Staining ability of henna (Lawsonia inermis) leaf paste as animal identification marker on Djallonke sheep

Peter T Birteeb¹ and Nashiru Alhassan

Department of Animal Science, Faculty of Agriculture, University for Development Studies, Box TL 1882, Tamale, Ghana.
bpetert2000@yahoo.com
¹ ICAR-Indian Agricultural Statistics Research Institute, Library Avenue, New Delhi – 110012, India.

Abstract

The traditional animal identification systems such as ear tagging, tattooing, branding, and ear notching are easy to apply, but they present certain disadvantages such as cost implications, problems of reading, loss or damage and animal welfare concerns. Henna leaf paste has been proposed as a suitable alternative for use in animal identification. This study aimed to determine the effects of water temperature (WT), paste preparation time (PPT) and coat colour (CC) of an animal on the legibility of henna paste used as an animal identification marker. Henna paste was prepared in a manner to form a 2x3 (i.e. cold water [CW] and hot water [HW] each at 6 [SH], 15 [FH] and 24 [TH] hours paste preparation time) factorial experiment with six treatment combinations (CWSH, CWFH, CWTH, HWSH, HWFH, HWTH). Each treatment (henna paste mixture) was applied on the coat of 10 matured Djallonke sheep to create identification marks, and the legibility of the marks read from a distance using GPS receiver. The experiment was conducted in both rainy and dry seasons, each lasting for 16 weeks.

From ANOVA, water temperature significantly (p < 0.05) affected the average legible distances from the 4^th and 7^th weeks for rainy and dry seasons applications respectively. Hot water preparations yielded significantly higher average legible distances irrespective of the season. Henna paste preparation time showed significant effect (p = 0.011) on average legible distances from 6 ^th week as 24 h preparation time yielded the highest distance of 68.53 m in dry season while the 6 h preparation time yielded the shortest distance of 38.75 m in rainy season. Animal’s coat colour significantly ( p < 0.001) affected average legible distances as white coats had longest while black coats had shortest average distances. The interaction of WT and PPT significantly (p < 0.01) affected average legible distances from the 4^th and 7^th weeks for rainy and dry seasons respectively. Henna paste prepared with hot water and used after 24 h yielded highest average legible distances of the identification marks especially on animals with white coat colour.

Keywords: coat colour, legible distance, livestock, preparation time, water temperature

Introduction

Animal identification is the basis for keeping accurate production records of the herd such as an animal's parentage, birth date, production records, health history, and a host of other important management information (Neary and Yager 2002). The main reasons for using an animal identification system in livestock production are to indicate property ownership, to identify individual animals as a prerequisite for individual performance recording in improved breeding and management systems and to allow a mechanism for disease and residue trace back to the property of origin (Caja et al 2004). However, for an identification system to be fully adopted and used, it should not be easy to copy (to prevent forgery), it should be durable, easy to apply to the animal without requiring expensive or sophisticated equipment, not cause pain or discomfort to the animal, have a high retention, be clearly readable at a distance, not damage the hide or the meat, and should not become a portal of entry for infection, as well as require an infrastructure beyond the means of the farming community using it (FAO/WHO 2004). Animals can be identified using ear tags, tattooing, skin branding, ear notching, nose prints, DNA analysis, microchip implants, rumen boluses radio frequency identification devices (RFID) and paint marks, and each method can be classified into biological, electronic or mechanical systems (Ekuam 2009). However, these methods have several limitations ranging from health, pain to animals, safety, cost implications, loss of tags as well as animal welfare concerns (Caja et al 2004; Evans et al 2005; Ekuam 2009; Silveira 2013).

The aforementioned limitations prompt the need to explore other methods of identifying animals. Lawsonia inermis (henna) can be used as an alternative method of animal identification (Saani 2011; Osman 2012; Osai-Oyete 2014). Application of henna leaf paste does not involve the use of special tools, it has no welfare concerns and the risk of introducing infectious agents to intact skin is null and also, it has the advantage of being easy, quick and painless in application (Urkin 2006) as well as being relatively harmless (Neri et al 2002). Though the use of henna paste as an animal identification marker is very legible at a distance and cost effective compared to the use of ear tags (Osai-Oyete 2014), the effects of temperature of the water used in paste preparation, the time period the paste is allowed to stand before application and the animal’s coat colour on the legibility of the identification marks have not been reported. Therefore, this experiment was conducted to assess the effects of water temperature, paste preparation time and coat colour of sheep on the legibility of henna paste used as animal identification marker on sheep.

Materials and methods

Study Area

The research was conducted at Pong-Tamale in the Savelugu/Nanton municipality in Northern Ghana. Pong-Tamale lies on latitude 9º 40’ N and longitude 0º 49’ W (Encarta 2009). The annual rainfall pattern is erratic with the annual average ranging from 750mm to 1050mm. The minimum, average and maximum temperatures are 16°C, 34°C and 42°C respectively with the low temperatures experienced from December to late February (Anonymous 2010).

Materials used and henna paste preparation

The materials used for this study were henna leaves powder, spatula/stirrer, water, fresh lemon juice (extracted from lemon fruit), 'frytol' vegetable cooking oil (Plate 1a), silver bowls/containers, Winsor and Newton colour chart, 2-inch paint brush, Garmin eTrex GPS receiver, electronic weighing scale and a 60ml syringe. The henna leaves powder was obtained from Savelugu market in northern Ghana. Both hot (100°C) and cold (32.5°C) tap water were used in preparing the henna leaf paste. About 900grams of henna powder was placed in two separate bowls each containing 450g of the henna powder. Then 1500ml of cold tap water was added to the powder in one bowl while 1500ml of boiled tap water was added to the other bowl. Each mixture was stirred to obtain uniform paste. Then, 250ml of lemon juice and 100ml of 'frytol' vegetable cooking oil was added to each bowl and stirred to attain a uniform mixture of the henna leaf paste (Plate 1b). The citric acid in lemon juice breaks down cellulose in the henna powder as it produces a chemical reaction which releases the dye making it available to stain a surface (David et al 2004). The mixtures were allowed to stand overnight (24 h) before application. Exactly 9h later after the preparation of the first mixture, another set (both cold and hot water) was prepared and allowed to stand for 15 h before application. The third set was prepared 18h after the first set and allowed to stand for 6 h before application. All pastes were applied on the sheep around the same time period.

Plate 1. The ingredients used to prepare the henna paste

Experimental animals and design

The experiment was conducted on-farm using animals (sheep) from three different farmers. The study was a 2 x 3 factorial experiment in a Randomized Complete Block Design (RCBD) which was blocked by season (rainy and dry). A total of sixty sheep aged between 6 months and 5 years were used in each season. The factors were water temperature (cold-32.5°C and hot-100°C) and henna paste preparation time (6, 15 and 24 h) which formed six treatment combinations namely cold water at 6 h henna paste (CWSH), cold water at 15 h henna paste (CWFH), cold water at 24 h henna paste (CWTH), hot water at 6 h henna paste (HWSH), hot water at 15 h henna paste (HWFH) and hot water at 24 h henna paste (HWTH) (Table 1). A 2-inch designer paint brush was used to administer four coats of henna paste on the lateral side of each animal (Plate 2) in accordance to the recommendation of Osman (2012). Each treatment was applied on ten sheep with seven having white coat colour, two having brown colour and one having black colour.

Plate 2. Henna paste as identification marker on sheep

Data collection and analysis

The Winsor and Newton Artists’ oil colour chart ( www.winsornewton.com) was used to identify the colours of the identification marks (Plate 3). The distance at which the identification mark was legible was measured using Garmin (eTrex) Global Positioning System (GPS) receiver. The coordinates for a particular spot near the pen where each animal was kept for reading legible distance was taken using the GPS receiver. Data collection was done weekly and the experiment lasted for 16 weeks. All data were analysed using SPSS version 20. ANOVA was used to test the effects of water temperature, henna paste preparation time and coat colour of animal on legible distances, and means separated using the Least Significant Difference at p <0.05.

Plate 3. Comparing the colour of identification mark with a colour chart

Results

Effects of water temperature on legibility of identification mark

The effect of water temperature on average legible distances is shown in Figures 1 and 2. The average legible distances of the identification marks seemed to decrease in a curvilinear pattern over time for both cold and hot water preparations. Whereas the average legible distances decreased continuously in the rainy season, those in the dry season rather inclined upward in the 7^th week before resuming a general decline to the end of the study. At the start of the study, water temperature had no significant effect (p > 0.05) on the average legible distances but significant differences (p < 0.05) occurred from the 4^th week for rainy season application (Figure 1) and the 7 ^th week for dry season application (Figure 2), with similar significant differences seen in the results of subsequent weeks. However, the lines got wider apart from the 5^th and 8^th weeks onward for rainy and dry season applications respectively, indicating greater differences in the average legible distances between cold and hot water preparations. In the rainy season application (Figure 1), higher variances were associated with the average legible distances in the first 4 weeks, while in the dry season application (Figure 2), higher variances were associated with the average legible distances up to the 7^th week. However, distances in the subsequent weeks had lower variances.

Figure 1. Effect of water temperature on legible distance during the rainy season

Figure 2. Effect of water temperature on legible distance during the dry season

Effects of preparation time of henna paste on legibility of identification mark

The result of preparation time of henna paste on the legibility of identification marks across seasons is shown in Table 2. A general decrease in average legible distances of the identification marks over time was recorded from the beginning of the experiment until the 6^th and 7^th weeks for dry season application when increase in the average legible distances were recorded. The decreasing trend resumed after the 7^th week and continued until the end of the experiment. At the start of the study to the 5^th week, the legibility of henna paste used as an animal identification marker was not significantly (p > 0.05) affected by the paste preparation time. From the 6^th week onwards, henna paste preparation time significantly (p ≤ 0.01) affected the average legible distances of the identification marks. From the 6^th week onwards, 24 h henna paste preparation time for dry season application recorded the highest average legible distances (Table 2). However, from the 6^th week onwards 6 h henna paste preparation time recorded the shortest average legible distances except in the 7^th and 11^th weeks when 15 h paste preparation time recorded the shortest average legible distances for dry and rainy season applications respectively.

Effect of coat colour of animal on legibility of identification mark

The effect of coat colours of animals on the legibility of the identification marks across seasons is shown in Table 3. The colours of the identification marks were generally Cadmium yellow and Winsor yellow deep on animals with white coat colours (Plate 2), Cadmium orange and Cadmium yellow deep on animals with brown coat colours and Winsor yellow deep and Cadmium yellow on animals with black coat colours. Legibility was highly significantly (p < 0.001) affected by the coat colours of animals, as those with white coat colours had the highest average legible distances irrespective of the season of application of henna paste. Within 24 h of application, animals with white coats had the highest legible distances followed by brown coats and then black coats. After one week of henna paste application, white coats in the dry season had the highest legible distances followed by white coats in the rainy season while black coats recorded the shortest legible distances irrespective of the season (Table 3). The legible distances of the identification marks decreased generally from the initial time of application to the end of the experimental period. Animals with black coats were the first to lose their identification marks as the marks faded after the 3^rd and 6 ^th weeks for rainy and dry seasons respectively. The identification marks of animals with brown coat colours became illegible after the 6^th and 7^th weeks for rainy and dry seasons applications respectively. By the 8^th week, only animals with white coat colours still had legible identification marks on their bodies. In the 15^th week and afterwards, there was no significant difference in legible identification marks on animals.

Interactive effects of water temperature and paste preparation time on legibility of the identification marks

The results (Figures 3 and 4) showed that, the initial measurements of all treatments (interaction of water temperature and paste preparation time) were generally similar and hence no significant differences (p > 0.05) among them within the first 3 weeks irrespective of the season. However, interaction effect on average legible distances of identification marks was significant (p ≤ 0.009) from the 4^th and 7 ^th weeks for rainy season (Figure 3) and dry season (Figure 4) applications respectively. The weekly measurements showed a generally decreasing trend in the average legible distances recorded for each treatment but some marginal increases were observed for CWTH, HWSH and HWFH during the 4^th week and for HWTH during the 5^th week for rainy season application (Figure 3). For dry season application, the marginal increase in average legible distances started after the 5 ^th week for all treatments except CWFH and HWTH (Figure 4). The legible marks of CWTH, HWSH, HWFH and HWTH lasted until the end of the experiment across seasons but CWSH lasted for 10 and 14 weeks for rainy and dry season applications respectively, whilst CWFH lasted for 13 and 15 weeks for rainy and dry seasons applications respectively. There was no significant (p>0.05) difference between CWTH and HWSH across seasons.

Figure 3. Interactive effect of water temperature and paste preparation time (treatments) on legibility of identification marks during the rainy season

Figure 4. Interactive effect of water temperature and paste preparation time (treatments) on legibility of identification marks during the dry season

Discussion

Effects of water temperature on legibility of identification marks

The significant effect of water temperature on the legibility of the identification marks suggests that hot water (high temperature) facilitated more release of the dye lawsone in the hot water preparation than in the cold water preparation. This finding agrees with the report on Henna page (Anonymous 2003) and that of Alam et al (2007) that, higher water temperature releases lawsone (dye) faster than lower temperatures. Verma et al (2008) also reported that boiled water preparation of henna produced good colour imprints compared to cold water preparation. This result however contradicts the report by Osai-Oyete (2014) in which water temperature had no significant effect on the colour and staining ability of henna paste.

The high variability associated with the average legible distances at the start of the experiment until the 4^th and 7^th weeks for rainy and dry seasons respectively may be due to the presence of animals with different (black, brown and white) coat colours since the legibility of the identification marks differed widely across the different coat colours. The lower variance after the 7^th week in dry season application may be as a result of the fading of the identification marks on animals with brown and black coat colours leaving only animals with white coat colours which resulted in more precise measurements. The marginal increases in average legible distance in the dry season were due to the early fading of identification marks on animals with black and brown coat colours, since measurements on those animals were mostly outliers (smaller values). The higher distances of hot water preparation implied that higher temperatures released more dye for better staining, hence it is better to use hot water in the preparation of henna paste.

Effects of preparation time of henna paste on the legibility of identification marks

he significant effect of preparation time of henna paste on the legibility of the identification marks was in line with the report of Dalglies (1949), which indicated that allowing henna paste overnight (24 h) before use released more lawsone and hence improved the stain. The marginal increases in average legible distances observed in dry season application were due to the early fading of the identification marks on animals with brown and black coat colours. The higher average legible distances and longer legibility (over time) of identification marks from 24 h paste preparation time may mean that the longer the paste is allowed to stand the more the release of the dye (lawsone). The highest concentration of lawsone then produced the sharpest colour contrasts hence the higher average legible distances recorded for 24 h compared to the 15 h or less paste preparation times.

Effects of coat colour of animal on legibility of identification marks

The significant effect of coat colours of animals on the legibility of identification marks is consistent with earlier works in which coat colours of animals affected the visibility of identification marks (Hall et al 2009; Anonymous 2013). The lower average legible distances recorded for brown and black coat coloured animals may be due to the fact that, the colours of the dye which included cadmium orange, cadmium yellow deep, winsor yellow deep and cadmium yellow on the animals, were less contrasting and less visible on the brown and black coat colours compared to the white coat colour . Also, the early fading of identification marks on brown and black coat coloured animals implied that the colour of the lawsone dye was not a suitable physical identification marker on such animals if the identification marks were intended to be used over a long period. Considering the legibility on each of the three coat colours across the seasons, the average legible distances were generally lower in rainy season than in dry season. This might be due to the effects of rainfall. The identification marks on animals could easily be washed off by rain water whenever it rained since most of the pens used to house the animals were not roofed hence exposing the animals to the rains compared to the dry season application. Earlier, Osai-Oyete (2014) reported that the legibility of henna paste used as an animal identification marker was affected by rainfall.

Effects of water temperature and preparation time on legibility of identification marks

The similarity between the legible distances of cold water preparation used after 24 h (CWTH) and hot water preparation used after 6 h (HWSH) suggests that longer periods of waiting time and higher water temperatures have similar effects on legibility of identification marks from henna paste. The early fading of legible identification marks of cold water preparations at 6 and 15 h (CWSH and CWFH) in the rainy season may be attributed to inadequate release of the dye lawsone or the inability of the dye to stain the coat of sheep firmly. The clearly legible marks of CWTH, HWSH, HWFH and HWTH till the end of the experiment suggest better staining of the dye from these treatment combinations. This suggests that, the interactive effect of water temperature and henna paste preparation time affected the release of lawsone. This finding agrees with previous reports (Anonymous 2003; Alam et al 2007; Verma et al 2008) in which high water temperatures increased release of lawsone and hence produced good colour imprints compared to cold water preparations. Also, Dalglies (1949) opined that the longer the paste preparation time, the higher the lawsone released. Therefore, when cold water is used to prepare henna paste for animal identification, the mixture should be allowed to stand for at least 24 h before use to ensure longer staining period. The marginal increases in the average legible distances were due to early fading of the identification marks on black and brown coat coloured animals as well as censoring (lost to follow up) during the study period.

Conclusion

• The temperature of water used in the preparation of henna paste significantly affected the staining ability and legibility of the identification marks as hot water preparations produced higher average legible distances than cold water preparations.
  
  
• The time that henna paste was allowed to stand after preparation before use also significantly affected the staining ability and legibility of the identification marks irrespective of the season of application.
  
  
• The coat colour of an animal highly significantly affected the legibility of the identification marks as white coat colour had the highest average legible distances followed by brown coat colour and then black coat colour.
  
  
• Boiling water should be used in preparing henna paste and the prepared paste should be kept for at least 15 h before it is applied on sheep.

Acknowledgement

The authors wish to acknowledge and thank the following farmers: Mr. Ibrahim Yakubu Lam, Mr. Alidu Inusah and Mr. Yahaya Abdul-Fatawu of Pong Tamale for providing their sheep for the study as well as being instrumental in the data collection process.

References

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Received 13 October 2016; Accepted 20 August 2017; Published 3 October 2017

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