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Copper sulphate and zinc sulphate--a topical remedy for management of sole ulcers in dairy cattle.

Introduction

Sole ulcer is one among the most important lesions causing clinical lameness in dairy animals. Nutrition especially feeding of more concentrates or increase of concentrate: forage ratio has been implicated as a major cause of this anomaly (Manson and Leaver 1987). Moreover, the dairy cows kept on concrete floors are more vulnerable to such ulcerative conditions (Lischer et al., 2002). The major cause of sole ulcer is the mechanical pressure operating in area of sole at the point at which it merges with heel. This pressure causes damage in vessels of corium due to development of thrombosis and ischemia of stratum germinativum and alter, production of horn within sole area (Greenough 1987, Holzhauer et al., 2006). It is a very painful condition which can cause a significant reduction in milk yield (Amory et al., 2008), because it adversely affects udder health and reproductive performance of dairy animals (Hultgren et al., 2004). Due to pain associated with them, the sole ulcers in dairy animals are of greatest concern as far as animal welfare is concerned. Sole ulcers have routinely been observed as a major problem in organized dairy farms having intensive feeding practices (personal observation).

Trace minerals play an important role in production and maintenance of healthy keratinized tissues (Mulling et al., 2000; Randhawa et al., 2012). Systemically, Zinc plays a key role in the formation of structural proteins during the keratinization process (Cousins 1996). Topically Zinc ions are known to induce synthesis of metallothionein, a sulfhydryl rich protein that protects skin and superficial tissues against free radicals (Rostan et al., 2002). In a recent study, Zinc sulphate along with fibroblast growth factor and granulocyte colony stimulating factor (G-CSF) significantly enhanced the wound healing probably through promotion of fibroblast proliferation and down regulation of MMP-1 (matrix metalloproteinase 1) expression in ulcerative wounds of diabetic rats (Shaoqiang et al., 2009). On the other hand, topical Copper sulphate has been shown to stimulate angiogenesis in wounds accelerating wound healing. Besides that, copper stimulates expression of integrins along with zinc and manganese (Tenaud et al., 1999) and causes stabilization of fibrinogen and up-regulation of copper dependant enzymes, such as lysyl oxidase, important for matrix remodelling (Kobayashi et al., 1994).

Copper has potent anti-bacterial (Borkow and Gabbay, 2005), anti-fungal (Weber and Rutal 2001) and antiviral (Borkow et al., 2007) properties. Recently the combination of Copper sulphate and Zinc sulphate has also been evaluated as footbath for laminitic lesions (Sarel et al., 2006, Speijers et al., 2010). But their disposal has always been observed to create toxicity at the site of disposal so in this experiment they have been used in minimum amount for dressing of sole ulcers.

[FIGURE 1 OMITTED]

Materials and Methods

This study was carried out in 8 Holstein Friesian crossbred cows from the University Dairy farm. The selection of animals was made by direct visual examination of claw soles, before and after claw trimming. Cows with sole ulcers (pododermatitis circumscripta) located either in lateral and medial hind digits were included in this study. In all eight animals, the lesions were treated after removal of necrotic tissue with the application of Copper sulphate and Zinc sulphate (1:1) and a light bandage was applied in such a way that weight bearing was not disturbed on the affected digit. The dressing and bandaging of sole ulcers was performed every 5th day until wound healing occurred.

Measurement of wounds

Length, width and depth of sole ulcer were measured on every subsequent dressing. On the day of first dressing, approximate length and breadth of respective digit was also measured to calculate the percentage area of digit affected with the sole ulcer. A relation between percentage area of digit affected with sole ulcer and healing period was calculated.

Statistical analysis

The effect of the combination on healing of sole ulcers and association between percentage area of digit affected with sole ulcer and healing period was analysed statistically using Student's t- test (paired t- test) by using SPSS version 17 software.

Results and Discussion

The length, width and depth of sole ulcers were recorded every 5th day while doing dressing with Copper sulphate and Zinc sulphate. Dimensions of sole ulcers considerably reduced on every subsequent dressing indicating significant improvement in wound status (Table 1, Fig 1). Different bacteriostatic dressings have been used by Veterinarians to bandage a claw affected with sole ulcers (Perusia, 2006). Copper is known for its wound healing properties as it plays an important role in proliferation and remodeling phases. It has been reported that copper induces number of factors in wound healing cascade. The vascular endothelial cells growth factor (VEGF) expression induced by copper is believed to stimulate angiogenesis in wounds thus accelerating wound healing. This angiogenesis or copper induced proliferation of endothelial cells is not inhibited by serum deprivation or by the presence of antibodies against a variety of angiogenic factors (Hu 1998). Other factors like Glycyl-L-histidyl-L-lysine (GHK) (Swaim et al., 1996, Canapp et al., 2003), multiprotein complexes containing S100A13 protein (Prudovsky et al., 2003) and Fibronectin, a large extracellular matrix cell adhesion glycoprotein (Ahmed et al., 1999) are known to play additive roles in wound repair mechanism. GHK-[Cu.sup.2+] has two main functions: (1) first as a potent tissue protective, anti-inflammatory agent that limits oxidative damage after tissue injury and (2) as a signal that activates tissue remodelling, that is the process for removal of damaged protein and scar tissue and its replacement by normal tissue. The formation of multiprotein aggregate containing the S100A13 protein enables the release of [FGF.sub.1] in response to stress (Prudovsky et al., 2003). Fibronectin helps in organization of matrix deposition and promotion of angiogenesis (Ahmed et al., 1999). Besides that, copper stimulates expression of alpha2beta1, alpha3beta1, alpha6beta4 and alphaVbeta5 integrins along with zinc and manganese (Tenaud et al. 1999), causes stabilization of fibrinogen and up-regulation of copper-dependant enzymes, such as lysyl oxidase, important for matrix remodelling (Kobayashi et al., 1994).

On the other hand, topical zinc in the form of divalent zinc ions has been reported to provide antioxidant protection to wounds. It is suggested that zinc ions induce synthesis of metallothionein that protects against free radicals thus providing antioxidant defense for skin (Rostan et al., 2002). Expression of metallothionein gene is up regulated following topical application of zinc and copper in wound margins particularly in regions of high mitotic activity (Lansdown 2002). In addition, zinc salts have been observed to accelerate the re-epithelization process besides having moderate antibacterial and anti-inflammatory properties. It has been observed that zinc salts activate endogenous zinc dependent metallo-proteinases which may facilitate keratinocytes migration (Agren 1999). Topical zinc oxide treatment has also been reported to enhance endogenous expression of insulin like growth factor 1 in granulation tissue from porcine wounds (Tarnow 1984). In a recent experimental study, Zinc sulphate in combination with recombinant human acid fibroblast growth factor and stem cell stimulator granulocyte colony stimulating factor has been observed to enhance wound healing possibly through proliferation and differentiation of fibroblasts, regeneration of blood vessels and up-regulation of tissue inhibitor of metalloproteinase 1 and down regulation of matrix metalloproteinase 1 expression in ulcerative wounds of diabetic rats (Shaoqiang et al., 2009).

It may be the synergistic effect of Copper sulphate and Zinc sulphate that have resulted in the significant improvement in wound status on every successive dressing in the present study. Both the salts are having excellent anti-bacterial and anti-fungal properties besides having their role in proliferation and remodeling of wounds. It seems that copper by stimulating angiogenesis and inducing various factors reduce the margins of wounds and zinc protects their further deterioration by its antioxidant action. Further, in the re-epithelialization process, zinc facilitates keratinocytes migration and copper promotes the process by forming disulphide linkages between Cys residues. There may be increase in local tissue concentrations of these elements which might be a stimulating factor in this wound repair mechanism. Copper sulphate as 7% solution has been commonly used for footbathing agent in dairy herds in many countries due to its disinfectant properties. Manske et al. (2002) found a peroxide compound of copper very effective against heel horn erosions and dermatitis. Copper sulfate has been shown to penetrate the horn quite extensively (Kempson et al., 1998). Bergsten et al. (2007) reported a reduction in the odds of having digital dermatitis (DD) in legs treated with copper sulfate. In addition, copper sulphate also has the property of destroying granulation tissue within the ulcer (Blowey, 2005). Zinc in the form of zinc sulphate powder has also been used as dry footbaths in some countries. Zinc salts are also known for their anti-bacterial and anti-fungal properties. In the present study, percent area of sole affected with sole ulcer had also been measured on first dressing and corresponding healing period was also noted upon (Table 2.). It was seen that most of sole ulcers took healing time corresponding to their size but there were few which were smaller ones but took more time as compared to other with comparatively bigger size. It may be due to the fact that healing period of sole ulcers does not depend only on size of ulcer but also varies with quality of wound. In this study, sole ulcers with more depth took more time as compared to less deep ulcers which could be the possible reason for their prolonged healing. Some authors consider that it is a major error to apply a bandage unless some method of relieving pressure on the sole of the affected claw has been used (Greenough 1987). In the present study, it was particularly seen that application of bandage would not alter the weight bearing of animals.

Conclusions

It was concluded that combination of Copper sulphate and Zinc sulphate can be effectively used for dressing of sole ulcers due to their variety of properties in the wound repair mechanism. Sole ulcers with more depth may take more time to heal irrespective of their approximate area. Further the wound healing properties of these elements may be utilized in future for management of topical lesions in animals.

References

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Ahmed, Z., Idowu, B.D. and Brown, R.A. (1999). Stabilization of fibronectin mats with micromolar concentrations of copper. Biomaterials 20: 201-09.

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D. Dewangan (1) and Swaran Singh (2)

Department of Veterinary Medicine College of Veterinary Science Guru Angad Dev Veterinary and Animal Sciences University (GADVASU) Ludhiana--141004 (Punjab).

(1.) Present address: Assistant Veterinary Field Officer, Anuppur, Madhya Pradesh

(2.) Professor and Corresponding author.

E-mail: drswaran68@gmail.com
Table 1: Measurements of sole ulcers and their Mean [+ or -] SE value
during dressing with copper sulphate and zinc sulphate

Measurements         Before                5 days

Depth          1.09 [+ or -] 0.11   0.875 [+ or -] 0.07 *
Length         1.65 [+ or -] 0.31   1.51 [+ or -] 0.30 **
Width          1.53 [+ or -] 0.26   1.38 [+ or -] 0.25 **

Measurements          10 days                 15 days

Depth          0.68 [+ or -] 0.07 **   0.56 [+ or -] 0.07 *
Length         1.34 [+ or -] 0.29 **   1.03 [+ or -] 0.23 **
Width          1.13 [+ or -] 0.18 **   1.00 [+ or -] 0.19 **

Measurements          20 days                 25 days

Depth          0.37 [+ or -] 0.06 *    0.27 [+ or -] 0.07 **
Length         0.83 [+ or -] 0.23 **   0.71 [+ or -] 0.22 **
Width          0.72 [+ or -] 0.20 **   0.60 [+ or -] 0.19 **

Measurements          30 days                 35 days

Depth          0.18 [+ or -] 0.08 **   0.08 [+ or -] 0.04 **
Length         0.55 [+ or -] 0.23 **   0.38 [+ or -] 0.20 **
Width          0.47 [+ or -] 0.19 **   0.28 [+ or -] 0.15 *

Measurements          40 days

Depth          0.05 [+ or -] 0.03 *
Length         0.16 [+ or -] 0.11 **
Width          0.15 [+ or -] 0.10 **

** Significant at 5% level of significance.

* Significant at 1% level of significance.

Table 2: Percentage area of sole affected with
sole ulcers vs healing period (days)

Animal No.         Percentage area of     Healing period
                   sole affected with         (days)
                    sole ulcers (%)

1164                     21.76                  45
1158                     9.167                  45
1341                      7.5                   35
1238                      8.4                   35
1151                      3.37                  30
1238                      1.94                  30
1287                      2.72                  20
1287                      4.82                  25

Percentage area               [Length x Width of sole
of sole affected with                 ulcer/
sole ulcer              =     Length x Width of claw]  x  100
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Title Annotation:Clinical Article
Author:Dewangan, D.; Singh, Swaran
Publication:Intas Polivet
Geographic Code:9INDI
Date:Jan 1, 2016
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