GHENT UNIVERSITY

FACULTY OF VETERINARY MEDICINE

Academic year 2010-2011

The copper soil-plant-animal cycle: factors associated with copper status of cattle in the

Gilgel Gibe Catchment, Ethiopia

By

Thomas VAN HECKE

Promoter: V. Dermauw Research Project in the context

Co-promoters: Prof. Dr. Ir. G. Janssens of the Master Thesis

Y. Kechero

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The author and the promoters agree this thesis is to be available for consultation and for personal

reference use. Every other use falls within the constraints of the copyright, particularly concerning the

obligation to specially mention the source when citing the results of this thesis.

The copyright concerning the information given in this thesis lies with the promoters. The copyright is

restricted to the method by which the subject investigated is approached and presented. The author

herewith respects the original copyright of the books and papers quoted, including their pertaining

documentation such as tables and illustrations. The author and the promoters are not responsible for

any recommended treatments or doses cited and described in this study.

May 2011, Merelbeke

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Acknowledgments

This dissertation was made possible by the efforts of many people who offered their support and

expertise. First of all, I would like to express my deepest gratitude to my promoter Prof. Dr. Ir. Geert

Janssens for giving me the chance of performing veterinarian research in Africa. Without his guidance,

suggestions and financial support, this project would not be possible. I would also like to thank

Veronique Dermauw who was always willing to advise and help when problems arose during

fieldwork. Her patience, corrections and suggestions contributed largely to the successful completion

of this thesis. Great appreciations go to Yisehak Kechero for his invaluable plant expertise and

company in the field. I am grateful to Prof. Dr. Eric Van Ranst for taking the time to explain me the

difference in soil types in the Gilgel Gibe Catchment. Also Dr. Moti Yohannes was greatly appreciated

for his help and assistance in the parasitology lab at JUCAVM (Jimma University College of

Agriculture and Veterinary Medicine).

I would like to express my gratitude to the workers in the Jimma Municipal Abattoir for their

cooperation. Fieldwork was not possible without the assistance of several community workers and

drivers and of course the cooperation of local farmers.

Last but not least, I would also like to thank all the other Belgian thesis students at JUCAVM and

Ethiopian students, Brooke Kebede and Shemita Misganaw in particular, for their companionship and

friendship. They made this research not only a unique scientific but also an unforgettable life

experience.

Thomas Van Hecke

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Table of Contents

Abstract ................................................................................................................................................ - 1 -

Introduction .......................................................................................................................................... - 2 -

PART 1: Literature Survey ................................................................................................................ - 3 -

1.1 Copper ........................................................................................................................................... - 4 - 1.1.1 Function of copper in cattle .................................................................................................... - 4 - 1.1.2 Copper metabolism ................................................................................................................ - 4 - 1.1.3 Deficiency symptoms .............................................................................................................. - 5 - 1.1.4 Diagnosis of copper deficiency ............................................................................................... - 7 -

1.2 Copper deficiency in the East of Africa .......................................................................................... - 8 - 1.2.1 The Rift Valley ........................................................................................................................ - 8 - 1.2.2 Ethiopia .................................................................................................................................- 10 - 1.2.3 Jimma ...................................................................................................................................- 12 -

1.3 The copper cycle in the soil – plant – cattle system ....................................................................- 12 - 1.3.1 Summary scheme .................................................................................................................- 12 - 1.3.2 Parent material .....................................................................................................................- 13 - 1.3.3 Soil ........................................................................................................................................- 14 -

1.3.3.1 Association soil pH and bioavailability molybdenum and copper .................................- 14 - 1.3.3.2 Soil types around Jimma ...............................................................................................- 14 - 1.3.3.3 Influence of altitude on mineral concentrations in nitisols .............................................- 15 - 1.3.3.4 Influence of altitude on mineral concentrations in plants ..............................................- 15 - 1.3.3.5 Influence of altitude on mineral concentrations in cattle ...............................................- 15 -

1.3.4 Plants ....................................................................................................................................- 16 - 1.3.4.1 General..........................................................................................................................- 16 - 1.3.4.2 Copper antagonists .......................................................................................................- 16 - 1.3.4.3 Tannins..........................................................................................................................- 16 - 1.3.4.4 Season ..........................................................................................................................- 16 -

1.3.5 Cattle ....................................................................................................................................- 17 - 1.3.5.1 Fysiological status .........................................................................................................- 17 - 1.3.5.2 Infection .........................................................................................................................- 17 - 1.3.5.3 Parasites .......................................................................................................................- 17 - 1.3.5.4 Geophagy ......................................................................................................................- 18 -

PART 2: Materials and Methods .....................................................................................................- 19 -

2.1 Prevalence of copper deficiency ..................................................................................................- 20 -

2.2 Evaluation of management practices ...........................................................................................- 20 - 2.2.1 Study area ............................................................................................................................- 20 - 2.2.2 Soil and geophagy ................................................................................................................- 21 - 2.2.3 Management practice ...........................................................................................................- 22 - 2.2.4 Plant intake ...........................................................................................................................- 22 - 2.2.5 Blood sampling .....................................................................................................................- 23 - 2.2.6 Statistical analysis ................................................................................................................- 23 -

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PART 3: Results ...............................................................................................................................- 24 -

3.1 Prevalence of copper deficiency ..................................................................................................- 25 -

3.2 Management practices.................................................................................................................- 27 - 3.2.1 General .................................................................................................................................- 27 - 3.2.2 Background information........................................................................................................- 28 - 3.2.3 Statistical analysis ................................................................................................................- 29 -

3.3 Plant composition of the diet ........................................................................................................- 30 - 3.3.1 General .................................................................................................................................- 30 - 3.3.2 Background information........................................................................................................- 32 -

3.3.2.1 Diet composition according to region ............................................................................- 32 - 3.3.2.2 Diets in Planosols/Vertisols ...........................................................................................- 32 - 3.3.2.3 Diets in Nitisols/Ferralsols .............................................................................................- 32 -

3.3.3 Statistical analysis ................................................................................................................- 32 -

3.4 Mineral composition of plant species ...........................................................................................- 33 - 3.4.1 General .................................................................................................................................- 33 - 3.4.2 Mineral composition plants according to region ...................................................................- 33 - 3.4.3 Mineral composition plants according to soil type ................................................................- 33 -

3.5 Mineral intake in selected cattle ...................................................................................................- 34 - 3.5.1 General .................................................................................................................................- 34 - 3.5.2 Statistical analysis ................................................................................................................- 35 -

3.5.2.1 Mineral intake in cattle according to region ...................................................................- 35 - 3.5.2.2 Mineral intake in cattle according to soil type ...............................................................- 35 - 3.5.2.3 Mineral intake in cattle according to feeding strategy ...................................................- 35 -

3.6 Mineral concentrations in plasma of selected cattle ....................................................................- 36 - 3.6.1 General .................................................................................................................................- 36 - 3.6.2 Statistical analysis ................................................................................................................- 37 -

3.6.2.1 Influence of management on mineral concentrations in plasma...................................- 37 - 3.6.2.2 Correlation between mineral content in diet and plasma ..............................................- 38 -

PART 4: Discussion .........................................................................................................................- 40 -

4.1 Prevalence of copper deficiency ..................................................................................................- 41 -

4.2 Management Practices ................................................................................................................- 41 -

4.3 The copper cycle in the soil-plant-cattle system ..........................................................................- 42 - 4.3.1 Summary scheme results .....................................................................................................- 42 - 4.3.2 The influence of soil-type on mineral concentrations in plants.............................................- 43 - 4.3.3 Mineral content of forages in the Gilgel Gibe Catchment .....................................................- 44 - 4.3.4 The influence of diet on mineral status of cattle ...................................................................- 46 -

4.3.4.1 Botanical composition ...................................................................................................- 46 - 4.3.4.2 Mineral composition ......................................................................................................- 46 -

4.3.5 The influence of soil-type on mineral concentrations in cattle..............................................- 47 -

4.4 Suggestions to improve copper status .........................................................................................- 48 -

PART 5: Addendum and References .............................................................................................- 49 -

5.1 Addendum ....................................................................................................................................- 50 -

5.2 References ...................................................................................................................................- 54 -

Abstract

In tropical and subtropical countries mineral deficiency, unbalance and toxicity are frequently

observed in grazing ruminants. Ethiopia is a landlocked country located in the Horn of Africa

and the country’s estimated cattle population of about 45 million in 2007 is believed to be

Africa’s largest (FAO, 2007). A study conducted by Dermauw et al. (2009) in the Gilgel Gibe

valley in SW Ethiopia proved altitude as an influencing factor on the mineral status of cattle.

This research was conducted in the same valley to evaluate the impact of different

management practices on the copper status of cattle and to improve the productivity and

reproduction of grazing cattle. Also estimation has been made on the prevalence of copper

deficiency in the region. From the 53 liver samples collected at Jimma municipal abattoir, 81%

had deficient copper levels, from which 58% were in the clinically deficient range.

The practice of communal- or individual herding and herd size did not influence copper status

in cattle. The botanical and mineral composition of diets was determined for 19 cattle. 87

different plant species belonging to 25 plant families were ingested and 52 different plant

species were analysed for mineral content. Analysis of plant species showed mineral

imbalances as a widespread phenomenon in the Gilgel Gibe Catchment. Cattle grazing on

planosols/vertisols and associated wetlands ingested significantly lower amounts of copper

and higher amounts of molybdenum than cattle grazing on nitisols/ferralsols. Higher

molybdenum concentrations in diet were significantly associated with lower copper status in

cattle. Also cattle grazing in the wetlands had lower copper status than cattle grazing in well

drained lands. These results indicate cattle grazing on wetlands are more likely to develop

severe copper deficiency than cattle grazing on well drained soils.

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Introduction

Ethiopia is a landlocked country located in the Horn of Africa and with over 80.7 million people it is the

second most populous nation in Africa. The country’s estimated cattle population of about 45 million in

2007 is believed to be Africa’s largest. Ethiopia is ranked number six on the list of highest numbers of

cattle in the world (FAO, 2007). The livestock sector contributes about 30% of agricultural GDP and

12% of total GDP excluding the value of draught power and manure. About 10 million oxen work as

draught animals in agricultural areas. Export of livestock counts for 14% of agricultural export and 11%

of total export. Hereby, the livestock sector is the second most important export product next to coffee.

Live animals are exported to Somalia for re-export to the Gulf countries and Yemen, but also to

Kenya, Sudan and Eritrea (GRM, 2007). Cattle production is distributed throughout the country with

the greatest concentration in the highlands.

Cattle in Ethiopia are almost entirely of the zebu type and are poor sources of milk and meat.

However, these cattle do relatively well under the traditional production system. Contagious diseases

and parasitic infections are major causes of death. Recurring drought takes a heavy toll on the animal

population, although it is difficult to determine the extent of losses. During the rainy seasons water and

grass are generally abundant but with the onset of the dry season forage is generally insufficient to

keep animals nourished and able to resist disease (Ofcansky and LaVerle, 1991).

In tropical and subtropical countries mineral deficiency, unbalance and toxicity are frequently observed

in grazing ruminants. Phosphorus is most likely to be deficient, followed by copper, cobalt, sodium,

iodine and selenium. Intoxication of selenium, molybdenum and fluorine are also widespread in

tropical countries. Furthermore, numerous reports are available about deficiencies and toxicities of

other minerals, such as calcium, magnesium, potassium, iron, zinc and manganese (McDowell et al.,

1983). The losses in production caused by clinical and subclinical shortage of minerals in livestock are

enormous (Thornton et al., 1972a, 1972b). Marginal deficiencies are more widespread than severe

shortages and affect large number of animals in tropical countries. Because marginal shortage is

associated with a reduction of productivity, growth rate and reproduction it is very important to detect

these deficiencies in cattle (Underwood, 1981).

Faye et al. (1986) performed a survey on the mineral status of ruminants in Ethiopia. Analysis of

plasma samples showed severe and marginal shortage of copper was widespread. Remarkably, all

severe deficiencies and a minority of the marginal shortages were prevalent in the Rift Valley.

A study conducted by Dermauw et al. (2009) in the Gilgel Gibe valley proved altitude as an influencing

factor on the mineral status of cattle. Cattle grazing in lower altitudes seemed to have a lower copper

status than cattle grazing in higher altitudes. Since there were significant differences in copper status

between different herds at the same altitude, the study concluded that there must be other factors

influencing copper status.

This research was conducted in the same valley to evaluate the impact of different management

practices on mineral status of cattle and to improve the productivity and reproduction of grazing cattle.

- 3 -

PART 1

- Literature Survey -

- 4 -

1.1 Copper

1.1.1 Function of copper in cattle

Copper is an essential trace element that is incorporated in a various amount of metallo-enzymes.

Shortage of copper leads to impaired function of these enzymes and results in several symptoms. A

summary of the most important copper enzymes and their functions and deficiency symptoms is given

by Table 1.

Table 1: physiological functions and deficiency symptoms of the most important copper

dependant enzymes (from McDowell, 2003; Underwood and Suttle, 1999)

Enzymes Physiological functions Deficiency symptoms

Cytochrome oxidase Terminal oxidase in the respiratory chain

Lysyl oxidas Adds a hydroxyl group to lysine residues

in collagen, allowing cross-linking between collagen fibres

Impaired wound healing and blood vessel integrity

Cu-Zn superoxide dismutase E.g., protection of erythrocytes against oxygen radicals Anaemia

Ceruloplasmin (Ferroxidase I) Ferroxidase II

Important in the flow of iron that supports hematopoiesis Anaemia

Dopamine-β-hydroxylase Dopamine-β-mono-oxygenase Hydroxylation of dopamine

Tyrosinase Melanin formation Lack of pigmentation (achromotrichia)

Superoxide dismutases Extracellular ceruloplasmin Intracellular Cu thioneins

Antioxidant defence

1.1.2 Copper metabolism

Monogastric and pre-ruminant animals absorb copper more efficiently than ruminants. High amounts

of copper released during rumen digestion are likely to be precipitated as copper sulphide (Underwood

and Suttle, 1999). When high amounts of molybdenum and sulfur are present in diet, thiomolybdates

are formed in the rumen, which reduce copper absorbability. Consequently, excess of molybdenum

and sulphur in diet have a negative influence on copper status (Underwood and Suttle, 1999;

McDowell, 2003). Increased dietary concentrations of iron and zinc can also have an inhibitory effect

on the utilisation of copper (Van Campen and Scaife, 1967; Hall et al., 1979; Humphries et al., 1983).

Table 2 shows ideal concentrations of minerals in forages. Values outside normal concentrations are

classified into either a marginal or high antagonistic likelihood. Maximum Tolerable Classification

(MTC) is defined as the maximum dietary level that will not impair animal performance when fed for a

limited period (Mortimer et al., 1999).

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Table 2: classification of trace elements in forage relative to their abilities to meet either dietary

requirements (A) or cause an antagonistic problem with copper (B) (Mortimer et al., 1999)

A. Trace mineral Severely deficient

Marginally deficient Adequate MTC

Copper (mg/kg) < 4 4 - 9,9 > 10 100 Manganese (mg/kg) < 20 20 - 39,9 > 40 1000 Zinc (mg/kg) < 20 20 - 29,9 > 30 500 Selenium (µg/kg) < 100 100 - 199,9 > 200 2000 Copper:Mo Ratio < 4 4,0 - 4,5 > 4,5 -

B. Copper antagonist Deficient Ideal Marginally antagonistic

Severely antagonistic MTC

Iron (mg/kg) < 50 50-200 200 - 400 > 400 1000,00 Molybdenum (mg/kg) - < 1 1 - 3 > 3 5 Sulfur (%) < 0,10 0,15-0,20 0,20 - 0,30 > 0,30 0,40

MTC= Maximum Tolerable Concentration

Suckling or milk fed animals absorb copper very well with an efficiency of 50% and more. The high

efficiency of copper utilisation and high amounts of copper in the liver of newborn animals, preserve

young animals from the consequences of low copper levels in milk. When a functional rumen

develops, absorbability of copper decreases to 1-10% (Suttle, 1975; Underwood and Suttle, 1999).

Absorption of copper occurs predominantly in the upper section of the small intestine (McDowell,

2003; NRC, 2005). Copper absorbed in the portal blood binds to albumin and transcuprein and is

transported to the liver. Subsequently, copper can be stored in the liver bound to metallothionein,

excreted in bile or used for the synthesis of copper metallo-enzymes. Copper binds to ceruloplasmin

for transportation to extrahepatic tissues. Biliary excretion is the major mechanism for copper

homeostasis (McDowell, 2003; NRC, 2005).

1.1.3 Deficiency symptoms

Depigmentation of hair or achromatrichia is one of the early clinical symptoms of copper shortage in

cattle. Typical is the depigmentation of the hairs around eyes (‘spectacle eye’) and ears (Figure 1A

and 1B), but it can affect all hairs (Figure 1C). Red hairs turn to yellowish-dun (Figure 1A and 1C)

colour while black turns to reddish-brown (Figure 1B). Hair also becomes dull and kinky (Allcroft and

Lewis, 1957; Davis et al., 1946; Harvey, 1952).

A delayed oestrus can be caused by shortage of this mineral and consequently, deficiency can have a

negative influence on fertility and reproduction. Infertility is more likely to be related to secondary

deficiencies due to excess of molybdenum than to primary deficiencies (Kendall et al., 2003). Growth

retardation and delayed puberty in copper deficient calves are also presumably more related to excess

of molybdenum than primary shortage. Cattle supplemented with iron did not alter growth even though

copper plasma levels were depressed (Phillippo et al., 1987a; Phillippo et al., 1987b; Gengelbach et

al., 1994; Underwood and Suttle, 1999). Also the presence of diarrhoea is more attributed to excess of

molybdenum rather than shortage of copper (Ward, 1978).

- 6 -

Figure 1: indications for copper deficiency; A: depigmentation of red hairs around the eyes –

B: depigmentation of black hairs around the eyes and horns – C: severe loss of hair colour

over the whole body, hair looks dull and kinky, diarrhoea on the hindquarters, small posture

Deficiency in copper leads to the dysfunction of ceruloplasmin, a cupro-protein responsible for the

release of iron from the liver. Since iron is a component of haemoglobin, copper shortage leads to an

iron resistant anaemia because of iron accumulation in the liver and to a lesser extent in other organs

such as the spleen (Evans and Abraham, 1973). Iron is next to haemoglobin also a component of

myoglobin, cytochrome and the enzymes catylase and peroxidase. Consequently, iron is responsible

for the transport of oxygen to the cells and cellular respiration.

Ceruloplasmin also acts as an acute phase protein by permitting the incorporation of iron in transferrin.

In this way, free ferrous iron concentrations decrease and thus, ceruloplasmin plays an important role

in anti-oxidant and antibacterial defence (Carver et al., 1982; Letendre and Holbein, 1984).

Copper status influences the non-specific immune function. Shortage of copper leaded to a decreased

ability of granulocytes to kill Candida albicans in sheep and cattle (Jones and Suttle, 1981). Also

depression of neutrophil viability, phagocytic ability and killing capacity were observed as

consequences of copper deficiency. Consequently, copper deficient cattle are more susceptible to

infectious diseases (Boyne and Arthur, 1986; Gengelbach, 1994). No differences were found in total

IgM and IgG between copper deficient and copper adequate calves. Consequently, it seems copper

status does not affect specific immune function (Stabel et al., 1993; Ward et al., 1997).

Copper deficiency can cause malformation of collagen- and elastin fibres (Owen, 1981). These fibres

support lung parenchyma and are necessary for the normal function of the lungs. Malformation of

collagen and elastin in the lungs consequently leads to pulmonary emphysema (O’Dell et al., 1978).

One of the most severe consequences of copper deficiency is loss of integrity and elasticity of arteries

or even the heart. Rupture of major arteries leads to the sudden death syndrome or falling disease

(Allen and Klevay, 1978; Bennetts, 1942).

Copper is essential for normal elastin and collagen production in cartilage, osteoid, ligaments and

tendons. Therefore, lameness and skeletal abnormalities such as osteochondrosis and growth plate

pathologies are also frequently observed due to copper shortage (Maas and Bradford, 1990).

- 7 -

The copper containing enzyme dopamine-β-mono-oxygenase converts dopamine into norepinephrine

(Friedman and Kaufman, 1965). Shortage can lead to disorders of the central nervous system

including ataxia, tremor and clonic seizure. Copper induced ataxia or swayback typically occurs in

newborn lambs (Barlow, 1963).

1.1.4 Diagnosis of copper deficiency

Most clinical signs as described above can also be caused by other factors such as parasitic infections

and Johne’s disease (Herd, 1994). Consequently, copper deficiency can not be diagnosed based only

on clinical symptoms. Determination of copper concentrations in blood plasma and liver lead to the

final diagnosis of copper shortage. Analysis of forage samples for copper, molybdenum, iron and

sulphate and water analysis for sulphate can also help diagnose the problem.

Plasma samples can be used as a general indicator for the copper status of cattle. Plasma copper

concentrations do not reflect dietary intake unless copper storage in the liver is severely depleted

(Underwood and Suttle, 1999). Release of hepatic copper is often sufficient to maintain normal plasma

copper levels until liver copper decreases below 30 mg/kg (Mills, 1987). By the time plasma copper

levels are below normal range, the animal is at least suffering from subclinical deficiency.

Measurement of plasma copper concentration is considered to be the best test available for confirming

cattle with clinical symptoms as deficient (Underwood and Suttle, 1999).

Laven et al. (2007) reported plasma copper concentrations suitable for the detection of marginal

copper status. On the other hand, analysis of serum copper concentrations is not recommended

because the loss of copper during clotting is too great.

Inflammation and infection increase plasma levels of the copper containing ceruloplasmin as part of

the acute phase response (Disilvestro and Marten, 1990; Stable et al., 1993). This increase may

complicate interpretation of plasma copper levels. Ceruloplasmin activity in serum and plasma was

found to be suitable to detect copper shortage in blood (Laven et al., 2007).

When plasma copper concentrations are in the normal range, the only way to determine copper status

is by liver biopsy. Analysis of liver tissue is the best method to evaluate current copper status. In late

pregnancy, plasma and liver copper levels decline drastically in cows (Xin et al., 1993). Values of

copper, iron and zinc in blood and liver are categorised in Table 3.

- 8 -

Table 3: categorisation of copper, iron and zinc levels in blood and liver (adapted from Puls,

1988, Wikse et al., 1992)

Copper Iron Zinc

Status Plasma (mg/l)

Liver DM (mg/kg)

Serum (mg/l)

Liver WM (mg/kg)

Plasma (mg/l)

Liver DM (mg/kg)

Clinically deficient < 0,2 <20 - - - -

Deficient 0,2 - 0,5 <33 0,15 - 1,2 < 40 0,2 - 0,4 < 20 - 40 Marginal 0,5 - 0,7 33 - 125 - - 0,5 - 0,8 25 - 40 Adequate 0,7 - 0,9 125 - 600 1,3 - 2,5 45 - 300 0,8 - 1,4 25 - 200

High 0,9 - 1,1 600 - 1250 4,0 - 6,0 53 - 700 2,0 - 5,0 300 - 600 Toxic > 1,2 > 1250 - - 3,0 - 15,0 > 1000

DM= Dry Matter, WM= Wet Matter

Kellaway et al. (1978) indicated copper levels in hair as a sensitive indicator of copper status when the

copper amount in liver is lower than 20 mg/kg. Hair analysis is not recommended by other authors.

Dirt and contamination lead to overestimation and washing of the hair leads to underestimation of

certain minerals (Herd, 1994).

1.2 Copper deficiency in the East of Africa

1.2.1 The Rift Valley

In the East of Africa different surveys were conducted about the mineral status of cattle and wildlife.

Research in Djibouti, Ethiopia and Kenya has proven that severe copper deficiency is associated with

the pedogeological area of the Rift Valley (Faye en Grillet, 1981; Faye et al., 1986; Faye et al., 1990;

Hedger et al., 1964). The Rift system forms a narrow, elongate fault in the earth’s crust that extends

from Djibouti to Mozambique (Figure 2). The volcanic activity in the Rift has a strong influence on the

present soil types and geochemistry of soil and water. The most important near-surface rock-types are

young, volcanic stones containing either very high or very low levels of certain trace elements (Morley,

1999). Consequently, the volcanic character of the Rift Valley influences the flow of trace elements in

the soil-plant-animal system.

Typical for the Rift are the high concentrations of molybdenum and sulfur in soil and vegetation. Since

these elements are strong antagonists of copper, copper deficiency can still develop in grazing cattle

even when grasses have normal copper values (Faye et al., 1991). Furthermore, other climatic

conditions prevail in the Rift in comparison with the surrounding regions. There is less rainfall in the

Rift en this relative dryness supports dust contamination on plants. Since iron particles are widespread

in African soil types, iron elements can easily land on the vegetation whereby the iron content of plants

is increasing (Faye et al., 1991). Next to molybdenum and sulfur, this mineral is also a strong

antagonist of copper and can induce symptoms of copper shortage (secondary copper deficiency).

- 9 -

Figure 2: illustration of the Rift Valley (adapted from Maps of the World, 2011)

Because of these specific mineral compositions of soil and vegetation and climatic conditions it is not

surprising swayback or enzootic ataxia occurs frequently in the Rift Valley. This nervous disease is

associated with low copper status and is characterized by lack of coordination of movement and

paralysis of the back legs. Swayback is an important problem in newborn ruminants, especially sheep

and goats. Local inhabitants recognise this disease and even have names for it: loïpsiepsiep (Masaï),

kipsiepsiep (Tugen) or degamaka (Afar) (Hedger et al., 1964; Faye et al., 1991; Roeder, 1980b).

The first survey about copper shortage in the Rift Valley was performed around Kenya’s Lakes

Baringo and Harrington in 1964. Through plasma analysis of cattle, sheep and goat widespread

copper shortage was detected in the region. Grazing pastures showed normal copper concentrations

(9.3-12.8 mg/kg) but high molybdenum- (0.5-5.6 mg/kg) and sulfur levels (0.6-6.5%) (Hedger et al.,

1964).

Copper deficiency was also detected in the impala in Lake Nakuru National Park in the Kenyan Rift

Valley. This shortage was caused by low copper status in soil in addition to high molybdenum levels in

grasses. Grazing pastures in the park contained average molybdenum concentration above 2 mg/kg

which was sufficient to induce copper shortage (Maskall and Thornton, 1991).

Similar research was conducted in the Amboseli National Park. This park is also situated in the Rift

close to the Tanzanian border at the foot of the Kilimanjaro. In this area very high molybdenum

concentrations (average of 86 mg/kg) and low copper levels (average of 3.6 mg/kg) were detected in

the grass species Sporobolus spicatus. This species is definitely capable of inducing copper shortage

in cattle and wildlife (Maskall en Thornton, 1996).

In a survey about the nutrition of the sable antelope in the Shimba Hills National Park mineral levels

were also determined in soil and forage samples. This area is situated close to the Kenyan coast and

outside the Rift. Even though soil was poor in copper, grasses had normal copper and molybdenum

levels (Sutton et al., 2002).

- 10 -

Djibouti, a neighbouring country at the Northwest of Ethiopia is largely occupied by the Rift. In 1988 a

survey was conducted on mineral status of cattle in this country. Hundreds of animals (n=310) were

involved and analysis of plasma samples indicated shortage of copper in 17% cattle, 30% goat, 31%

sheep and 46% camels. Furthermore grasses had low copper levels (2.1-5.6 mg/kg) while woody

plants contained higher values (5.4-14.1 mg/kg). In most forage samples concentrations of

molybdenum and sulfur were very high and even reached toxic levels. For example Tamarix nilotica

contained 10.8 mg/kg molybdenum and 1% sulfur (Faye et al., 1990).

Sudan is not crossed by the Rift. Faye et al. (1991) suggested cases of copper deficiency in Sudan

are rare and mild even though soil and vegetation have low copper levels. An explanation is that,

through the nomadic lifestyle of the herders, the animals graze from time to time in copper rich areas,

so they don’t develop shortage. However, several articles report cases of copper deficiency indicating

copper deficiency as a widespread problem in the country (Mukhtar, 1970; Tartour, 1975; Abu Damir

et al., 1983; Abdelrahman et al., 1998).

Furthermore, shortage of copper was detected in forages in the East of Congo (Mandiki et al., 1986)

and Malawi (Mtimuni 1982).

1.2.2 Ethiopia

In 1980, Roeder described for the first time copper shortage in Ethiopia. In 2 isolated locations in the

Rift (Adami Tullu and Metahara) enzootic ataxia was observed in lambs. Plasma copper levels in

ataxic lambs were very low with an average of 0.15 mg/l. Soil contained sufficient available copper but

molybdenum and sulfur concentrations in soil and vegetation were not determined (Roeder, 1980a;

Roeder, 1980b).

During a survey (Faye et al., 1986) on the mineral status of ruminants in Ethiopia 1082 plasma

samples (432 cattle, 425 sheep, 173 goats en 52 camels) from 89 different locations were analysed.

At the same time 59 forage samples from 54 locations were collected. Copper, iron and zinc levels

were determined in the plasma samples. The feed samples were analysed for cobalt, copper, iron,

zinc and manganese. Concentrations of molybdenum and sulfur were not measured.

Analysis of the feed samples indicated shortage of copper and zinc was the most important problem in

mineral nutrition. In 28 locations a severe copper shortage was determined and in 9 locations a

marginal deficiency. Zinc shortage was severe in 28 locations and marginal in 17. Deficiency in

manganese was more rare and was determined in 9 locations. The areas where severe copper and

zinc shortage was observed were situated in the Rift Valley, the Omo valley, the province of Wollega

and the area around Lake Tana (Figure 3A).

- 11 -

Figure 3: A. Mineral deficiency in forage samples – B. Mineral deficiency in plasma samples

(Faye et al., 1986)

Analysis of plasma samples showed severe shortage of copper in 11 locations and marginal shortage

in 24 locations. Remarkable is that the severe deficiencies and a minority of the marginal shortages

were situated in the Rift Valley (Figure 3B).

Based on pedogeological maps of the country and the results of the survey a sketch was drawn,

describing the mineral status of cattle in Ethiopia and Eritrea (Figure 4). This map is only a rough

representation of the situation and naturally more research needs to be performed to complete the

map (Faye et al., 1986).

Figure 4: zones of mineral shortages in cattle in Ethiopia and Eritrea (Faye et al., 1986)

- 12 -

1.2.3 Jimma

Jimma is comfortably the largest settlement in western Ethiopia, with a population estimated at around

175.000 in 2008 (Briggs, 2009). The Gilgel Gibe dam is situated about 50 km in the West of the city. In

this region Dermauw et al. (2009) investigated the influence of altitude of grazing areas on the mineral

status of cattle. Cattle grazing in lower altitudes had a lower copper status than cattle grazing in higher

altitudes. Since there were significant differences in copper status between different herds at the same

altitude, the study concluded that there must be other factors influencing copper status (Dermauw et

al., 2009).

During a research around subclinical mastitis 11 dairy farms were visited in Jimma. Analysis of plasma

samples showed that 37% of the dairy cows (crossbreeds Holstein Friesian x local zebu) had a

shortage of copper (Belay et al., 2009).

1.3 The copper cycle in the soil – plant – cattle system

1.3.1 Summary scheme

Figure 5: the copper cycle in the soil – plant – cattle system (adapted from Howard et al., 1962;

Hall et al., 1979; Brennan et al., 1980; Humphries et al., 1983 Adriano, 1986; CDA, 1988; Butler,

1989; Bank et al., 1990; Faye et al., 1991; Stable et al., 1993; Xin et al., 1993; Underwood and

Suttle, 1999; Whitehead, 2000; Baissa et al., 2003; Baissa et al., 2005)

- 13 -

1.3.2 Parent material

The complex relations between the geochemical nature of soils and their parent materials, mineral

composition of plants and the occurrence of mineral deficiencies and excesses in grazing livestock

have been documented since the 1960’s (Webb, 1964).

In the Rift Valley, volcanic formations are mainly composed of basalt, ryolithe and trachyte and these

formations have high molybdenum and sulfur content. High concentrations of copper have been

reported in soils derived from basalts in Nigeria and Chad (Cottenie et al., 1981; Pias, 1968). Soils

developed on volcanic ash have low copper and zinc content (Reuter, 1975). West (1981) observed

that proportions of copper and molybdenum in soils derived from trachyte are such that molybdenum

induced copper deficiency is likely (West, 1981).

Jimma is situated in the Ethiopian volcanic plateau (FAO/UNESCO, 1975) and so the parental

materials in Jimma region are composed of basalt, ryolithe and trachyte. The Geological map of

Ethiopia (UN, 1972) shows the parental materials in Jimma and surrounding areas (Figure 6). The

region is divided in the magdala group and the ashangi group. The magdala group consists of

rhyolites, trachytes, rhyolitic and trachytic tuffs, ignimbrites agglomerates and basalts. In the Ashangi

group, the dominant parental materials are alkali olivine basalt and tuffs and rare rhyolites.

Unfortunately, the geological map is not very precise and so geological information in Jimma region

might be inaccurate and incomplete.

Figure 6: parental materials in Jimma and surrounding areas (adapted from UN, 1972)

Magdala group: rhyolites, trachytes, rhyolitic and trachytic tuffs; ignimbrites agglomerates, basalts

Ashangi group: alkali olivine basalt and tuffs, rare rhyolites

- 14 -

1.3.3 Soil

1.3.3.1 Association soil pH and bioavailability molybdenum and copper

Bioavailability of trace elements in soils is strongly influenced by the soil pH and appears to be related

to the concentrations of sodium and calcium (Maskall and Thornton, 1996). Alkali soils generally show

increased bioavailability of molybdenum while availability of copper decreases (Adriano, 1986).

Copper levels in wheat were found to decrease with increasing pH in several Kenyan soils (Nyandat

and Ochieng, 1976). On the other hand, strongly acid conditions can damage roots and interfere with

copper uptake by plants (CDA, 1988).

At Lake Nakuru National Park, the high molybdenum content of several plant species was linked with

the elevated pH of soils. The alkali tolerant grass species Sporobolus spicatus which grows on

alkaline solonetz soils had exceptional high concentrations of molybdenum (Maskall and Thornton,

1991). Particularly high molybdenum concentrations in Sporobolus spicatis in the Amboseli National

Park were also associated with alkaline soils (pH 9.2-10.7) (Maskall and Thornton, 1996).

1.3.3.2 Soil types around Jimma

The most common soil types around Jimma are nitisols and planosols. Less common are vertisols and

ferralsols (Deckers et al., 2008).

Nitisols are the most intensively cultivated soils in Ethiopia with area coverage of 12% of the total area

of the country and nitisols rank first in terms of area coverage of arable lands (23%) (FAO, 1984). This

soil type is a well drained, red, tropical soil (Figure 7A) that is rich in iron and is predominantly found in

level to hilly land in tropical rain forest or savannah vegetation. They are generally considered to be

fertile soils and have favourable physical properties. Nitisols belong to the most productive soils of the

humid tropics and are widely used for plantation of coffee and for food crop production (Yerima and

Van Ranst, 2005). Nitisols in Kenya had relative low pH (4.2-6.4) and relative low concentrations for

sodium and calcium. At the same time the superficial horizon of the nitisols was relatively rich in trace

elements (37.9 mg/kg Cu and 4.2 mg/kg Mo) (Maskall and Thornton, 1996). The low pH and relative

high amount of copper in nitisols might be favourable characteristics for copper content in the

vegetation but high amounts of iron might interact with availability of copper. Ferralsols have similar

geochemistry as nitisols (Bowell, 1993).

Figure 7: A. Cow in fertile nitisol area – B. Cattle grazing in poor wetland planosol area

- 15 -

Planosols have bleached and light coloured surface soil abruptly overlying finer subsoil. They have

light forest or grass vegetation and are seasonally or periodically wetlands (Figure 7B). These lands

soak up water during the raining season and than slowly release it. Wetlands cover approximately

1.4% of Ethiopia’s land surface and the most dominant wetland types in the country are swamps and

marshes (FAO, 1984; Hillman, 1993).

In the Ethiopian highlands, planosols occur in association with vertisols in lower parts of the landscape

and with nitisols in higher areas. Planosols are poor soils that are typically used for brick making and

extensive grazing. Generally, they are not used for agriculture. Mature planosols are chemically

strongly degraded and have low cation exchange capacity (Yerima and Van Ranst, 2005). The

superficial horizon of planosols has a pH around 6.3 (Zhiyi, 1989). Planosols in Slovakia had an

average copper content of 23.5 mg/kg and 0.9 mg/kg molybdenum (Lastincova et al., 2003) but these

amounts might not represent the composition in planosols around Jimma.

1.3.3.3 Influence of altitude on mineral concentrations in nitisols

Baissa et al. (2005) examined the effects of altitude on status of minerals in nitisols in Western

Ethiopia. Nitisol is also the dominating soil type around Jimma. Concentrations of extractable copper,

zinc and molybdenum were determined. Values of extractable copper were below the critical level in

18, 3.6 and 1.8% of the soil samples collected in respectively low, mid and high altitude. Although

there seems to be a trend to more deficient soil samples in the lower areas, altitude had no significant

influence on amount of extractable copper. Altitude had also no significant effect on molybdenum and

zinc status in nitisols (Baissa et al., 2005).

On the other hand altitude had a significant influence on concentrations of iron and manganese in

nitisols. The amounts of extractable iron and manganese are increasing with increasing altitude

(Baissa et al., 2003).

1.3.3.4 Influence of altitude on mineral concentrations in plants

Research to evaluate the effect of altitude and parent material on the mineral composition of forages

was conducted in the Mt. Elgon region. Mt. Elgon is an extinct volcano on the border between Kenya

and Uganda. This study showed a significant influence of altitude on concentrations of copper and

selenium in grasses. At lower areas only low copper (<6 mg/kg) and selenium values (<0.1 mg/kg)

were found in forages but values ranged widely at higher altitudes. Altitude had no significant influence

on other trace elements (Jumba et al., 1995).

1.3.3.5 Influence of altitude on mineral concentrations in cattle

The study conducted by Dermauw et al. (2009) has proven that altitude has an influence on the

mineral status of cattle. During this research, the Gilgel Gibe Catchment was divided in 3 regions,

based on their altitude. Cattle originating from the area on medium and high height had higher copper

concentrations in plasma than cattle in the lower areas. Because herds also differed significantly in the

same region, this parameter seemed to be very variable. Next to the altitude of the grazing lands,

there must be other factors that have an influence on copper status of cattle. Furthermore, zinc levels

- 16 -

were significantly higher in the middle area than in the lower and higher areas. Sulfur and iron showed

no significant difference between the 3 regions.

1.3.4 Plants

1.3.4.1 General

Trace element concentrations in tropical pastures can fall as the plant matures (Gomide et al., 1969)

and during periods of rapid growth (Fleming, 1973). According to available data copper contents are

extremely variable between plant species in tropical areas, particularly between grasses and browse

plants. In Lake Nakuru National Park grasses tend to contain higher levels of copper than browse

plants (Maskall and Thornton, 1991). In irrigated areas or in swamps, some grass species were

severely deficient in copper, whereas woody species and grass plants had copper concentrations

above normal (Faye, 1985). Shrub species draw their nutrients from deeper down and consequently

have a different trace element composition (Faye and Tisserand, 1989).

1.3.4.2 Copper antagonists

Higher concentrations of iron, molybdenum, zinc and sulfur decrease bioavailability of copper in plants

(see 1.1.2 Copper metabolism).

1.3.4.3 Tannins

Tannins in plants form insoluble mineral complexes and this can lead to reduced availability of

minerals (South and Miller, 1998). These substances are phenolic compounds and are subdivided in

condensed and hydrolysable tannins. The hydroxyl groups on the aromatic rings are chemically

reactive and form indigestible complexes with proteins. Condensed tannins have been reported to

interfere with iron absorption (Butler, 1989). Cattle and sheep are sensitive to condensed tannins,

while goats are more resistant (Kumar and Vaithiyanathan, 1983). In general, woody plants contain

higher levels of tannins than grasses (Iason and Van Wieren, 1999).

In the Gilgel Gibe Catchment, samples were collected from the main consumed plants in 6 regions.

The average tannin concentration per region ranged between 0 and 3.8% on dry matter base. High

tannin levels were associated with a decrease in copper plasma concentration in cattle while plasma

iron levels were not affected (Kechero et al., 2010).

1.3.4.4 Season

An increase in trace element concentrations of plants has been observed in the wet season for

pastures in the Kenyan highlands (Howard et al., 1962), for grass species adjacent to Lake Nakuru

(Maskall, 1991) and for several grass and browse species in Mole National Park in Ghana (Bowell and

Ansah, 1993). In Western Sudan, forages were adequate in copper during the wet season and

deficient during dry season. Serum copper levels were low throughout the year but particularly low in

the mid rainy season and late dry season (Abdelrahman et al., 1998).

- 17 -

Contamination of vegetation by soil dust increases in the dry season. Because iron and iron salts are

abundant in African soil types there is an increase of iron concentration in plants (Faye et al., 1991).

Grass cover becomes scarcer as the dry season progresses. In this way, the amount of ingested soil

increases in the diet of grazing cattle (Abrahams and Thornton, 1994).

1.3.5 Cattle

1.3.5.1 Fysiological status

Liver copper levels decline drastically in late pregnancy. Xin et al. (1993) reported a decrease of liver

copper by 51% from 8 weeks pre-partum until parturition. This decline is attributed to the drainage of

copper by the foetal liver at an exponential rate. Liver copper concentration is still lower 2 months after

parturition compared to copper levels before the pre-partal decline. Xin et al. (1993) observed lowest

copper levels in plasma 5 weeks pre-partum which does not coincide with changes in liver copper.

1.3.5.2 Infection

Inflammation and infection increase plasma levels of the copper containing ceruloplasmin as part of

the acute phase response (Disilvestro and Marten, 1990; Stable et al., 1993). Ceruloplasmin permits

the incorporation of iron in transferrin and consequently free ferrous iron concentrations decrease. In

this way, ceruloplasmin plays an important role in anti-oxidant and antibacterial defence (Carver et al.,

1982; Letendre and Holbein, 1984).

Mastitis can result in lowered copper serum concentrations. Mastitis leads to secretary disorders in the

mammary glands and increased permeability of blood capillaries. After E. coli infection, the mean

serum concentration of copper measured 52% of pre-challenge concentration. Experimental cows

were however marginal deficient in copper before infection and decline in mean serum copper

concentration was not significant (Erskine and Bartllet, 1993). Infection with S. aureus resulted after

24h in a serum copper concentration of 89% compared to concentration before infection. However,

mean copper concentration did not differ significantly in time (Middleton et al., 2004). Another study

showed no significant differences in serum copper levels between healthy cows and cows with

subclinical and clinical mastitis (Ibtisam El Zubeir et al., 2006).

1.3.5.3 Parasites

A depression of blood copper levels has been reported in ruminants infected with nematodes

(Bremner, 1959; McCosker, 1968; Frandsen; 1982). Bank et al. (1990) concluded that gastrointestinal

nematodes interfered with copper metabolism by causing an increase in pH in the abomasal and

duodenal part of the digestive tract. Other studies have proven that parasitism enhances copper

deficiency by influencing the molybdenum and sulfur antagonism (Ortolanie et al., 1993; Frandsen,

1982). The results from Adogwa et al. (2005) suggest that interaction with the absorption of copper in

the gastrointestinal tract is not the only mechanism by which the gastrointestinal parasites interfere in

the copper metabolism. In their study parasitism had a negative effect on blood copper levels even

when copper was administered parentally.

- 18 -

Next to gastrointestinal parasites, also blood parasites can decrease blood copper levels. Animals

infected by Babesia show lower iron and copper serum levels, while iron and copper serum

concentrations increased one month after treatment of infected animals (Askar et al., 2008). Infection

with Theileria annulata can also depress iron and copper levels in cattle (Omer et al., 2003). Copper

deficient animals could also be more susceptible to trypanosome infection than animals with normal

copper levels (Crocker et al., 1992).

1.3.5.4 Geophagy

Soil ingestion or geophagy is a phenomenon that is widespread in ungulates and other animals

although this behaviour is not exhibited everywhere (Kreulen and Jager, 1984; Mahaney and

Hancock, 1990). The geophagic soil contains high amounts of clay and fine silt with little sand

(Krishnamani and Mahaney, 2000). Ingested soils are usually situated on older, stable landscape sites

where they had ample time to reach maturity and even old age. In this way they have passed through

many stages of weathering where primary minerals have been transformed into secondary minerals

(Mahaney, 1999).

The reason why animals deliberately ingest soil is not completely known. One theory suggests

geophagy serves a detoxification function in relation to plants secondary compounds (Johns and

Duquette, 1991). Another explanation concerns the contribution of ingested soil to dietary mineral

intake. Studies on soil consumption by people in Africa and Central America indicated that in certain

cases these clays are important sources of supplementary minerals such as calcium, copper, iron,

magnesium and zinc. Stoszek (1976) observed that while three grass-fed cattle became severely

copper deficient, a fourth animal, which had the habit of eating soil, accumulated copper.

Furthermore, animals seem to prefer ingesting soil from termite mounds. Termite mounds seem to

contain higher concentrations of minerals than surrounding soils and a higher amount of plant species

is present close to the termitaria (Holdo and McDowel, 2004). It is likely that termites, in their

mobilisation of the soil, concentrate metals including copper (Kebede, 2004).

However, when accidentally ingested soil contributes 10% in the diet of sheep, Cu absorption and

utilisation are reduced as much as 50%. Even when the diet contains only 2% of soil the utilisation of

copper is significantly reduced. The antagonism between copper and ingested soil is strongly

suggested to be caused by iron amounts in soil (Suttle et al., 1975; Suttle et al., 1984; Underwood and

Suttle, 1999). Soil ingestion was measured in heifers by determining soil concentrations in faeces.

Calculations showed an intake of 0.73 and 0.99 kg soil per animal each day (Mayland et al., 1977).

Nitisols around Jimma are rich in iron and therefore it is likely that copper utilisation is reduced in cattle

grazing on nitisols by accidental nitisol ingestion.

- 19 -

PART 2

- Materials and Methods -

- 20 -

2.1 Prevalence of copper deficiency

A survey was conducted at Jimma municipal abattoir to estimate the prevalence of copper deficiency

in the Gilgel Gibe valley. Jimma zone is situated in the south western part of the country in the

regional state of Oromia. The town is located at approximately 350 km south west of the capital Addis

Ababa. In 2003, Jimma district had a livestock population of 18354 cattle (CSA, 2003). Slaughter

animals are coming to Jimma municipal abattoir from different areas surrounding the town.

During meat inspection of slaughtered cattle, 53 animals were selected. All animals were adult, male

cattle from local breed, further selection was at random. Since copper values in the liver are the

golden standard for determining copper deficiency in cattle, approximately 50 g of liver tissue was

collected of all selected animals. The presence of parasitic infections was determined in these

sampled livers by inspection. Samples were placed on plastic petri dishes and were oven dried at 65°

C for 72 hours. Afterwards, dried samples were grinded in a mechanical grinder and stored in closed

tubes.

Destruction of liver samples happened by an accelerated wet digestion method. One gram of each

dried and grounded sample was weighed after which 10 ml of HNO3- was added. Samples were stirred

in an ultrasonic bath for 15 minutes at room temperature. Subsequently, samples were destructed by

open microwave method. After digestion was complete, dilutions were made to meet analytical

requirements (Campbell and Whitfield, 1991). Levels of Zn, Cu and Fe were determined through ICP-

OES.

2.2 Evaluation of management practices

2.2.1 Study area

The study was conducted in the Gilgel Gibe Catchment in the south western part of the country in the

regional state of Oromia. The catchment has an altitude ranging between 1059 and 3259 meter above

sea level. The two raining seasons in the region are the kiremt and the belg. The kiremt lasts from

mid-June to mid-September and during this season 90-95% of the nation’s total cereals output is

produced. The belg receives rainfall from February to May and only provides 5-10% of the cereal

output (USDA/FAS, 2008). The survey was started at the end of the kiremt, from September to

October.

The study area, located at the southern part of the Gilgel Gibe Catchment and within a radius of 35 km

from Jimma, was divided in 3 regions based on their altitude. Region 1 was situated between 1700

and 1800m, region 2 between 1800 and 2000 meters and region 3 between 2000 and 2200 meters. In

each region, 6 herds were selected at random (in region 3, one extra herd was included in the study).

In each herd, one representative animal was selected: female, between the age of 4 to 6 years, not

pregnant and preferably not lactating. Because farmers in the region don’t have the habit to wean their

- 21 -

calves, animals lactating for more than 4 months were allowed in the survey. Cows that were lactating

less than 4 months were not included because liver copper concentration is still lower 2 months after

parturition compared to copper levels before the pre-partal decline (Xin et al., 1993). The selected

animals were numbered according to the date of observation (Figure 8).

Figure 8: locations of the selected cattle in the Gilgel Gibe Catchment in association with the

altitude of their grazing lands (Adapted from Van Ranst et al., 2010)

2.2.2 Soil and geophagy

The most dominating soil types around Jimma are the nitisols and the planosols. Less occurring types

are the ferralsols - associated with the nitisols - and the vertisols - associated with the planosols.

Because of similar appearance of the soils, no distinction was made between nitisols and ferralsols

and between planosols and vertisols.

Soil types were distinguished by colour in two groups. Group PV includes the planosols and the

vertisols and this group had a soil colour from grey to black. Planosols and vertisols become wetlands

during the raining season and are covered with light forest or grass vegetation. These areas are poor

soils that are generally used for brick making and extensive grazing.

The NF group contains the nitisols and the ferralsols and colour is red to brown. These soils are

productive, fertile soils that are widely used for plantation of coffee and food crops. The NF group is

found in higher areas than the PV group and are rich in iron.

During the observation of the selected animals, attention was given whether or not animals were

ingesting soil deliberately.

- 22 -

2.2.3 Management practice

Different management practices were determined by selecting different criteria. According to their

grazing strategy, herds were classified in 2 groups. The first group contained herds grazing on

communal areas. Farmers can leave their cattle to the communal grazing lands where one or more

herders look out for their cattle. In the evening farmers return to the communal grazing areas to collect

their cattle and return their animals to the stables. In this way, a large herd belonging to different

farmers is formed every day and herded by a limited amount of people. The second group contained

small herds belonging to an individual farmer.

Herd size was classified in 3 groups: less than 10 animals (1), between 10 and 40 animals (2) and

more than 70 animals (3). BCS of observed animals was determined using an adapted condition

scoring for zebu cattle, scaled from 1 to 9 (Nicholson and Butterworth, 1986).

Route and distance covered by the observed animals were monitored by GPS tracking. All

observations were entered into a geographic information system (GIS) as text files and referenced

according to their distance bearing from home. Total covered distance was measured from the

moment cattle left their stable in the morning until return in the evening. Herding radius was defined as

the maximum distance from home at which a herd was observed during that day. By comparing the

total covered distance and the herding radius, different herding practices could be determined and

influence on mineral status could be evaluated.

2.2.4 Plant intake

Estimation on the plant composition of the diet was realised by observation. During one grazing day

the selected animal was observed from the moment leaving the stable in the morning until return in the

evening. Every 10 minutes the ingestion of plant species was recorded. In case a bite was taken from

several plant species at the same moment, the involved plant species were recorded. Based on these

results a score sheet of the ingested plant species could be calculated for each observed cow. One

plant observation received 1 point. When more species were recorded during one observation, the

involved plant species received 1 point divided by the amount of species recorded at that moment.

When these scores were divided by the number of total observations, estimation could be made on

the proportions of the ingested plant species.

Samples of the most important plant species were collected in paper bags and oven dried at 65° C for

72 hours. Afterwards, dried samples were grinded in a mechanical grinder. Samples were stored in

plastic bags until Zn, Mo, Co, Cu, Mn and Fe were determined with ICP-OES and Se and S through

ICP-MS analysis.

Destruction of plant samples happened by an accelerated wet digestion method. One gram of each

dried and grounded sample was weighed after which 10 ml of HNO3- was added to each sample.

Samples were stirred in an ultrasonic bath for 15 minutes at room temperature. Unlike destruction of

liver samples, destruction of plant samples happened in a microwave utilizing closed vessels. By the

- 23 -

combination of heat and pressure in a closed vessel, reaction rate increases and digestion time

decreases (Vigler et al., 1980; Okamoto and Fuwa, 1984).

To determine the estimated mineral intake we could use the data on the proportions of the ingested

plant species. The percentage of each ingested plant was multiplied by the mineral content of the

species. When mineral concentrations were not known for all species a correction factor was

introduced to extrapolate the mineral concentration of the known percentage of ingested plants to

100%. In this way mineral intake by cattle could be compared between the different regions.

2.2.5 Blood sampling

To prevent stress during the observation, blood samples were taken when animals returned to their

stable. After fixating the animal, blood was collected from the jugular vein in two heparin tubes of 6 ml.

The blood samples were immediately transported in ice water to the Agricultural Campus of Jimma

University.

First, the presence of blood parasites was determined in the blood, more specifically for Babesia and

Trypanosoma. Giemsa stained blood smears were used to examine infection with Babesia. Giemsa

colouring was obtained by air drying a thin blood smear, after which fixation of the sample occurred in

methyl alcohol for 2 minutes. After drying, the sample was stained by Giemsa stain 10% for 30

minutes. Finally, the blood smear was examined by microscope for the presence of intracellular

parasites in the red blood cells.

Infection with Trypanosoma was checked by the haematocrit centrifugation technique described by

Woo (1970). Capillary tubes were filled with blood sample and sealed at one end using plasticin, than

centrifuged for 7 minutes at 3500 rates per minute. Afterwards, the buffy coats of the tubes were

examined for the presence of trypanosomes using a microscope.

The remaining blood samples were centrifuged for 10 minutes at 3500 rates per minute. Plasma was

collected and samples were temporarily stored in the deep freezer. Copper, iron and zinc were

analysed by ICP-OES and manganese through ICP-MS analysis. Similar to the destruction of plant

samples, an accelerated wet digestion method utilising closed vessels was used for the destruction of

plasma samples.

2.2.6 Statistical analysis

Statistical analysis was performed on all data using SPSS17 (SPSS Inc., Illinois, Chicago).

Independent t-tests and one-way ANOVA with Tukey post-hoc tests were used to determine

differences between groups. Pearson correlations were used to determine interrelationships between

selected parameters.

- 24 -

PART 3

- Results -

- 25 -

3.1 Prevalence of copper deficiency

Liver samples collected at Jimma municipal abattoir were subdivided in different classes, dependant

on their copper concentrations. Classification occurred according to the criteria of Puls (1988) and

Kincaid (1999): clinically deficient (<20 mg/kg), deficient (<33 mg/kg), marginal (33-125 mg/kg),

adequate (125-600 mg/kg) and high (>600 mg/kg) (Table 4).

Based on these data, estimation was made on the prevalence of copper deficiency in the area. On the

total of 53 inspected bulls, 43 animals were in the deficient range while only 10 cattle had adequate

concentrations. An overall prevalence of copper deficiency in the area was estimated at 81% (43/53).

In the deficient cattle, a distinction was made between clinically deficient, deficient and marginal

deficient. Clinically deficient copper concentrations were measured in 25 cattle, 7 cattle had deficient

levels and 11 animals were in the marginal range. These results indicate shortage of copper as a

severe problem in the region.

Liver of cattle normally contains between 45 and 300 mg/kg iron based on wet matter (Puls, 1988). In

the 53 sampled animals, 3 animals or 6% had iron levels above 300 mg/kg. All other samples were in

the adequate range. Except for one sample, all animals had zinc levels between 25 and 200 mg/kg

and were in the normal range (Puls, 1988).

Table 4: classification liver samples (Adapted from Kincaid, 1999; Puls, 1988)

Cu Fe Zn

n % Range DM (mg/kg) n % Range WM

(mg/kg) n % Range DM (mg/kg)

Clinically deficient 25 47 <20 - - - - -

Deficient 7 13 <33 - - <40 - - <20-40

Marginal 11 21 33-125 - - - - 25-40

Adequate 10 19 125-600 50 94 45-300 52 98 25-200

High - - >600 3 6 53-700 1 2 300-600

Total 53 100 53 100 53 100

DM= Dry Matter, WM= Wet Matter

Because parasites can influence mineral status, presence of parasitic infections in sampled livers was

determined by inspection. No distinction was made in the extent of infection. Only 8 samples were

uninfected. Fasciola spp. was the most common parasite found in sampled livers. Six cattle were

infected with Echinococcus granulosus and one animal had Ascaris spp. in the liver (Table 5).

- 26 -

Table 5: parasitic infections in sampled livers

Parasite n %

Ascaris spp. 1 2

E. granulosus 6 12

Fasciola spp. 38 71

Uninfected 8 15

Total 53 100

An independent t-test was conducted to compare mineral levels of uninfected livers with infected

livers. No significant differences were calculated. Figure 9 shows concentrations of copper in relation

to concentration of iron. Remarkable is that high iron levels in liver only occurred when copper values

were deficient. Higher amounts of copper were typically accompanied by lower iron values.

.

Figure 9: correlation between copper (mg/kg) and iron (mg/kg) values in livers collected in the

Jimma abattoir, Ethiopia

- 27 -

3.2 Management practices

3.2.1 General

Management practices in the different regions are shown in Table 6. The selected animals were

numbered according to the date of observation. Herds were subdivided in communal (C) or individual

(I) herds and were classified in 3 groups according to the number of animals in the herd. A difference

was made between grazing (G) animals and cattle that were fed with crop residues (CR). The

percentage of crop residues in diet was estimated by observation. Stovers from Saccharum

officinarum, Sorghum spp., Zea Mays and Pisum sativum, leaves from Musa spp. and Ensete

ventricosum and residues from Eragrostis tef and Linum usitatissimum were considered as crop

residues.

Total distance (TD) and herding radius (HeRa) are expressed in km. The amount of ingested plant

species (AIPS) is a factor expressing diversity in diet.

Table 6: management practices in the different height regions around Jimma, Ethiopia

Reg Cow ID SOIL Wet

Land Man Herd size

Feeding strategy

Crop Residue TD HeRa AIPS BCS

Observed supplementation

by farmer 1 1 PV 1 C 3 G 0,0 3,7 0,5 9 2 - 1 2 PV 0 I 1 G 2,3 4,4 1,1 18 6 - 1 3 PV 1 C 3 G 0,0 5,6 1 9 5 -

1 4 PV 1 I 1 G 0,0 1,5 0,26 8 5 Around 2 kg of corn enriched with salt

1 6 PV / NF 1 I/C 2 G 0,0 5,9 1 11 5 - 1 7 PV 1 C 3 G 0,0 6,3 0,73 14 4 -

2 5 NF 0 C 2 G 2,3 6,1 1,3 12 3 One leaf from Ensete ventricosum and one leaf from Musa spp.

2 10 NF 0 I 1 G 2,7 0,9 0,35 18 4 - 2 11 NF 0 I 1 G/CR 13,2 0,7 0,22 20 6 -

2 12 NF 0 I 1 CR 50,0 0,05 0,02 13 5

Stover from Zea mays, leaves from Rhus

glutinosa and Premna schimperi

2 16* PV 1 C 1 CR 66,7 1,6 0,36 6 7 Bulule once a week

2 17* NF 0 I 1 CR 52,1 0,55 0,17 9 7 Bulule tree times a week

3 8 NF 0 I 1 G 0,0 1,6 0,35 11 5 -

3 9 NF 0 I 1 G 0,8 1,8 0,38 26 4 Herbal mixture from pea field

3 14 NF 0 I 1 G/CR 27,5 0,36 0,06 14 4 Pennisetum purpureum, Saccharum officinarum, Stover from Zea mays

3 15 NF 0 I 1 G/CR 21,9 1,5 0,33 18 5

Melinis repens, Pennisetum purpureum,

stover Zea mays, Erythrina brucei

3 18 NF 0 I 1 G/CR 39,5 4,1 0,79 22 5 - 3 19* NF 0 I 1 G/CR 27,3 1,7 0,58 21 5 - 3 13 NF 0 I 1 G 1,0 3,2 0,88 22 5 -

Reg= Region (1= 1700-1800m; 2= 1800-2000m; 3= 2000-2200m), *= changed feeding strategy few days before observation,

PV= Planosol/Vertisol, NF= Nitisol/Ferralsol, Man= Management (C= Communal grazing, I= Individual herds), Herd size (1=

<10; 2= 10-40; 3= >70 cattle) G= Grazing, CR= Crop Residues, Crop residues (% of total diet), TD= Total Distance (km),

HeRa= Herding Radius (km), AIPS= Amount of Ingested Plant species, BCS= Body Condition Score (1-9)

- 28 -

3.2.2 Background information

From the 19 observed farmers, 8 farmers presented supplementary food to their animals. Cow4

received every day around 2 kg of corn enriched with salt before the animal was herded on grazing

pastures.

One large leaf from Ensete ventricosum (false banana) and from Musa spp. (banana) was given daily

to cow5 (Figure 10A). Ensete ventricosum is locally known as ‘kocho’ and the pseudo stems and

underground rhizomes from this plant serve as an important food source for people from the region.

One farmer supplemented his animals with a herbal mixture cut from his pea field (Figure 10B). In the

morning around 0.4 kg from this mixture was presented to each of his animals. Early in the afternoon,

the farmer brought his animals back home from the grazing pasture. In stable, approximately 5 kg

from the same mixture was again presented to each animal.

Farmers 12, 15 and 16 offered a lot of forages to their animals in the morning. These forages include

crop residues such as Saccharum officinarum (sugar cane) (Figure 10D) and Zea mays but also cut

grass from Melinis repens (Figure 10E), Pennisetum purpureum (elephant grass) (Figure 10C) and cut

branches from Premna schimperi, Rhus glutinosa and Erythrina brucei.

Figure 10: Supplementary food presented by farmers; A: Leaf from Ensete ventricosum and

Musa spp. – B: Herbal mixture from pea field – C: Pennisetum purpureum – D: Chopping

Saccharum officinarum – E: Melinis repens, cut from a private field

- 29 -

Two animals (cow16 and 17) were supplemented with a local product known as ‘bulule’. This product

consists mainly of sorghum and corn and a small amount of teff. After grinding this grain mixture,

water is added to the powder. The obtained flour is packed first in the leaves from Ensete ventricosum

after which the product undergoes fermentation during one night. Bulule was given once every week

to cow16 and 3 times a week to cow17.

Not a single cow on the PV- or NF pastures ingested soil deliberately (geophagy).

3.2.3 Statistical analysis

When herders applied communal grazing, a significant increase in total distance (4.87 km +/- 0.76)

and herding radius (0.82 km +/- 0.14) was observed, compared to individual herds (respectively 1.72

km +/- 0.38 and 0.42 km +/- 0.09). Animals fed with crop residues covered significant lower total

distance (1.32 km +/- 0.45) and herding radius (0.32 km +/- 0.09) than grazing animals (3.73 km +/-

0.62 and 0.71 km +/- 0.11).

No residues from crops were present in diets of cattle from region 1. Due to local circumstances,

animals were first observed in region 1 when local crop fields were not yet harvested. The percentage

of crop residues in diet is strongly correlated with the date of observation (R = 0,744**) using Pearson

correlations. For this reason it is more likely that the percentage of crop residues in diet depends on

the date of observation and not related to region.

BCS did not differ significantly between cows from communal or individual herds. Cow16, 17 and 19

changed management and feeding strategy few days before observation. In stead of grazing on

pastures, these cattle were fed with crop residues on the moment of observation. Consequently, the

management of these animals could not be related to their BCS. When these animals were not

included in statistical analysis, BCS tended to be lower in communal herds. The BCS from cattle that

received supplementary food by farmers did not differ significantly from cattle without

supplementation.

Statistical analysis was conducted to compare management practices between the different regions

(Table 7A) and the different soil types (Table 7B). Management of cattle grazing in Region 1 and

associated planosols/vertisols differed significantly from cattle grazing in Regions 2 and 3 and

associated nitisols/ferralsols. On PV-soils, cattle were typically held in larger herds grazing on

communal lands while cattle on NF-pastures typically grazed in individual smaller herds. The total

distance covered by cattle on PV was higher than cattle held on NF. The amount of ingested plant

species was lower in the PV-areas and higher in NF. These results indicate that PV-areas have a

poorer diversity in plant species and cattle held on NF-pastures have a more varied diet.

Table 7: the link between management practices in the different regions (A) and the different soil types (B)

A. REGION 1 REGION 2 REGION 3 SEM P C 0,67 0,33 0,00 0,11 0,037 Herd 2,17 1,17 1,00 0,18 0,007 TD 4,57 1,65 2,04 0,49 0,022 HeRa 0,77 0,40 0,48 0,09 0,217 AIPS 11,50 13,00 19,10 1.31 0,026 BCS 4,50 5,33 4,71 0.28 0,490

- 30 -

B. PV NF SEM P C 0,71 0,08 0,11 0,002 Herd 2,00 1,08 0,18 0,008 TD 4,14 1,88 0,49 0,029 HeRa 0,71 0,45 0,09 0,152 AIPS 10,71 17,17 1.31 0,009 BCS 4,86 4,83 0.28 0,972

Region (1= 1700-1800m; 2= 1800-2000m; 3= 2000-2200) PV= Planosol/Vertisol, NF= Nitisol/Ferralsol, SEM= Standard Error of

the Mean, C= Communal grazing, Herd= Herd size (1= <10; 2= 10-40; 3= >70 cattle), TD= Total distance (km), HeRa= Herding

Radius (km), AIPS= Amount of ingested plant species, BCS= Body Condition Score (1-9)

3.3 Plant composition of the diet

3.3.1 General

Estimates of the plant composition of the diet were realised by observation. In total, 87 different plant

species from 25 families were observed. Four plant species could not be identified by their scientific

name but were recognised by local farmers as lima, matane, mito and tokomo. One plant species was

not identified and was not recognised by farmers. The proportions of ingested plant species by the

observed cows are shown by Table 15 (Addendum). A 100% stacked bar chart was made on the plant

species composition of the diet for each region for better interpretation of the data (Figures 11, 12 and

13).

Figure 11: plant composition diet region 1 (1700-1800m)

- 31 -

Figure 12: plant composition diet region 2 (1800-2000m)

Figure 13: plant composition diet region 3 (2000-2200m)

- 32 -

3.3.2 Background information

3.3.2.1 Diet composition according to region

The botanical composition of diets according to region is shown by Figures 11, 12 and 13. Since

region is strongly associated to soil type, reference about botanical composition is made to 3.3.2.2

(Diets in Planosols/Vertisols) and 3.3.2.3 (Diets in Nitisols/Ferralsols).

3.3.2.2 Diets in Planosols/Vertisols

All observed cows in region 1 and cow16 in region 2 grazed on planosols/vertisols (PV), all others

were herded on nitisols/ferralsols (NF). Each grazing pasture in the PV-area was wetland except the

pasture where cow2 was herded. This pasture was situated at a higher altitude than a nearby marsh

so water in this pasture could easily be drained to the swamp. From all PV-areas, diversity of ingested

plant species was highest by the observed animal grazing on this pasture.

Remarkable was the one-sided diet in the PV areas where Cyperus- and Cynodon spp. were the most

dominant grazed vegetation. Cow16 also grazed on a planosol pasture but no ingestion of Cyperus- or

Cynodon spp. was observed. This is because this animal was observed in a later period when several

wheat fields were already harvested. In stead of grazing the Cyperus- and Cynodon dominated

grasslands, cattle feed themselves with crop residues on yielded crop fields in a later period of the dry

season. The most common ingested herbs were Centella asisetica and Trifolium spp. Only very small

bites were taken from the leaves of shrubs and trees such as Eucalyptus camaldulensis, Psidium

guajava (guava) and Sesbania sesban. Ingestion of the leaves from Eucalyptus camaldulensis was

prevented by farmers because of the economic value of Eucalyptus trees as construction material.

3.3.2.3 Diets in Nitisols/Ferralsols

Next to Cynodon spp., 18 other ingested grass species were observed in the nitisol area. Just like the

observed animals on the planosols, only small amounts were eaten from shrubs such as Maytenus

obscura, Premna schimperi, Sesbania sesban, Vernonia adoensis etc. The leaves from the trees

Erythrina brucei and Rhus glutinosa were offered in small amounts to two animals.

In the nitisol areas, the proportion of crop residues in the diet increased when animals were observed

in a later period. Most presented crop residues were stovers from Zea mays, followed by Saccharum

officinarum (sugar cane), Sorghum spp. and Pisum sativum (pea). Also crop residues from Eragrostis

tef (teff) and Linum usitatissimum (flax) were digested by 1 observed animal. Leaves from Musa spp.

(banana) and Ensete ventricosum (false banana or ‘kocho’) were digested by 2 animals.

3.3.3 Statistical analysis

An independent t-test was conducted for the comparison of diets from PV and NF. Cattle grazing on

PV had significant higher percentage of Cyperaceae in their diet (35.64 +/- 6.32) compared to diets

from NF (5.76 +/- 1.46). Diversity in plant species was significantly lower in PV (10.71 +/- 1.54) than in

NF (17.17 +/- 1.53). A significant higher amount of herb species was ingested in NF (8.42 +/- 0.94)

than in PV (4.86 +/- 0.99). These results indicate a more varied diet in the nitisol/ferralsol areas.

- 33 -

3.4 Mineral composition of plant species

3.4.1 General

In total, 87 ingested plant species from 25 families were observed. Four plant species could not be

identified by their scientific name but were recognised by local farmers as lima, matane, mito and

tokomo. One plant species was not identified and was not recognised by farmers.

To evaluate the mineral composition of the most important plant species, a total of 66 plant samples

were taken from observed regions. Mineral concentrations were determined in 52 different plant

species. From the planosols, 13 samples were collected while 53 samples were collected in nitisol

areas. More plant samples were taken in the nitisols because plant variation was higher in these

areas. Samples from Cyperus spp., Cynodon spp. and Centella asiatica were taken in both planosols

and nitisols.

Mineral concentrations in plant samples are shown in Table 16 (Addendum). Generally, Table 16

indicates mineral imbalances in plant species as a widespread phenomenon in the Gilgel Gibe

Catchment.

3.4.2 Mineral composition plants according to region

A one-way ANOVA using the Tukey post hoc test was conducted to evaluate the mineral

concentrations of plant species between the 3 regions. Plant species from region 2 had significantly

lower molybdenum (0.58 mg/kg +/- 0.12) levels and higher copper:molybdenum ratio (28.2 +/- 4.62)

than species from region 3 (respectively 1.51 mg/kg +/- 0.33 and 9.68 +/- 2.03). Zinc concentrations

were significantly higher in region 1 (50.99 mg/kg +/- 9.31) than region 2 (27.90 mg/kg +/- 2.73).

3.4.3 Mineral composition plants according to soil type

An independent t-test was used to compare mineral concentrations in plant species between PV and

NF. No significant differences were detected. However, same plant species seem to differ in mineral

concentrations according to the soil type they grow on. Samples from Cynodon spp, Cyperus spp and

Centella asiatica were collected from both PV and NF (Table 8). Remarkable were the lower copper

and higher molybdenum concentrations in Cynodon- and Cyperus spp. collected in wetland PV,

compared to same species collected in NF. Mineral concentrations of Centella asiatica were similar in

well drained PV and NF.

- 34 -

Table 8: mineral concentrations of Cynodon spp., Cyperus spp. and Centella asiatica in

Planosol/Vertisols and Nitisol/Ferralsol

SOIL WET LAND Plant Species Cu

(mg/kg) Mo

(mg/kg) Cu:Mo Fe (mg/kg)

Zn (mg/kg)

S (%)

PV 1 Cynodon spp. 4,2 1,63 2,56 911 31,5 0,121 PV 1 Cynodon spp. 5,0 1,76 2,85 260 38,4 0,215 NF 0 Cynodon spp. 7,1 0,70 10,09 2398 36,4 0,145 PV 1 Cyperus spp. 4,2 1,56 2,70 338 42,2 0,133 PV 1 Cyperus spp. 4,6 2,28 2,02 3020 19,7 0,111 NF 0 Cyperus spp. 7,8 0,67 11,55 4240 69,1 0,189 PV 0 Centella asiatica 10,1 0,76 13,36 2809 88,4 0,431 NF 0 Centella asiatica 11,1 0,67 16,44 1827 105 0,573

Severely deficient

Marginally deficient

Marginally antagonistic

Highly antagonistic

Above the MTC

PV= Planosol/Vertisol, NF= Nitisol/Ferralsol, MTC= Maximum Tolerable Concentration

3.5 Mineral intake in selected cattle

3.5.1 General

Based on the plant composition of the diet of observed animals and based on the mineral composition

of sampled plant species, estimation has been made on the average mineral intake in selected cattle

(Table 9).

Table 9: estimated mineral intake in selected cattle per kg DM

REGION CowID SOIL Wet Land

Feeding strategy

Cu (mg/kg)

Mo (mg/kg) Cu:Mo Fe

(mg/kg) Zn

(mg/kg) S

(%) 1 1 PV 1 G 4,21 1,53 2,75 749 36,39 0,13 1 2 PV 0 G 5,59 1,13 4,95 639 40,35 0,15 1 3 PV 1 G 5,34 1,43 3,74 956 47,97 0,19 1 4 PV/NF 1 G 4,18 1,36 3,07 692 37,65 0,14 1 6 PV 1 G 5,12 1,46 3,51 828 43,16 0,16 1 7 PV 1 G 4,59 1,66 2,76 1094 36,64 0,14 2 5 NF 0 G 7,98 0,88 9,03 1676 49,70 0,27 2 10 NF 0 G 7,85 1,19 6,61 2084 49,58 0,25 2 11 NF 0 G/CR 6,49 0,54 12,04 1621 39,83 0,20 2 12 NF 0 CR 5,05 0,51 9,99 1577 49,29 0,15 2 16 PV 1 CR 4,10 1,71 2,39 400 32,29 0,14 2 17 NF 0 CR 4,52 0,62 7,33 682 26,43 0,13 3 8 NF 0 G 7,56 1,25 6,02 3855 51,12 0,21 3 9 NF 0 G 7,03 1,96 3,58 2300 42,68 0,18 3 14 NF 0 G/CR 7,41 0,73 10,20 1551 41,82 0,18 3 15 NF 0 G/CR 6,15 0,89 6,92 1212 33,31 0,13 3 18 NF 0 G/CR 4,99 0,61 8,12 1555 33,41 0,14 3 19 NF 0 CR 4,69 0,97 4,83 1000 37,69 0,15 3 13 NF 0 G 7,38 1,39 5,33 955 41,95 0,24

Severely deficient

Marginally deficient

Marginally antagonistic

Highly antagonistic

Above the MTC

Region (1= 1700-1800m; 1800-2000m; 2000-2200m), PV= Planosol/vertisol, NF= Nitisol/ferralsol, G= Grazing, CR= Crop Residue, MTC=

Maximum Tolerable Concentration

- 35 -

Table 9 indicates marginal intake of copper in all animals, ranging from 4.10 to 7.98 mg/kg per kg DM.

The amount of zinc was sufficient in all animals except one marginal shortage in cow17. Iron particles

were present in antagonistic concentrations in all diets. From the diets of 19 cattle, 11 had iron levels

even above the MTC. Molybdenum was present in marginal antagonistic concentrations in all diets

from cattle grazing on planosols/vertisols and in 4 of 12 cattle grazing on nitisols/ferralsols. Marginal

shortage of copper and marginal antagonistic concentrations of molybdenum resulted in a severe

disbalanced copper:molybdenum ratio in all diets of cattle grazing on planosols/vertisols, except cow2.

Cow9 is the only animal on nitisols/ferralsols with disbalanced copper:molybdenum ratio. Sulfur levels

were adequate in region 1 and in planosols/vertisols but marginally antagonistic in 5 cattle grazing on

nitisols/ferralsols.

3.5.2 Statistical analysis

3.5.2.1 Mineral intake in cattle according to region

A one-way ANOVA using the Tukey post hoc test was conducted to evaluate the mineral

concentrations in total diets between the 3 regions. The copper:molybdenum ratio of the total diet was

significantly lower in region 1 (3.46 +/- 0.34) than region 2 (7.89 +/- 1.36).

3.5.2.2 Mineral intake in cattle according to soil type

An independent t-test was conducted to compare mineral levels in total diet from cattle herded on PV

with cattle herded on NF. Mineral concentrations of total diet seem to be more associated with soil

type than with region. Statistical analysis indicates mineral intake by cattle is strongly influenced by

type of soil (Table 10). Cattle grazing on PV ingested significantly lower amounts of copper than cattle

herded on NF. On the other hand, molybdenum was significantly higher in the PV-group. Iron particles

were significantly higher in NF and lower in PV. No significant influence from soil type was present on

the amounts of zinc and sulfur in estimated diets, although there was a trend for sulfur.

Table 10: link between mineral concentrations in diet and soil type

PV NF SEM P Cu 4,73 6,43 0,31 0,005 Mo 1,47 0,96 0,1 0,003

Cu:Mo 3,31 7,5 0,66 0 Fe 765 1672 183 0,003 Zn 39,21 41,4 1,57 0,47 S 0,15 0,19 0,01 0,082

PV= Planosol/Vertisol, NF= Nitisol/Ferralsol, SEM= Standard Error of the Mean

3.5.2.3 Mineral intake in cattle according to feeding strategy

Pearson correlations were calculated to evaluate the impact of the proportion of ingested crop

residues, poaceae and herbs on mineral concentrations in total diet (Table 11).

- 36 -

Stovers from Saccharum officinarum, Sorghum spp., Zea Mays and Pisum sativum, leaves from Musa

spp. and Ensete ventricosum and residues from Eragrostis tef and Linum usitatissimum were

considered as crop residues. Higher percentages of crop residues in diet were significantly correlated

with lower amounts of zinc in total diet. Also total molybdenum and sulfur tends to be lower when

higher amounts of crop residues were included in diet.

Higher proportions of poaceae in diet are associated with lower molybdenum and zinc levels and a

more balanced copper:molybdenum ratio in total diet while higher percentages of herbs are

significantly correlated with higher molybdenum and zinc levels and a lower copper:molybdenum ratio.

Table 11: correlation between total mineral concentrations and % of crop residues, poaceae

and herbs in diet

Cu Mo Cu:Mo Fe Zn S Crop

Residues (%) Pearson

Correlation -0,389 -0,433 0,278 -0,248 -0,49 -0,435

P 0,1 0,064 0,249 0,306 0,033 0,063

Poaceae (%) Pearson Correlation -0,093 -,557 ,520 0,042 -,533 -0,22

P 0,704 0,013 0,023 0,863 0,019 0,365

Herbs (%) Pearson Correlation 0,148 ,571 -,512 0,011 ,461 0,234

P 0,546 0,011 0,025 0,965 0,047 0,335

3.6 Mineral concentrations in plasma of selected cattle

3.6.1 General

Plasma concentrations of copper, iron, zinc and manganese were measured in all observed animals

(Table 12). Ten observed cows had a severe shortage whereby cow19 was almost completely

depleted in copper (0.07 mg/l). A marginal deficiency was found in 6 animals. Only 3 animals had

adequate copper amounts in plasma. Even though cow12 had typical symptoms of copper deficiency

such as depigmentation around the eyes and back legs, adequate copper levels were found in this

animal. This may be caused by infection, in this case trypanosomiasis. For statistical analysis, plasma

copper concentrations were recoded in 3 groups: severely deficient (0), marginally deficient (1) and

adequate (2).

Iron concentrations were in the normal range for all animals except cow17 (4.09 mg/l). Marginal

deficient concentrations in zinc were measured in 7 cows. Normal concentrations of manganese were

measured in all animals.

Because infection can influence copper values in plasma, attention was given on the presence of

clinical signs of disease. Cows1 and 11 suffered from severe diarrhoea. Because dysuria and

- 37 -

stranguria were noticed in cow6, this animal was suspected of urinal infection. Trypanosomis was

diagnosed by microscope in cows5, 12 and 18. No Babesia was found in blood smears.

Table 12: mineral concentrations in plasma of selected animals

REGION CowID SOIL Wet Land

Cu (mg/l)

Cu (code)

Fe (mg/l)

Zn (mg/l)

Mn (mg/l) Extra

1 1 PV 1 0,41 0 2,28 0,75 0,047 Diarrhoea 1 2 PV 0 0,70 2 2,98 0,90 0,052 - 1 3 PV 1 0,32 0 3,21 0,88 0,035 - 1 4 PV 1 0,28 0 2,07 0,74 0,047 - 1 6 PV/NF 1 0,66 1 1,63 0,71 0,045 Urinal infection 1 7 PV 1 0,31 0 2,33 1,39 0,099 - 2 5 NF 0 0,53 1 2,32 0,98 0,051 Tryps 2 10 NF 0 0,66 1 2,15 0,71 0,037 - 2 11 NF 0 0,32 0 1,69 0,76 0,017 Diarrhoea 2 12 NF 0 0,73 2 1,80 0,78 0,018 Tryps 2 16* PV 1 0,45 0 1,78 0,96 0,035 - 2 17* NF 0 0,77 2 4,09 0,87 0,062 - 3 8 NF 0 0,33 0 3,49 0,78 0,030 - 3 9 NF 0 0,36 0 2,42 0,90 0,033 - 3 14 NF 0 0,61 1 1,98 1,15 0,037 - 3 15 NF 0 0,64 1 2,54 1,06 0,037 - 3 18 NF 0 0,56 1 2,29 1,12 0,043 Tryps 3 19* NF 0 0,07 0 2,49 1,01 0,045 - 3 13 NF 0 0,39 0 2,31 0,89 0,041 -

Region (1= 1700-1800m; 2=1800-2000m; 3=2000-2200m), *= changed feeding strategy few days before observation, PV=

Planosol/Vertisol, NF= Nitisol/Ferralsol, Tryps= trypanosomiasis

3.6.2 Statistical analysis

3.6.2.1 Influence of management on mineral concentrations in plasma

An independent t-test was conducted to evaluate the impact of communal grazing and herd size on

mineral concentrations in plasma. No significant differences were calculated.

Pearson correlations were calculated to evaluate the impact of crop residues in diet on mineral

concentrations in plasma. The percentage of crop residues in diet was significantly correlated with the

plasma copper concentration while no correlations were determined with plasma iron, zinc or

manganese (Table 13). Proportions of poaceae and herbs in total diet were not correlated with mineral

concentrations in plasma.

Table 13: correlation % crop residues in diet and mineral concentrations in plasma

Cu Fe Zn Mn Pearson

Correlation ,517 -0,343 0,214 -0,382

P 0,04 0,194 0,427 0,145

Severely deficient

Marginally deficient High

- 38 -

A one-way ANOVA using the Tukey post hoc test and an independent t-test was conducted to detect

significant differences in plasma mineral concentrations between regions and different soil types. No

significant associations were calculated even when infected animals were excluded from statistical

analysis.

To compare mineral status of cattle grazing on wetlands with animals grazing on well drained lands,

an independent t-test was conducted. The recoded copper concentration in plasma was significantly

lower in animals grazing on wetlands (0.17 +/- 0.17) than cattle grazing on well drained lands (0.85 +/-

0.22).

3.6.2.2 Correlation between mineral content in diet and plasma

Pearson correlations between mineral content in diet and plasma were calculated. A strong trend was

detected between plasma copper values and molybdenum concentrations in diet (R = -0.472 and P =

0.065) (Figure 14). Correlations between other mineral concentrations in diet and plasma copper

levels were not significant. These results indicate molybdenum as an important factor influencing

copper plasma concentrations.

Figure 14: Pearson correlation between plasma copper levels (mg/l) and molybdenum concentrations in diet (mg/kg)

To evaluate the mutual effect of Cu, Mo, Fe, Zn and S in the diet on plasma copper, the data from

mineral concentrations in diet were recoded. The more a certain mineral contributes to the

development of copper deficiency, the more points were given to the diet (Table 14). Copper values in

diets were all marginally deficient and vary from 4.18 to 7.98 mg/kg. Because variation in copper

concentration was wide, values were divided in 2 groups. Diets between 4 and 6 mg/kg copper

received two points; diets containing more than 6 mg/kg received one point. Respectively 1, 2 and 3

points were added when molybdenum, iron or sulfur were present in marginal- and severe

antagonistic concentrations and levels above the MTC. When the copper:molybdenum ratio was

marginally or severely disbalanced, respectively 1 and 2 points were granted. Concentrations of zinc

were not included in the mutual effect analysis because all estimated zinc concentrations in diet were

not in the antagonistic range.

- 39 -

Table 14: recoded mineral concentrations in diet

Severely deficient

Marginally deficient

Marginally antagonistic

Highly antagonistic

Above the MTC

*= changed feeding strategy recently and was not included in statistical analysis when plasma minerals were evaluated

Statistical analysis showed no significant correlation between the total points of the diets and copper

concentrations in plasma. Cow 16, 17 and 19 changed feeding strategy from grazing to feeding with

crop residues few days before observation. When these animals were not included in statistical

analysis, significant correlation was present (R = -0.542* and R = 0.030). These results indicate a

mutual effect of copper shortage and molybdenum, iron and sulfur overload to the development of

copper deficiency in the region.

Diet scores were significantly higher in the PV group (6.714 +/- 0.360) compared to NF (5.083 +/-

0.260). Diets in wetlands had a higher score (7.000 +/- 0.258) than diets in well drained soils (5.080

+/- 0.239). Pearson correlations indicated higher scores were correlated with higher percentages of

cyperaceae in diet (R= 0.750, P= 0.000).

CowID Cu Mo Cu:Mo Fe Zn S Total 1 2 1 2 2 0 0 7 2 2 1 0 2 0 0 5 3 2 1 2 2 0 0 7 4 2 1 2 2 0 0 7 6 2 1 2 2 0 0 7 7 2 1 2 3 0 0 8 5 1 0 0 3 0 1 5 10 1 1 0 3 0 1 6 11 1 0 0 3 0 1 5 12 2 0 0 3 0 0 5 16* 2 1 2 1 0 0 6 17* 2 0 0 2 0 0 4 8 1 1 0 3 0 1 6 9 1 1 2 3 0 0 7 14 1 0 0 3 0 0 4 15 1 0 0 3 0 0 4 18 2 0 0 3 0 0 5 19* 2 0 0 3 0 0 5 13 1 1 0 2 0 1 5

- 40 -

PART 4

- Discussion -

- 41 -

4.1 Prevalence of copper deficiency

From the 53 liver samples collected at Jimma municipal abattoir, 81% had deficient copper levels,

from which 58% were in the clinically deficient range. The sample size of animals might be too small to

calculate a correct prevalence of copper deficiency in the region but these results indicate copper

shortage in cattle as a severe problem. It is very likely that copper deficiency leads to a severe loss of

production in the Gilgel Gibe Catchment.

During a survey conducted by Faye et al. (1986), copper status of cattle was evaluated in 89 locations,

spread over all Ethiopia. All severe copper deficiencies were only situated in the Rift Valley while

marginal copper deficiencies were present over the whole country. Our results show severe copper

deficiency in 47% of sampled cattle in Jimma, located outside the Rift.

Remarkable is that high iron levels in liver only occurred when copper values were deficient. Higher

amounts of copper were typically accompanied by lower iron values. Several articles are available

reporting same results (Elvehjem and Sherman, 1932; Marston et al., 1938; Dent et al., 1956). Dietary

deficiency of copper or iron leads to hypochromic anaemia (Lahey et al., 1952) and increased

concentrations of the other mineral in liver. Several theories about this mechanism are suggested. The

most popular theory explaining these results suggests that iron release is dependent on the function of

ceruloplasmin. This plasma cupro-protein is responsible for the enzymatic oxidation of Fe2+ (Osaki et

al., 1966). Some authors however do not support this theory that plasma iron is decreased in copper

deficient animals. Mice lacking ceruloplasmin were not usually anaemic and even though increased

hepatic iron levels were observed, plasma iron was in the normal range (Harris et al., 1999).

Our results show an association between deficient copper levels and higher iron levels in liver and

vice versa. Even though copper shortage leads to a decreased iron release from the liver, our results

and observations by Dermauw et al. (2009) indicates iron levels in plasma are not deficient in the

region.

4.2 Management Practices

Management practices differed significantly according to region and associated soil type. The study

area was divided in the planosol/vertisol group (PV) and the nitisol/ferralsol group (NF). PV areas were

found in lower altitudes and NF in higher regions. In wetland PV, cattle were typically held in large,

communal herds while cattle in NF grazed in smaller, individual herds. Because of the larger herds in

PV, cattle covered more distance compared to herds in NF. Cattle grazing in PV areas had a more

one-sided diet while cattle grazing on NF ingested a higher diversity in plant species.

BCS did not differ significantly between cattle herded on different soil types but tended to be lower in

communal herds. It is likely that the lower BCS is the result from higher competition between animals

in communal, large herds than in individual herds. The practice of communal- or individual herding or

- 42 -

herd size did not influence copper status. Also total covered distance and herding radius were not

correlated with copper plasma levels.

As the season progresses, higher proportions of crop residues were present in the diet. Observed

crop residues were stovers from Saccharum officinarum, Sorghum spp., Zea Mays and Pisum

sativum, leaves from Musa spp. and Ensete ventricosum and residues from Eragrostis tef and Linum

usitatissimum. Higher concentrations of crop residues in diet correlated with significant lower zinc

amounts in total diet. Also molybdenum and sulfur tended to be low when higher proportions of crop

residues were present. Higher proportions of crop residues were associated with a better copper

status. This association is probably biased since PV pastures were observed first when crop fields

were not yet harvested and thus no crop residues were present in diet of the PV group. The total

proportions of poaceae and herbs in total diet did not influence copper status.

4.3 The copper cycle in the soil-plant-cattle system

4.3.1 Summary scheme results

Figure 15: the copper cycle in the soil-plant-cattle system in the Gilgel Gibe Catchment,

Ethiopia

AIPS= Amount of Ingested Plant Species

- 43 -

4.3.2 The influence of soil-type on mineral concentrations in plants

Statistical analysis of all collected plant samples showed no significant differences between mineral

content of samples collected in PV and NF. The absence of significant differences might be the result

from unequal samples taken in PV (13) and NF (53). Also no distinction was made between samples

taken from grass-, herb-, shrub- and tree species. For these reasons, statistical analysis was not

suspected to show significant differences in trace elements.

Cynodon spp, Cyperus spp and Centella asiatica were the most dominant plant species in PV

pastures. Samples from these species were collected from both PV and NF (Table 8). Remarkable

were the lower copper- and higher molybdenum concentrations in Cynodon- and Cyperus spp.

collected in wetland PV, compared to same species collected in NF. Mineral concentrations of

Centella asiatica were similar in well drained PV and NF. These results indicate that plant species in

wetland PV might have lower copper and higher molybdenum levels than well drained soils.

The importance of mycorrhizal fungi associated with roots of plants (Taiz and Zeiger, 2002) is reduced

in organic rich wetlands. Because these fungi play a role in transporting minerals such as copper, iron

and zinc (Banfield et al., 1999) to the roots of the plants, copper is less mobile in wetlands

(Ragnarsdottir and Charlet, 2002).

Several articles confirm the increased availability of molybdenum in poorly drained soils and higher

molybdenum concentration in plants growing on these soils (Davies, 1956; Kubota et al., 1961). Peat

develops essentially under wet anaerobic conditions and has been associated with molybdenum

toxicity (Kubota, 1972).

Estimated mineral concentrations were calculated in total diet. Cattle grazing on PV ingested

significantly lower amounts of copper and higher amounts of molybdenum than cattle grazing on NF.

The presence of iron particles in plants was high in both soil types but was significantly higher in the

NF areas. Sulfur levels were adequate in all PV diets but marginal antagonistic sulfur concentrations

were found in some NF diets. Zinc concentrations did not differ significantly according to soil type.

The copper:molybdenum ratio in diet was severely out of proportion for all cattle grazing in the wetland

PV pastures. Remarkable was that the diet from cow2 who grazed on a well drained PV had an

adequate ratio. Except for one cow, all cattle in the NF areas had adequate ratios in diet. Again, these

results indicate cattle grazing in the wetland PV pastures ingest lower amounts of copper and higher

amounts of molybdenum.

Acid soils that contain high amounts of free iron oxides can remove the largest quantities of available

molybdenum from aqueous solutions (Jones, 1957; Karimian and Cox, 1978). Nitisols are well drained

soils that are rich in iron and have relative low pH (4.2-6.4) (Maskall and Thornton, 1996). This might

be an explanation for the significantly lower molybdenum content in total diet from cattle grazing in NF.

Our results indicate cattle herded on wetland PV are more likely to develop severe copper deficiency

than cattle held on NF pastures because shortage of copper and excess of molybdenum is more

distinct in wetland PV.

- 44 -

4.3.3 Mineral content of forages in the Gilgel Gibe Catchment

Of the 66 plant samples, only 17 samples had adequate copper levels. The grass family or the

Poaceae were the main component of the diet for most animals. All 24 samples from the Poaceae

were deficient for copper, 9 samples were even severely deficient. The 3 samples from the

Cyperaceae, the most common herbs in the planosols, were also deficient in copper.

Most samples taken from crop residues had a severe shortage in copper. All supplementary forages

presented by farmers were deficient in copper. Next to Mito (0.8 mg/kg), Saccharum officinarum and

bulule even had the lowest concentrations of all samples, respectively 1.7 and 1.8 mg/kg.

Highest concentrations of copper were measured in Vernonia adoensis (26.5 mg/kg), a not

determined species from Aspilia (22.6 mg/kg), Sida rhombifolia (19.5 mg/kg), Psidium guajava (19.3

mg/kg) and Ipomoea batatas (17.4 mg/kg). Because concentrations of antagonistic minerals were

quite favourable in Psidium guajava (Figure 16A) and Vernonia adoensis (Figure 16B), these plant

species might be good candidates to supplement cattle in the region. P. guajava produces the guava

fruit and is a common food source in the region. Analysis of copper content in guava fruit showed

relative low concentrations (4.61 mg/kg +/- 0.26) (Ang and Ng, 2000). The tannin-rich leaves from P.

guajava are proven to have antidiarrheic, antispasmodic, antibacterial and antifungal effects

(Sudarisman et al., 1988; Dalimartha, 2001). Possibly, tannins present in V. adoensis and P. guajava

may depress copper availability. Further research is needed to evaluate the potential of these plants in

preventing or overcoming copper deficiency.

Figure 16: candidates for copper supplementation in the Gilgel Gibe Catchment – A: Psidium

guajava (19.3 mg/kg Cu) with fruit (Forum Natura Italiana, 2011a) – B: Vernonia adoensis (26.5

mg/kg Cu) (Forum Natura Italiana, 2011b)

Iron was present in high concentrations in most samples. Concentrations far above the MTC (1000

mg/kg) were found in 19 samples with highest levels found in Persicaria nepalensis (8427 mg/kg). Of

other samples, 21 had marginal and 17 severe antagonistic levels of iron. These results were

expected since iron particles are abundant in African soils (Faye et al., 1991). The high

concentrations of iron are most likely suspected of having antagonistic interactions with absorption of

copper.

- 45 -

Marginal high concentrations of molybdenum were found in 21 samples and 3 samples had high

antagonistic levels. Remarkable were the high concentrations of molybdenum in the leaf of Ensete

ventricosum (16.60 mg/kg) and the herbal mix supplement (6.71 mg/kg). E. ventricosum (Figure 17A)

can grow up to 12 meters and 1 meter in diameter (Birmeta et al., 2002). Local farmers grow the plant

as a crop (Figure 17B) and the starchy pulp in its stem and corm serves as human staple food and is

locally known as ‘kocho’. Nurfeta et al. (2009) determined severe deficient copper amounts (2.2

mg/kg) in the leaves from E. ventricosum. Another study showed copper levels varying between 6.7

and 15.7 mg/kg (Nurfeta et al., 2008). The toxic values of molybdenum in the leaves from E.

ventricosum may form a major risk for developing severe copper deficiency in cattle.

Figure 17: A: Cow with diarrhoea and depigmentations all over the body ingesting leaves from

E. ventricosum - B: Plantation of E. ventricosum (UNCCD News, 2010)

Sulfur concentrations seem to be very variable in collected samples. Seven samples had shortage in

sulfur from which 6 samples were taken from the Poaceae. Marginal – and severe antagonistic

concentrations were shown in respectively 23 and 5 samples. Two samples from Centella asiatica,

collected at 2 different locations on different soil types had one of the highest sulfur concentrations in

all plants (0.573% and 0.431%). Other samples with sulfur levels above the MTC were Sida

tenuicorpa (0.401%) and Physalis peruviana (0.486%).

Zinc was deficient in 29 samples, from which 14 severely deficient. Generally, selenium tends to be

very low in collected plant species. Only 10 samples were in the adequate range, all others were in the

deficient range. Most samples were adequate in cobalt and manganese. Remarkable were the high

concentrations of manganese in Persea spp. (1662 mg/kg) and Sesbania sesban (1159 mg/kg).

- 46 -

4.3.4 The influence of diet on mineral status of cattle

4.3.4.1 Botanical composition

A higher amount of crop residues was correlated with higher copper status in cattle. This association

is probably not reliable since PV pastures were observed first when crop fields were not yet harvested

and thus no crop residues were present in diet of the PV group.

4.3.4.2 Mineral composition

All diets from observed cows had marginal deficient copper levels ranging from 4.10 to 7.98 mg/kg per

kg DM. African soils are abundant with iron particles in soil (Faye et al., 1991) and plant samples in

the Gilgel Gibe Catchment also had high iron concentrations. Molybdenum had antagonistic

concentrations in all PV- and some NF-pastures. Sulfur concentrations were in the marginal

antagonistic range in some NF diets while zinc was in the adequate range. Zinc was marginally

deficient in only 1 diet.

To evaluate the mutual effect of Cu, Mo, Fe, Zn and S in the diet on plasma copper, the data from

mineral concentrations in diet were recoded. The more a certain mineral contributes to the

development of copper deficiency, the more points were given to the diet (Table 14). Higher points

were associated with a lower copper status. These results indicate the mutual effect of copper,

molybdenum, iron and sulfur on copper status. Zinc was not included in the mutual effect analysis

because no antagonistic concentrations were present in total diet.

A strong trend was detected between plasma copper values and molybdenum concentrations in diet.

Correlations between other mineral concentrations in diet and plasma copper levels were not

significant. These results indicate molybdenum as an important factor influencing copper plasma

concentrations.

Ward and Spears (1997) compared the long-term effects of supplemental molybdenum with a low

copper diet on copper status. The group of steers with supplemental molybdenum had decreased

plasma copper levels. Diets low in copper that were supplemented with 5 mg/kg molybdenum per kg

DM resulted in depressed growth in heifers (Phillipo et al., 1987; Gengelbach et al, 1994). The

depressed growth was attributed to the effect of molybdenum because a control group that was

supplemented with iron did not alter growth even though copper plasma levels were depressed.

All diets had marginal copper- and high iron levels but according to our results, it seems that cattle

ingesting marginally antagonistic concentrations of molybdenum are more likely to develop severe

copper deficiency and presumably show a decreased growth.

- 47 -

4.3.5 The influence of soil-type on mineral concentrations in cattle

Diets from PV contained significant lower copper- and iron- and higher molybdenum particles than

diets from NF. At the same time higher molybdenum concentrations in diet were correlated with

decreased copper plasma levels. Consequently, statistical analysis occurred to evaluate the effect of

soil type on plasma copper concentrations. No significant differences were calculated between cattle

grazing on PV and NF.

PV pastures become wetlands in the wet season because stagnation of rain water. Because these

wetlands are not suitable to grow crops, local farmers use these areas to herd their cattle. These

wetlands are typically used as communal grazing areas where farmers from the environment leave

their cattle in the morning and return in the evening. Consequently, a large herd is formed every day

whereby the wetlands are typically overgrazed. All observed PV pastures were wetlands except

pasture2. This area was situated at some higher altitude and was well drained by a near swamp.

Statistical analysis showed significant lower plasma copper levels (recoded) in cattle grazing on these

wetlands (M= 0.17 +/- 0.17) compared to cattle herded on well drained lands (M= 0.85 +/- 0.22). Used

codes for plasma copper concentration were 2 (adequate), 1 (marginal deficient) and 0 (severely

deficient). Remarkable was the adequate copper status of cow2 that grazed on well drained PV area.

Analysis indicates cattle herded in wetlands are more likely to develop severe copper deficiency.

These results can be explained by the higher availability of molybdenum in wetlands (Davies, 1956;

Kubota et al., 1961, Mitchell, 1974). This increased availability corresponds with the observed higher

molybdenum levels in plants from PV pastures and associated wetlands. Also there was a strong

trend to lower copper plasma concentrations with higher molybdenum concentrations in diet.

The statement that cattle grazing in wetlands have more decreased copper levels also corresponds

with the observations by Dermauw et al. (2009). During this research blood samples were taken in the

Gilgel Gibe Catchment from 90 cattle from 3 regions, based on altitude. Cattle from low areas had

significantly lower copper plasma levels than cattle herded in medium and high altitude. Since PV

pastures and associated wetlands are situated in the lower parts of the valley, both studies seem

compatible. There were however significant differences in copper status between cattle grazing in

same regions. Not all PV pastures are wetlands so this might explain the differences in copper status

for the lower region.

Several report cases about copper deficiency in wetlands are available. A particular copper deficiency

syndrome, formerly called “peat scours”, was reported by Cunningham (1944). Most characteristic

symptoms of the acute form affecting dairy cattle were loss of condition, severe diarrhoea and

scouring. The symptoms occurred in cattle grazing on peat land whenever there was a flush growth of

pasture. A decreased copper status was determined in these cattle. Later, Cunningham (1950)

reported that the presence of peat scours was attributed to low copper- and higher molybdenum

concentrations in forages. Allcroft (1947), Allcroft and Parker (1949) and Henderson (1957) reported

similar cases of peat scours. The long standing use of syndromes called “peat scours” or “scouring

- 48 -

disease” to describe molybdenum induced copper deficiency, indicates that the problem was

frequently observed on wet soils.

Similar syndrome of copper deficiency on wet soils, known as “polder disease”, was described in the

Netherlands (Sjollema, 1938; Brouwer et al., 1938). High molybdenum levels were detected in the

wetland areas of the Florida Everglades (Kretschmer and Beardsley, 1956).

In the NF group, cattle were found with varying copper status from almost depleted to adequate. This

variation might be explained by the varying amounts of molybdenum in diets from NF-pastures. Local

enrichments or depletions of molybdenum can be present in parent material (Gupta, 1997). The

presence of intestinal parasites or differences in tannin content in diet may be other causes for the

varying copper status in NF areas.

4.4 Suggestions to improve copper status

Our results indicate molybdenum as an important factor influencing copper status in cattle in the Gilgel

Gibe Catchment. Molybdenum is present in higher concentrations in diets from cattle grazing on

wetlands leading to more severe shortage of copper. Because there are too many cattle in the region

and crops are usually grown on better drained areas, avoiding these wetlands is not an option for local

farmers. Since copper levels decline drastically in late pregnancy because of drainage of copper by

the foetal liver (Xin et al., 1993), it might be a good idea for pregnant cows to avoid the wetlands.

Supplementing cattle with copper sulphate or spraying copper sulphate on pastures or crops might not

be realistic due to the severe poverty of farmers in the region.

Hay-making can reduce molybdenum toxicity drastically. It was suggested that protein content and

solubility decreases when forages are dried. Consequently, less sulphide is formed in the rumen which

reduces availability of copper. When forages were harvested, the problems in “swayback” areas in the

British Isles, North Holland and South Florida disappeared (Kubota, 1975; Ward, 1978). The

harvesting of hay may be an alternative practice to utilize forages from the molybdenum rich

vegetation in the wetlands. The problem with these areas is that these lands are typically used as

communal grazing areas. Too many cattle graze these lands whereby severe overgrazing is a

common problem. The production of hay might be difficult on these overgrazed wetlands.

Because availability of molybdenum increases in wet soils, drainage of wetlands might decrease

molybdenum levels. Cow2 was the only one grazing on a well drained PV. Remarkable hereby was

the adequate copper level in plasma as the only cow grazing on PV. Because fasciolosis is also a

common problem in the region, drainage of the wetlands is likely to improve the health of livestock.

Drainage is however expensive and hard to realise in practice.

Mineral analysis of collected plant samples showed favourable concentrations in Psidium guajava and

Vernonia adoensis. Feeding these plants to cattle might improve their copper status. Ensete

ventricosum had toxic levels of molybdenum and feeding this plant is likely to have a negative

influence on cattle when given in high proportions. Further research is needed to evaluate the impact

of feeding these plants.

- 49 -

PART 5

-Addendum and References-

- 50 -

5.1 Addendum

Table 15: proportions ingested plant species by observed cows

REGION REGION 1 (1700-1800) REGION 2 (1800-2000) REGION 3 (2000-2200)

COW 1 2 3 4 6 7 5 10 11 12 16 17 8 9 14 15 18 19 13

Acanthaceae 1,2 0,5 6,0 0,7 2,6

Acanthacea spp. 2,6 Hygrophila auriculata 1,2 0,5 6,0 0,7

Anacardiaceae 3,8

Rhus glutinosa 3,8

Asteraceae 5,4 2,2 1,7 11,0 7,0 5,1 9,3 10,4 0,9 3,8 7,8 11,0 12,1 17,3 Ageratum conyzoides 1,5

Aspilia africana 3,4 0,9 2,6 Aspilia mossambicensis 4,7 3,8

Aspilia spp. 1,3 2,1 3,1 Bidens macroptera 0,7 3,9 4,2 2,6 7,6 12,2

Bidens pilosa 4,9 Bidens presentinatia 3,0

Bidens spp. 0,8 2,2 1,4 0,9 5,1 9,3 2,1 5,7 5,7 2,0 Solanecio gigas 1,7 Solanecio spp. 0,7 0,9

Vernonia adoensis 4,2

Brassicaceae 4,5 1,0 Brassica spp. 4,5 1,0

Celastraceae 1,1 2,7

Maytenus obscura 1,1 2,7

Commelinaceae 0,9 8,7 2,5 0,8 1,0 Commelina spp. 0,9 8,7 2,5 0,8 1,0

Convolvulaceae 0,9

Ipomoea batatas 0,9

Cyperaceae 50,7 31,4 42,5 39,7 43,2 41,9 3,0 7,8 7,0 2,2 17,7 11,1 7,1 6,5 4,6 1,0 1,0 Carex spp. 10,4 2,3

Cyperus (giant) 11,8 Cyperus alternifolius 8,3

Cyperus spp. 32,0 29,1 42,5 39,7 43,2 30,1 3,0 7,8 7,0 2,2 17,7 11,1 7,1 6,5 4,6 1,0 1,0

Euphorbiaceae 1,6 Phyllanthus sepialis 1,6

Lamiaceae 2,3 0,7 5,6 8,2 1,3 3,0 12,4

Plectranthus punctatus 2,1 1,3 Salvia leucantha 2,2

Satureja paradoxa 2,3 0,7 Satureja spp. 5,6 4,0 3,0 12,4

Leguminosae 1,4 8,7 9,2 0,8 1,9 3,4 9,3 1,3 1,3 11,1 10,3 19,1 18,8 3,1 14,2 1,2

Desmodium intortum 5,2 Desmodium repandum 0,7 4,7 Desmodium uncinatum 1,0

Erythrina brucei 3,1 Indigofera spicata 1,3 4,3

Pisum sativum 10,9 Senna spp. 2,3

Sesbania sesban 0,8 11,1 Trifolium spp. 1,4 6,4 9,2 0,8 1,2 3,4 8,6 1,3 10,3 13,8 3,1 14,2 1,2

Linaceae 3,9

Linum usitatissimum 3,9

Loganiaceae 8,0 Buddleja polystachya 8,0

Mackinlayaceae 3,1 14,6 3,4 5,6 3,2 24,8 15,2 7,1 3,8 7,7 0,8 9,6 1,8 1,5 5,1

Centella asiatica 3,1 14,6 3,4 5,6 3,2 24,8 15,2 7,1 3,8 7,7 0,8 9,6 1,8 1,5 5,1

- 51 -

REGION REGION 1 (1700-1800) REGION 2 (1800-2000) REGION 3 (2000-2200)

COW 1 2 3 4 6 7 5 10 11 12 16 17 8 9 14 15 18 19 13

Malvaceae 6,1 0,8 4,5 5,4 7,0 0,5 7,5 1,6 3,0 Grewia spp. 1,6

Sida rhombifolia 0,5 7,5 Sida tenuicorpa 6,1 0,8 7,0 3,0

Musaceae 2,2 2,7 Musa spp. 1,1

Ensete ventricosum 1,1 2,7

Myrtaceae 2,3 2,6 Eucalyptus camaldulensis 2,6

Psidium guajava 2,3

Oxalidaceae 2,0 Oxalis radicosa 2,0

Phytolaccaceae 2,0

Phytolocca spp. 2,0

Plantaginaceae 0,5 0,9 Plantago lanceolata 0,5 0,9

Poaceae 47,9 40,3 24,8 53,4 42,4 50,0 54,5 48,4 76,5 60,2 74,1 83,3 59,4 45,6 64,6 63,0 69,5 55,0 50,0

Andropogon gajanus 1,3 Andropogon spp. 7,0 0,7 1,3 4,2 2,5 1,7 Aristida adoensis 3,2

Botriochloa insculpta 8,8 2,1 3,1 5,3 2,5 Bracharia spp. 2,3 12,3 4,8 26,1 0,8 1,3 Cynodon spp. 30,5 6,8 21,4 39,7 28,1 29,2 35,2 37,7 20,6 4,5 11,8 29,3 18,3 18,5 10,7 3,5 7,3 Digitaria spp. 4,2 2,2 9,4 1,3 3,8 2,1 5,5 6,6

Echinochloa colona 0,8 Eleusine floccifolia 2,0 7,8 8,8 5,5 3,0

Eragrostis paniciformis 0,6 1,0 Eragrostis spp. 0,7 1,0 Eragrostis tef 0,8 7,9

Hyparrhenia hirta 11,4 Lolium temulentum 2,0

Melinis repens 7,4 6,1 Melinis spp. 25,0 7,8 1,3

Panicium coloratum 1,3 Pennisetum clandestinum 2,7 1,3 0,9 13,2 7,7 0,9 1,0 12,9 Pennisetum purpureum 12,5 7,8

Pennisetum sphacelatum 8,3 2,0 8,9 0,7 19,3 2,6 5,3 4,7 Pennisetum thunbergii 2,1 2,3 0,9 2,3 1,0 5,7 1,0 3,1 4,4 1,3 8,1 3,9 12,2

Saccharum officinarum (S) 5,3 12,5 Sacciolepsis africana 0,9 1,0 Sorghum bicolour (S) 2,6

Sorghum halepense (S) 57,4 Sporobolus spp. 4,2 5,8 2,6

Zea Mays (S) 2,3 5,3 50,0 9,3 52,1 15,0 10,9 27,6 27,3 1,0 Zea Mays (F) 10,3 5,3 4,2 7,6

Polygonaceae 1,4 2,1 3,1 4,2 5,5 6,1

Persicaria nepalensis 3,1 4,2 Persicaria spp. 1,4 2,1 3,0 2,0

Rumex nepalensis 2,5 4,1

Solanaceae 4,2 Physalis peruviana 4,2

Verbenaceae 3.8

Premna schimperi 3,8

Zygophyllaceae 0,7

Tribulus terrestris 0,7

Unknown 0,7 4,5 3,8 0,9 5,0 1,3 Lima 2,5

Matane 0,9 Mito 3,4

Tokomo 3,8 1,3 Unknown 0,7 1,1 2,5

Herbal mix supplement 10.0

S= Stover, F= Fruit

- 52 -

Table 16: mineral concentrations of plant samples collected in the Gilgel Gibe Catchment

SOIL REG Cu Zn Fe Mo Cu:Mo S Se Co Mn

(mg/kg) (mg/kg) (mg/kg) (mg/kg) (%) (µg/kg) (mg/kg) (mg/kg)

Acanthaceae

Hygrophila auriculata PV 1 11,6 60,4 1136 1,05 11,02 0,278 190 2,54 611 Anacardiaceae

Rhus glutinosa NF 2 8,4 16,1 181 0,39 21,26 0,123 70 0,19 87 Asteraceae

Aspilia mossambicensis PV 1 11,2 45,8 1112 1,15 9,80 0,287 62 1,45 480 Aspilia spp. NF 2 22,6 39,9 301 0,33 69,00 0,304 82 0,37 180 Aspilia africana NF 3 8,1 34,4 1491 0,48 16,80 0,293 167 1,07 101 Bidens macroptera NF 2 5,4 27,1 540 0,34 15,91 0,272 494 0,42 63 Bidens pilosa NF 2 9,4 36,7 1551 0,83 11,42 0,203 152 1,17 117 Vernonia adoensis NF 2 26,5 34,6 302 0,47 56,38 0,276 90 0,31 132 Vernonia amygdalina NF 3 8,8 28,2 218 0,26 34,35 0,208 80 0,19 183 Celastraceae

Maytenus obscura NF 2 11,4 30,3 170 0,26 44,46 0,398 379 0,28 161 Convolvulaceae

Ipomoea batatas NF 2 17,4 21,3 617 0,35 49,64 0,360 93 0,48 226 Cyperaceae

Cyperus spp. PV 1 4,2 42,2 338 1,56 2,70 0,133 227 0,22 288 Cyperus spp. NF 3 7,8 69,1 4240 0,67 11,55 0,189 112 2,02 347 Giant Cyperus PV 1 4,6 19,7 3020 2,28 2,02 0,111 87 1,48 326 Lamiaceae

Satureja paradoxa NF 2 13,1 46,2 3300 1,05 12,49 0,381 125 2,85 269 Satureja spp. NF 3 9,7 61 947 3,90 2,48 0,286 90 0,48 119 Lauraceae

Persea spp. (avocado) NF 2 6,5 29,4 235 0,09 70,57 0,310 30 0,23 1662 Leguminosae

Desmodium uncinatum NF 2 5,2 20,7 1025 2,43 2,14 0,168 30 0,55 161 Erythrina brucei NF 2 6,0 36,7 518 0,08 74,22 0,216 70 1,03 387 Indigofera spp. NF 3 11,0 28,4 602 0,97 11,28 0,210 50 0,34 75 Pisum sativum NF 3 3,3 19 255 1,28 2,59 0,069 40 0,18 38 Senna didymobotrya PV 1 3,9 16,4 995 0,52 7,38 0,179 187 0,52 152 Sesbania sesbon PV 1 5,6 46,6 461 1,50 3,70 0,212 104 1,12 1159 Trifolium spp. NF 1 4,1 30,6 6398 0,81 5,02 0,150 170 1,15 306 Trifolium spp. NF 3 5,2 26,6 1120 2,79 1,86 0,115 86 0,84 55 Loganiaceae

Buddleja polystachya NF 2 11,3 37,4 407 2,06 5,49 0,246 200 0,50 55 Mackinlayaceae

Centella asiatica NF 1 11,1 105 1827 0,67 16,44 0,573 173 0,95 426 Centella asiatica PV 1 10,1 88,4 2809 0,76 13,36 0,431 69 1,25 752 Malvaceae

Grewia ferruginea NF 2 14,8 61,2 261 0,22 66,77 0,233 40 0,89 114 Sida rhombifolia NF 3 19,5 52 2388 1,07 18,22 0,285 70 1,19 140 Sida tenuicorpa NF 1 11,5 191 549 0,68 16,91 0,401 160 0,58 529

- 53 -

Unknown Mito NF 2 0,8 14,6 526 0,19 3,97 0,274 27 0,07 79 Supplement Bulule NF 2 1,8 21,1 189 0,17 10,59 0,089 40 0,50 55 Herbal mix supplement NF 3 8,6 52,4 1044 6,71 1,28 0,221 213 0,60 124

Severely deficient

Marginally deficient

Marginally antagonistic

Highly antagonistic

Above the MTC

Reg= Region (1= 1700-1800m, 2= 1800-2000m, 3= 2000-2200m), PV= Planosol/Vertisol, NF= Nitisol/Ferralsol, S= Stover,

MTC= Maximum Tolerable Concentration

Musaceae Ensete ventricosum NF 2 5,8 11,5 190 16,60 0,35 0,289 158 0,15 252 Musa spp. NF 2 7,0 9,04 249 0,15 47,03 0,182 76 0,15 980 Myrtaceae

Eucalyptus camaldulensis PV 1 5,1 20,9 134 0,04 115,98 0,127 102 0,23 825 Psidium guajava PV 1 19,3 31,6 210 0,65 29,55 0,228 62 0,19 151 Poaceae

Andropogon abyssinicus NF 3 4,4 34,5 295 0,19 23,37 0,137 40 0,16 101 Brachiaria spp. NF 1 4,9 59 3343 1,52 3,22 0,168 152 1,15 787 Brachiaria spp. NF 3 9,5 54,9 8026 1,78 5,36 0,224 303 3,68 299 Cynodon spp. PV 1 4,2 31,5 911 1,63 2,56 0,121 91 0,73 281 Cynodon spp. NF 2 7,1 36,4 2398 0,70 10,09 0,145 178 1,78 197 Cynodon spp. (young) PV 1 5,0 38,4 260 1,76 2,85 0,215 268 0,23 376 Hyparrhenia hirta PV 1 3,8 37,7 351 0,24 15,87 0,079 36 0,31 145 Melinis repens NF 3 5,3 31,5 259 2,63 2,01 0,244 90 0,15 90 Melinis spp. NF 3 3,4 28,8 166 1,48 2,29 0,148 70 0,15 71 Panicium coloratum NF 2 6,6 18,9 339 0,74 8,88 0,162 60 0,27 224 Pennisetum clandestinum NF 3 6,6 42,5 774 0,84 7,83 0,248 167 0,49 118 Pennisetum purpureum NF 3 9,3 40,5 391 1,88 4,91 0,149 60 0,32 43 Pennisetum sphacelatum NF 2 5,5 16,9 749 0,32 17,06 0,115 77 0,62 103 Pennisetum sphacelatum NF 3 3,1 18,4 280 0,65 4,81 0,069 134 0,13 111 Pennisetum sphacelatum NF 3 6,0 28 404 0,65 9,28 0,121 250 0,26 85 Pennisetum thunbergii NF 1 5,3 31,9 956 0,21 25,13 0,163 39 0,50 179 Pennisetum thunbergii NF 3 4,7 21,6 655 1,00 4,71 0,117 100 0,39 268 Saccharum officinarum (S) NF 2 3,4 13,6 322 0,08 45,05 0,170 58 0,15 128 Saccharum officinarum (S) NF 3 1,7 5,51 94 0,06 27,38 0,027 380 0,04 46 Sorghum bicolour NF 2 4,3 21,5 285 0,15 29,03 0,131 39 0,18 60 Sorghum halepense PV 2 3,3 28,2 255 1,66 2,01 0,124 160 0,54 393 Zea Mays (S) NF 2 2,9 10,6 205 0,34 8,45 0,077 10 0,12 65 Zea Mays (vrucht) NF 1 1,8 31,7 631 0,18 10,11 0,123 71 0,33 30 Zea Mays (S) NF 2 3,7 23,9 1015 0,27 13,76 0,066 69 1,00 97 Polygonaceae

Persicaria nepalensis NF 3 5,2 42,5 8427 1,70 3,03 0,155 223 4,09 513 Rubiaceae

Coffee leaf NF 2 11,1 7,63 282 0,26 42,64 0,212 20 0,17 114 Solanaceae

Physalis peruviana NF 2 11,4 29,8 196 0,64 17,81 0,486 90 0,33 113 Verbenaceae

Premna schimperi NF 2 5,8 63,6 150 1,06 5,53 0,164 30 0,22 172

- 54 -

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