception of TM 20  3 ppm) were added to LTM and HTM treatments but not to NTM treatment. Each treatment had 9 replicate floor pens with 12 birds per pen. The same amount of chopped wheat straw (3 cm depth) was used as bedding material (litter) and applied uniformly in each pen across all treatments at the first day of the experiment. Litter samples to measure litter moisture (per pen basis in all treatments) were collected from similar spot at least 1 foot away from feeder and waterer/nipple drinker. The sample size was from 2-5 inches across with a depth of 3-9 cm (from the top to the hard floor). Sampling was done on d6, 16 pads from all birds in each pen were examined by the same scorer throughout the study. Footpads were scored for ulcer and scabs after dipping the feet in a bucket of water and removing all adhering material from the footpad, and scored using 5-point scoring system. The scoring system was as follows: Score 1 = no lesions, dermal ridges intact within central plantar footpad surface, with or without discoloration; Score 2 = mild lesions, dermal ridges not intact with a small scab (≤2 mm in diameter) on the central plantar footpad surface; Score 3 = moderate lesions with a scab of 2-7 mm in diameter on the central plantar footpad surface; Score 4 = severe lesions with a large scab of ≥7 mm in diameter in the central plantar footpad surface but no scabs on the toes; Score 5 = most severe lesions with a large scab of ≥7 mm in diameter in central plantar footpad surface and small scabs on the toes 8 (Supplemental Figure 1). At 15, 22, 29, 36 and 43 d-of-age, one representative chicken from each pen with lesions closest to the pen average lesion score was chosen to collect footpad skin samples for total collagen protein and gene expression analyses.
Total Collagen Analysis. One piece of the left footpad of each bird per pen was collected and flash frozen on dry ice for collagen analysis. Footpad skin hydroxyproline (Hyp) content was measured to estimate collagen content 22 with some modifications. For the assay, 100 mg of tissue was homogenized in 6 ml of 6 N HCl in a glass tube using a Cole Parmer Lab Gen 700 (Cole-Parmer North America, Vernon Hills, IL) for about 30 sec, and hydrolyzed at 110 °C for 24 h after adding an additional 6 ml of HCl. One ml of hydrolyzed sample was neutralized with 2 ml of 3 N NaOH and diluted 2× with distilled H 2 O. Serial dilutions of 0.1 mg/ml trans-4-hydroxy-L-proline were used as standard to generate standard curve. A 0.25-ml aliquot of 0.01 M CuSO 4 , 0.25 ml 2.5 N of NaOH, and 0.25 ml of 6% H 2 O 2 were added in succession into glass tubes with 0.5 ml of diluted samples or standards. After vortexing for 1 min, tubes were incubated in water bath at 80 °C for 5 min with frequent shaking. After cooling tubes on ice, 1 ml of 3 N H 2 SO 4 and 0.5 ml of 4-dimethyl-amino-benzaldehyde were added in succession into glass tubes followed by incubation in water bath at 70 °C for 16 min with slight shaking. The optical density of supernatant was measured at 560 nm. Hyp content per gram of tissue was determined according to the standard curve. Total collagen content was calculated by multiplication of the Hyp content by 7.5 22,23 . Statistical Analyses. All data were tested for normality and subjected to analysis of variance as a completely randomized design using the proc mixed procedure of SAS 9.4 24 . Each pen was used as the experimental unit for the analysis. Growth performance including BW, cBWG, cFI and cPI used the average data per pen. Gene expression and total collagen protein used individual measurement from one representative bird per pen. Area  under the curve (AUC) for footpad lesion scores and relative mRNA levels of genes associated with FPD wound healing was calculated using the trapezoid method. Statistical differences among means were determined by using Duncan's multiple-range test.

Results
Growth Performance. Growth performance of the broilers at 14, 28 and 42 d-of-age is summarized in Table 3. Both LTM and HTM improved BW, cBWG, cFI and cPI on d14 (P < 0.002); however, only the chickens that received HTM had higher BW, cBWG (P < 0.01) and cFI (P < 0.04) than NTM chickens on d28, and better cFCR (P < 0.03) than NTM on d42 (Table 3).
Footpad Lesions. Figure 1 shows the results of footpad lesion scores among NTM, LTM and HTM treatments in broiler chickens at different ages. On d 21, 71% birds in all groups developed mild to moderate FPD, fresh and dry litter was top-dressed on existing litter on d21, 28 and 35 to promote FPD healing. TM did not alter litter moisture content in this study (Supplemental Table 1). Compared to NTM, LTM reduced (P = 0.06) FPD  Means within a column with different superscripts differ at P < 0.05. Reported standard error of the mean (SEM) is the average SEM of the individual least-square means. scores on d28, both LTM and HTM reduced (P = 0.08) FPD scores on d42 (Fig. 1). HTM reduced AUC of FPD lesion scores during d7-21 (P = 0.036), both LTM and HTM reduced AUC of FPD lesions scores during d21-42 (P = 0.043) and d7-42 (P = 0.074) ( Table 4).
No differences were detected in the AUC of total collagen protein, type I alpha 1 collagen (COL1A1) and type III alpha 1 collagen (COL3A1) mRNA during d15-43 (Table 5) and their levels at individual time points (data not shown).

Discussion
FPD is characterized by inflammation and necrotic lesions, ranging from superficial to deep, on the plantar surface of footpads and toes of chickens. Deep ulcers may lead to abscesses and thickening of underlying tissues and structures of the skin 1, 4 . Following a cutaneous lesion, the innate host defense initiates inflammation. This multifaceted process includes the cooperative efforts of different cell lineages during phases of proliferation, migration, matrix synthesis, and contraction, as well as the growth factors and matrix signals present at a wound site 25 . If the wound is not infected, the inflammatory response decreases and is substituted by angiogenesis and tissue remodeling 26 . Some of these cells, including immune and endothelial cells, keratinocytes, and fibroblasts, undergo changes in gene expression and phenotype 27 . In mammals, genome-wide transcriptional analysis tools have been developed to study the progression of changes during wound healing 13,28 .
In the present study, high levels of TM improved growth performance on d14, d28 and d42 and reduced AUC of FPD lesion score during d7-21 and d21-43, low levels of TM improved growth performance on d14 and reduced AUC of FPD lesion score during d7-21. These results suggest that high levels of TM were slightly more effective than low levels of TM in improving growth performance and reducing footpad lesions.
To understand the role of TM in wound healing, the relative mRNA levels of 13 genes previously described to be involved in the development and wound healing of footpad lesions in chickens 19 were measured at various time points in this study.

MMP13 and TIMP.
Lower levels of TM increased mRNA levels of MMP13 on d15 and d43. Lower and/or higher TM also increased mRNA levels of TIMP3 and TIMP4 on several days of the study, and AUC of TIMP3 and TIMP4 mRNA levels during d13-43. MMP13 is collagenase 3 that degrades collagen. TIMPs regulate extracellular matrix remodeling, especially cell migration in wound healing by modulating the activity of specific MMP 29 . TIMPs, especially TIMP3, also play an integral role in the regulation of inflammation 30 . In normal connective tissue, MMP and TIMP are balanced in order to maintain structural integrity. The balance between MMP and TIMP is also critical for the normal wound-healing process 31,32 . The increase of TIMP3, TIMP4 and MMP13 indicates that active matrix remodeling was occurring in footpads, suggesting that TM improved cell migration and matrix remodeling as described previously 33-36 . Collagen and integrins. Collagen is the main and most abundant structural protein in the connective tissue of the extracellular space. It is also the primary protein component of structural tissues such as tendons, ligaments and skin in animals. Collagen contributes up to 35% of the whole-body protein content 13,28,37 . Type I collagen is the most abundant collagen in skin. Integrins are heterodimeric glycoproteins that contain non-covalently associated α and β subunits. They are expressed in basal layer keratinocytes in vivo and responsible for intercellular or cell-substrate adhesion and for sensing mechanical stress [38][39][40] . Integrin α2 (encoded by ITGA2) and integrin α3 (encoded by ITGA3) bind type I, II, III, and XI collagen 41 , forming a heterodimer with integrin β1 subunit, hence acting as a functional cellular receptor for type I collagen fibrils to facilitate both cell spreading on type I collagen matrix and contraction of type I collagen gel 35,40 . In the present study, low and/or high levels of TM did not affect  Table 4. Area under the curve (AUC) of footpad lesion scores in broilers fed three supplemental trace mineral levels during d 7 to 21, d 21 to 42, and d 7 to 42 of age. a,b Means within a row with different superscripts differ at P < 0.05. Reported standard error of the mean (SEM) is the average SEM of the individual least-square means.
the amount of total collagen content, but increased the mRNA levels of ITGA2 and ITGA3. This finding further underlined the important role of TM in promoting collagen organization and remodeling by upregulating integrins during the re-epithelialization and cell migration phase of wound healing in chicken footpads.
CD40. CD40, expressed by fibroblasts, regulates the communication between tissue fibroblasts and T cells, which is necessary for wound healing. Increase of CD40 gene expression by high TM on d43 and increase of  There was no interaction between treatments and age, treatment differences in birds at same age were shown in the graphs. Means with different letters differ at P < 0.05. TNC and VEGF. TNC is produced by migrating and proliferating epidermis and plays an important role in wound healing. TNC gene expression is regulated by mechanical stress 42 , and it is expressed at high levels in wounds 44 . The mRNA levels of TNC were up-regulated by high levels of TM on d29 and down-regulated by both low and high levels of TM on d36, and were not different on d43. Tenascins are extracellular matrix glycoproteins that are abundant in the extracellular matrix of various organs and around healing of wounds 43,44 . Induction of TNC gene expression on d29 suggests that TM promoted cell migration and proliferation during the early phase of wound repair.
In the present study, lower levels of TM increased VEGF expression on d15, and both low and high levels of TM increased VEGF expression on d29. VEGF is one of the most potent proangiogenic growth factors in the skin that functions in angiogenesis, formation of granulation tissue, wound closure and re-epithelialization 35 . Angiogenesis is required following the formation of granulation tissue to provide vascular support for the newly formed tissue during wound repair 45,46 . Therefore, the amount of VEGF present in a wound can impact healing process. TM improved wound healing by increasing the levels of VEGF during the early wound healing phase. The reduction or lack of difference of TNC and VEGF gene expression after d29 suggests the decreased need of active wound repair, which indicates that footpad lesions are successfully healing, and is consistent with the reduction of footpad lesion scores.
TNFα and IL-1β. Inflammation is required to remove injured tissue and cell debris during wound healing 47 , and this inflammatory phase typically lasts for the first 4 days post injury 48 . Proinflammatory cytokines, such as IL-1β and TNFα, are raised right after skin wounding 13,28 . During normal wound healing, the highest levels of TNFα arise anywhere from 12 to 24 h after wounding 49 . After completion of the proliferative phase of wound healing, TNFα returns to basal levels 27 .
The mRNA levels of TNFα were increased in LTM treatment at d15 when the birds started to develop footpad lesions, but returned to basal levels by d22 and remained at basal levels afterwards. The mRNA levels of IL-1β were also increased in LTM treatment at d15, but were lower than control in both LTM and HTM treatments on d22, and were not different from control after d22. These results suggest that low levels of TM improved wound healing by promoting inflammation to remove injured tissue/cells during the inflammation phase (early wound healing phase, d15) and reducing further inflammation and tissue damage during the proliferation and maturation phase (late wound healing phase, d22 onwards), which helps to promote the wound healing process.
Summary. There were no differences between low and high levels of TM in most of parameters measured in this study except for 1) growth performance where high levels of TM were slightly more effective on d28 and d43; 2) AUC of footpad lesion scores where high levels of TM were slightly more effective during d7-21 and d15-43; 3) gene expression of MMP13 and CD40 where low levels of TM were slightly more effective.
In summary, supplementation of TM not only improved growth performance, but also reduced footpad lesions by improving the wound healing process via promotion of collagen synthesis, deposition and organization, cell migration, matrix remodeling, angiogenesis; and regulation of inflammation. The role of individual minerals in FPD wound healing will be the subject of future studies.  Table 5. Relative mRNA levels of the genes potentially associated with footpad dermatitis wound healing as measured by Area under the curve in broilers fed three supplemental trace mineral levels during d15 to 43 of age. a,b Means within a row with different superscripts differ at P < 0.05. Reported standard error of the mean (SEM) is the average SEM of the individual least-square means.