Upaya pencegahan mastitis subklinik dengan menggunakan homeopatikum secara kombinasi dan pengaruhnya terhadap kualitas dan produksi susu

CONTROL OF SUBCLINICAL MASTITIS USING

CERTAIN

HOMEOPATHIC COMBINATION

DRUGS

ITS

INFLUENCE

ON MILK

QUALITY

AND

PRODUCTION

AGATHA

WINNY

SANJAYA

THE GRADUATE

PRUGRAM

BOGOR A G ; R l i C U L T W

IJNWEMITY

BOGOR

Just as my non-violence will never fail homaeopathy never fails But the followers of homeopathy may fail owing to faulty application of the principles.

ABSTRAK

AGATHA WINNY SANJAYA. Upaya Pencegahan Mastitis Subklinik dengan Menggunakan Homeopatikum secara Kombinasi dan Pengaruhnya terhadap Kualitas dan Produksi Susu: Dibimbing oleh 1 WAYAN TEGUH WIBAWAN, MIRNAWATI SUDARWANTO, HEINRICH ENBERGS, SETYO WIDODO, MASDUKI PARTADIREDJA (aim), HElNER SOMMER (alm)

Tigapuluhtiga ekor sapi perah penderita mastitis subklinis dikelompokkan dalam grup A ( I 0 ekor), grup B (12 ekor) dan grup C (1 1 ekor). Sapi diobati dengan homeopatika dalam bentuk kombinasi dan plasebo, diobati pada minggu ke-4 dan 3 sebelum partus (a-p.) dan setiap minggu selarna 4 kali berturut-turut setelah partus (p.p.). Grup A diobati dengan Coenzyme

camp@

(minggu ke-4 dan 3 a.p.), Lachesis comp@ dan ~raumeep (minggu ke-I dan 2 p.p.), Coenzyme

camp@

dan Carduus comp@ (minggu ke-3 dan 4 p.p.). Grup B diobati dengan ~ r a u m e e l ~ dan Mucosa comp@ (minggu ke-4 dan 3 a.p.), Lachesis

camp@

dan iraumeel@(minggu ke-1 dan 2 p.p.), Coenzyme comp@ dan Carduus comp@ (minggu ke-3 dan 4 p.p.). Grup C adalah plasebo.

Pengujian dengan reagens IPB-1 di Lapangan memperlihatkan bahwa insidensi mastitis subklinis pada grup B adalah sebanyak 33,3% dan bertahan konstan sampai akhir pengamatan, sedangkan grup A dan C kasus mastitis sangat bervariasi. Kenaikan jumlah sel somatik susu dari grup A dan B satu minggu setelah terapi terakhir selama 2 minggu (minggu ke 5 - 7 p.p.) menuryukkan adanya suatu respon yang nyata terhadap terapi homeopathy kemudian diikuti penurunan jumlah somatik sel mendekati nilai normal.

Respon setelah disuntik Coenzyme comp. (minggu ke-3 a$.) memperlihatkan adanya peningkatan jumlah sel darah merah (RBC) untuk grup A (17,8%), selanjutnya diikuti penurunan RBC sampai akhir pengamatan. Gambaran haemoglobin dan pack cell volume tidak mengalami perubahan (konstan). Limfosit grup A dan C meningkat menjelang proses kelahiran, ha1 sebaliknya terjadi bagi grup B. Kadar haptoglobin (Hp) pada fase peripartal meningkat bagi grup B 0,86

mglml dan bagi grup A maupun plasebo 1,44 mglml serta 1,42 mglml. Pengamatan

5 minggu p.p. grup B mengalami penurunan jumlah Hp menjadi 0,09 mglml sampai akhir pengamatan adalah 0,07 mglml. Kadar Hp darah grup A menurun secara perlahan, kadar terendah dicapai pada minggu ke-5 p.p. yakni 0,18 mglml.

ABSTRACT

AGATHA WlNNY SANJAYA. Control of Subclinical Mastitis Using Certain Homeopathic Combination Drugs and Its Influence on Milk Quality and Production. Under the direction of I WAYAN TEGUH WIBAWAN, MlRNAWATl SUDARWANTO, HEINRICH ENBERGS, SETYO WIDODO, MASDUKI PARTADIREDJA (late), HEINER SOMMER (Late).

A total of 33 dairy lactating cows suffered from subclinical mastitis were classified into group A (10 cows), group B (12 cows) and group C (11 cows) which were treated with the combination of homeopathic drugs and placebo, applied at the 4'h and 3* week antepartum (a.p.) and every week for four times postpartum (p.p.). Group A received Coenzyme compQD (given in the 4'h and 3'C' week a.p.), Lachesis compQD with ~raumeel' (I& and 2nd week p.p.) and

Coenzyme compQD with Carduus compQD (3rd and 4'h week p.p.). Group B received ~ r a u m e e l ~ with Mucosa compQD (4" and 3rd week a.

i?

.), Lachesis comp@ with ~raurneel~(1" and 2nd week p.p.) and Coenzyme comp with Carduus compQP (3* and 4'h week p.p.) and group C as a placebo.

Incidence of mastitis in group A and C appeared irregularly. In contrast, group B showed a constant percentage (33.3%). Group A and B showed significant response to the homeopathic drugs, expressed as an increasing of the somatic cell count value after the last therapy for two weeks (5'h

-

week p.p.) and at the last observation decreased nearly to the normal value.

At the puerperal phase, the red blood

cell

of group A increased (17.8%) after the first therapy. Hemoglobine and pack cell volume showed no comparable results. Lymphocyte of group A and placebo increased during puerperal phase, contrary happened to group B. At peripartal phase haptoglobin increased for group B 0.86 mglml and group A as well as placebo 1.44 mglml. After birth, group B expressed a constant value (0.09

-

0.07 mg/ml), while group A and placebo raised significantly.

Statement of originality

The studies described in this dissertation were performed in accordance with the

regulations for the degree of Doctor in

the

Bogor Agricultural University.

This dissertation consists entirely of original research which has not been

submitted in whole or in part for any other degree at this university or at any other

CONTROL OF SUBCLINICAL MASTITIS USING

CERTAtN HOMEOPATHIC COMBLNATION DRUGS AND

ITS INFLUENCE ON MILK QUALITY AND PRODUCTION

AGATHA WlNNY SANJAYA

Doctoral Dissertation

submitted in fulfilment of the requirements to achieve a Doctor degree in Veterinary Sdence Study Program

THE GRADUATE PROGRAM

BOGOR AGRICULTURAL UNIVERSITY

B O G O R

CURRICULUM VITAE

Agatha W~nny Sanjaya was born in MEDAN, June

7*

1946 as the fifth child from the five children of lgnatius Dyan Kumiatan and Veronica Sukawati. The author

married with Dominicus Setyasusila Sanjaya and has two children Benedictus

Adhi and Martina Citra.

In 1964 entered the Faculty of Veterinary Medicine Bogor Agricultural

University and inaugurated as doctor of Veterinary Medicine in 1973. Master

degree was achieved in Veterinary Science in 1990 and in 1997 February,

entered Doctor Program at the Graduate Program in Bogor Agricultural University

and was funded by the Beasiswa Pendidikan Pascasarjana. A part of the

research was funded by the Project of University Research for Graduate

Title of Dissertation : CONTROL OF SUBCLINICAL MASTITIS USING CERTAIN HOMEOPATHIC COMBINATION DRUGS AND ITS INFLUENCE ON MILK QUALITY AND

PRODUCTION

Name of Student : Agatha Winny Sanjaya

Registration Number : 965104

Study Program : Veterinary Science

Approved by: 1. Advisory Committee:

Dr.drh.1 Wavan Tenuh Wibawan M.S. Prof.Dr.drh.Hi.Mimawati Sudawanto

chairman member

Prof. Dr. Heinrich Enberns member

2. Head of Study Program: 3. Director of the Graduate Program:

Acknowledgements

I would like to express my gratitude to my chairman advisor, Dr. drh. I

Wayan Teguh Wibawan, MS, Department of Animal Diseases and Veterinary

Public Health, member advisory, Prof. Dr. drh. Hj. Mimawati Sudawanto, Prof.

Dr. H. Enbergs, Dr. drh Setyo Widodo, for all his invaluable input, assistance and

support throughout the course of this work. Special my honour are due to the

late: Prof. Dr. Heiner Sommer and Prof. Dr. drh. H. Masduki Partadiredja for their

encouragement.

I am very grateful to the Beasiswa Pendidikan Pascasarjana, Project of

University Research for Graduate Education, for their sponsorship which enabled

me to study in Germany. My thanks also go to the Rector IPB, Director of PPS-

IPB, Dean of Veterinary Medicine Faculty, the head of Department of Animal

Diseases and Veterinary Public Health at IPB, Bogor, lnstitut fiir Anatomie,

Physiologie und Hygiene der Haustiere der Rheinischen-Friedrich-Wilhelms-

Universiet at Bonn, Fa. HEEL Baden-Baden Germany and the chief of Animal

Quarantine Service, Jakarta International Airport, for their support.

I would also like thank to the director of TAURUS Dairy Farm, Ir. Nugroho

and his crew, all my co-workers, who were very helpful and sympathetic during

the completion of the work in the field and laboratory.

I am also grateful to the staff members of Veterinary Public Health

Laboratory, University of Bonn where I had done my research work and to all of

my colleagues who supported me in various ways: Dr D.W. Lukman; Drh R.K.

Achyadi MS; Ir E. Sudamika M.Si; Dr Fachrudin; Drh E.S. Pribadi, MS; Drh Lukas

Tonga, Ir Agustine Santosa MS, Dr Linscheid; Dr U. Miiller; Dr S. Knura and my

friend Fr. Rosemarie Schattevoy. Last but not least, I would like to thank my

LlST OF CONTENTS

LIST OF TABLES LIST OF FIGURES LlST OF APPENDICES LlST OF ABBREVIATION

1. INTRODUCTION 1.1 Background

1.2 General Objectives of this Study 1.3 Hypothesis

2. LITERATURE STUDY 2.1 Milk

2.2 Defence Mechanism of Mammary gland 2.3 lrnmunological Defence at Peripartal Phase 2.4 Subclinical Mastitis

2.4.1 Prevalence and Characteristic of Subclinical Mastitis 2.4.2 Diagnosis of Subclinical Mastitis

2.5 Homeopathy as an Alternative Therapy

2.6 Advantageous of using Combination Form of Homeopathic Drugs 2.7 Control of Subclinical Mastitis by Homeopathic Treatment

2.8 Homeopathica Substances 2.8.1 Coenzyme compositum 2.8.2 Traumeel

2.8.3 Mucosa compositum

3. MATERIAL AND METHODS

3.1 Materials

3.1.1 Milk and Blood Samples 3.1.2 Instruments

3.1.3 Chemicals. 3.2 Animal contingent 3.3 Methods

vii vi i ix

3.3.1 Determination of Health Status of Cows Concerning to Subclinical Mastitis

3.3.2 Milk Quality Analysis

3.3.3 Blood Analysis

3.3.4 Clinical Chemistry

3.4 Data Collection

3.5 Research Design

3.6 Data Analysis

4. RESULTS AND DISCUSSION

4.1 Milk

4.1.1 Incidence of Subclinical Mastitis

4.1.2 Somatic Cell Count

4.1.3 pH of Milk

4.1.4 Milk Electrical Conductivity

4.1.5 Fat Content

4.1.6 Protein Content

4.1.7 Lactate dehydrogenase Enzyme in the Milk

4.2 Blood Profile

4.2.1 Red Blood Cells and Pack Cell Volume

4.2.2 Hemoglobin

4.2.3 Leukocyte Profiles.

4.2.4 Lactate dehydrogenase and Aspartate Aminotransferase Enzymes

4.2.5 Cholesterol

4.2.6 Urea in Blood

4.3 Haptoglobin

4.4 Milk Yield

5. CONCLUSIONS AND RECOMMENDATIONS

A. Conclusions B. Recommendations

LlST OF TABLE

1. Relationship between somatic cell count-score, somatic cell count and daily production loss of dairy cows

2. Estimated infection prevalence and looses in milk production Associated with elevated bulk tank somatic cell counts

3. Determination of CMT-score associated with an average somatic cell count

4. A comparison of methods used in subclinical mastitis test and their correlation with somatic cell counts

5. An overview of the use homeopathic drugs in mastitis control

6. Combination of homeopathic drugs used in the treatments

7. Application schedule of respective homeopathic combination

(A, B and C) and time of milk and blood sampling

LlST OF FIGURE

I. The incidence of subclinical mastitis by cows treated with a respective homeopathic drug combinations determined with IPB-I test

2. The effect of homeopathic treatments to the profile of somatic cell counts with a respective homeopathic drug combinations

3. The pH value of milk from subclinical mastitis treated with a respective homeopathic drug combinaiions

4. Milk electrical conductivity of subclinical matitis cows treated with a respective homeopathic drug combinations

5. Fat contents of milk from subclinical mastitis cows which pretreated with difference homeopathic combinations

6. Protein content of milk from subclinical mastitis cows treated with a respective homeopathic drug combination

7. Lactate dehydrogenase in milk of subclinical mastitis cows before and after treatments

8. Red blood cell profile of milk from subclinical mastitis cows treated with a respective combination of homeopathic drugs

10. The leukocyte profile of subclinical mastitis cows treated with respective

combinations of homeopathic drugs 48

I I. The neutrophil profile of subclinical mastitis cows treated with

respective combination of homeopathic drugs 49

12. The lymphocyte profile of subclinical mastitis cows treated with

respective combination of homeopathic drugs 49

13. The eosinophil profile of subclinical mastitis cows treated with

respective combination of homeopathic drugs 50

14. The LDH profiles of blood from subclinical mastitis cows treated with

respective combination of homeopathic drugs 52

15. The ASAT profiles of blood from subclinical mastitis cows treated with

respective combination of homeopathic drugs _52-

16. The cholesterol profile of blood from subclinical mastitis cows treated

with respective combination of homeopathic drugs 53

17. The urea profile of blood from subclinical mastitis cows treated with

respective combination of homeopathic drugs 54

18. Haptoglobin profile of subclinical mastitis cows treated with respective

combination of homeopathic drugs 56

19. Milk yield observed in normal lactation at the 3'Cl-p month before

,LIST OF APPENDICES

1. Mean values of somatic cell count in milk of subclinical mastitis cows treated with respective combination of homeopathic drugs

2. Mean values of pH milk of subclinical mastitis cows treated with respective combination of homeopathic

3. Mean values of electrical conductivity of subclinical mastitis cows treated with respective combination of homeopathic drugs

4. Mean values of milk fat from subclinical mastitis cows treated with respective combination of homeopathic drugs

5. Mean values of milk protein from subclinical mastitis cows treated

with respective combination of homeopathic drugs 68

6. Mean values observations of red blood cell, pack cell volume and

hemoglobin from subclinical mastitis cows 69

7. Mean values observation of leukocyte and its differentiation picture from subclinical mastitis cows

8. Mean values observation of blood enzyme and metabolite from

subclinical mastitis cows 71

9. Mean values of haptoglobin concentration in blood plasma from

subclinical mastitis cows 72

10. Total 5 months production from cows before and after treatment

with respective combination of homeopathic drugs 72

11. Statistical Analysis of Varians of 5 months milk production in normal

LIST OF ABBREVIATION APRP C CMT comp. CRP de n o w D DHlA Dl DNA et a!.

Fig. Hb HP IU LDH min. mg ml mS NAD PCV p.a. pH PMN rpm RBC

=

degree Celsius

=

registered

=

weighheight

=

unitllitre

=

ante partum

=

post partum

=

Aspartate Aminotransferase enzyme

= Acute Phase-Reactant Protein

=

centesimal scale

=

California Mastitis Test

=

compositum

=

C-Reactive Protein

=

remedy made from the products of a disease

=

decimal scale

=

Dairy Herd Improvement Association

= decilitre

=

Desoxyribonucleic acid

=

et alia

= Figure

=

hemoglobin

= haptoglobin

=

international unit

=

Lactate dehydrogenase

=

minute

=

milligram

=

millilitre

= millimetre Siemens

=

Nicotine Adenine Denucleotide

=

Pack Cell Volume

=

pro analyse

=

hydrogen ion concentration

=

Polymorphonuclear Neutrophil

=

rotation pro minute [image:15.593.81.443.48.781.2]

sec SCC

VLDL WMT WST

IJL

= second

= Somatic

cell counts contains leukocyte cells and mammary epithel cell debris

= very low density lipoprotein

= Wisconsin Mastitis Test

=

Whiteside Test

CONTROL OF SUBCLINICAL MASTITIS USING CERTAIN HOMEOPATHIC COMBINATION DRUGS AND ITS INFLUENCE ON MILK QUALITY AND PRODUCTION

1. INTRODUCTION

Subclinical mastitis is one of the most important diseases in dairy cattle in

lndonesia with the prevalence of 85-90% (Sudarwanto, 1995). It shows no

significant changes of udder and in milk appearance but causes a significant

economic loss (10-18%) mainly because of losses of milk production (15-40%)

per day, poor quality of milk, milk discard, drug costs and veterinary fee (Kirk et

a/., 1994; Lee, 1996; Sudarwanto, 1999). Similar results had also shown by Hirst

et a/. (1 984)

.

The decrease of milk production is felt to be more important than that

caused by clinical mastitis. The recent data show that the amount of dairy cattle

in Indonesia is 343,000 heads from which 260,000 are in lactation. They

produced 405,000 tons of milk/ lactation period. By assumption that 60% of the

lactated group suffer from subclinical mastitis, the economic loss due to the

decrease of milk production is approximately 108 billion rupiahs per year

(Sudarwanto, 1999).

1 .I Background

Antibiotics have been used in controlling mastitis. The use of inappropriate

antibiotics cause bacterial resistance, residue problems, allergy, metabolic

disorders, toxicity and influenced in milk processing (Kiehvein, 1976). Previous

Jakarta, Bogor and Bandung contained significantly high antibiotic residues

(Sudarwanto et a/. 1992).

Nowadays, greater emphasis has been placed on antibiotic residue

avoidance to ensure a residue-free milk for the consuming public. The major

change in this direction is the reduction in the use of antibiotics in the treatment

of mastitis.

As an alternative, homeopathic drugs are to be taken into consideration.

Their advantages are that they cause no toxic side effects and no residues in

animal's products. Missing corresponding placebo-controlled studies it was the

aim of the following studies to test the effect of certain homeopathic drugs in

prevention of subclinical mastitis in dairy cows.

1.2 General Objectives of this Study

1). To study the most effective combination of the homeopathic drugs in

prevention and therapy of subclinical mastitis.

2). To study the effect of the homeopathic drugs in enhancing milk production

and quality.

3). To study the influence of homeopathic drugs to the blood profile of subclinical

mastitis-treated cows.

1.3 Hypothesis

1). Homeopathic drugs depress the incidence of subclinical mastitis.

2). Homeopathic drugs enhance the milk quality and milk yield.

3). The effective combination of homeopathic drugs for subclinical mastitis

2.

LITERATURE STUDY

2.1 Milk

Milk is composed of water, carbohydrate (lactose), fat, protein, minerals

and vitamins and is secreted as a complex mixture of these components. The

properties and importance of milk are greater and more complex than the sum of

its individual component parts.

Water content of milk is about 87% and dependent upon the synthesis of

lactose. If water is added to the milk, the additional water is easily detectable by

several methods. These methods are based upon changes in freezing point of

the milk (cryoscopic method) or on changes in refraction light of the whey

component of milk after precipitation and removal of the casein and fat

components. These methods and other standard methods of milk testing are

described in the Association of Official Agricultural Chemists (AOAC), Official

Laboratory Analytical Procedures (National Academy of Science).

Carbohydrate substance of milk is mostly lactose. Lactose is a disaccha-

ride composed of the monosaccharides D-glucose and D-galactose, joined in a

13-1,4-glycosidic linkage. The chemical name of lactose is 4-0-13-D-galacto-

pyranosyl-D-glucopyranose and is essentially unique to milk. Lactose plays an

important role in milk synthesis. Lactose is responsible for maintaining the

osmolarity of milk and for drawing water in milk synthesis. Lactose is not as

sweet as other disaccharides such as sucrose or monosaccharides fructose or

glucose. The lactose cleaved to glucose and galactose in the neonate intestine

is done by an enzyme activity called lactase or 13-galactosidase. Other carbo-

hydrates are found in milk at low concentrations, such as free glucose (0.1 mM)

The fat component of milk is composed of a complex mixture of lipids.

Triglycerides or triacylglycerides are the major type of lipid in milk fat, 98% of the

total milk fat (wtw). Triglycerides are composed of 3 fatty acids covalently bound

to a glycerol molecule by ester bonds. Milk fat is secreted from mammary

epithelial cells as fat globules which are primarily composed of a globule of

triglyceride surrounded by a lipid bilayer membrane similar to the apical

membrane of the epithelial cells. This fat globule membranes helps to stabilize

the fat globules in an emulsion within the aqueous environment of milk. Other

milk lipids include diacylglycerides (0.25-0.48%), monoacylglycerides (0.02-

0.04%), phospholipids (0.6-1.0%), cholesterol (0.2-0.4%), glycolipids (0.006%)

and free fatty acids (0.1-0.4%).

The fatty acids used for the synthesis of triglycerides arise from the break

down of blood lipids and from de novo synthesis within the mammary epithelial

cells. About 40-60% of the fatty acids come from the blood. These are primarily

derived from very low density lipoproteins (VLDL), which are synthesized in the

intestine or liver. VLDL are composed of 90-95% on the inner core and 5-10%

protein at the outer surface. Chylomicrons, containing ingested fatty acids from

the intestine, also can act as a source of blood-derived fatty acids for the

mammary gland. Synthesis of short or medium chain of fatty acids occurs by de

novo synthesis in the cytoplasm of the mammary epithelial cells. The sources of

carbon the acetate and 13-hydroxybutyrate are mainly used.

The total protein component of milk is composed of numerous specific

proteins. The primary group of milk proteins are the caseins. Caseins have an

appropriate amino acid composition which is important for growth and develop-

ment of the nursing young. This high quality protein in cow milk is one of the key

reasons why milk is such an important human food. Casein is one of the most

other protein component of milk is whey or plasma phase of milk. The major

whey protein in cow milk are a-lactalbumin and 13-lactoglobulin. Alpha lactal-

bumin is an important protein in the synthesis of lactose and its presence is

central to the process of milk synthesis. Other whey proteins are immuno-

globulins (especially high in colostrum), serum albumin, enzymes, hormones,

growth factors, nutrien transporters, and disease resistance factors.

The major minerals found in milk are calcium and phosphorus. These

minerals are required in large quantities by the rapidly growing neonate for bone

growth and development of soft tissues. They are both mostly associated with

the casein micelle structure. Milk also contains most other minerals found in the

body.

Milk contain all the major vitamins. The fat soluble vitamins A, D, E, and K

are found primarily in the milk fat, and has limited amounts of vitamin K. The B

vitamins are found in the aqueous phase of milk. Milk always contains leukocyte

cells and mammary epithelial cell debris, also known as somatic cells in cow

milk. The concentration of leukocytes in milk varies with the species, infection

status of the gland and the stage of lactation.

2.2 Defence Mechanism of Mammary gland

Physical barriers of mammary glands play an important role in the defence

mechanisms of the mammary glands. This included the teat shape, teat

sphincter, streak diameter, streak canal, the length and the diameter of streak

canal and the keratin lining in streak canal, which contains factors that seem to

be bacteriostatic. Keratin is a meshlike substance, formed from descquamated

epithelial cells, fatty acids and cationic proteins and play as a physical

desquamated during milking which removes bacteria in the streak canal. The

keratin's fatty acids are bactericidal and bacteriostatic and it has proteins which

bind to and cause lysis of Gram positive bacteria. Furstenberg's rosette is

situated at the internal end of the streak canal, contain a protective leukocytes

population, which are thought to leave teat wall and enter the cistern via

Furstenberg's rosette. This rosette contains also ubiquitin a cationic protein

which has bactericidal activities.

lmrnunological defence of mammary glands includes all physiological

mechanisms allowing the body to identify and neutralise foreign bodies and is

comprised of: (1) cellular Immunity and (2) humoral Immunity. The immune cells

include the role of leukocytes such as granulocytes (neutrophils, basophils and

eosinophils) and lymphocytes, especially T-killer cells, and monocytes or macro-

phages. These cells are mainly responsible for the phagocytosis process in milk

and are considered as a second line of defence in the mammary gland. Humoral

immunity includes the role of antibody as opsonin, neutralisation of antigens or

activation of the complement system.

2.3 lmrnunological Defence at Peripartal Phase

lmmunological defence must be understood as an integrated system of

non-specific and specific immune responses, which has an important function to

maintain the physiological homeostasis. Peripartal period (about 4 weeks before

and after birth) is considered as a critical point and unstable physiological

condition of the dairy cows due to the sudden changes or switch over of many

processes including hormonal, metabolic, stress and cytologic performance.

Especially during the early lactation stage problems may arise to maintain the

cow in a physiological balance. This might influence the whole system of

(1) exhaustion of reserves of carbohydrates, (2) conversion of the body's protein into sugars, (3) mobilisation of lipid depots and decomposition of the lipids to produce energy with a risk of a fatty fiver and fmlly (4) increase of so called aceton-bodies. Interference between aceton bodies and leukocyte function decreased the interferon (IFN-Y) production. Interferon involved in the regulation of T cells, 6 cells, macrophages and granulocytes, so that the disturbance of IFN production caused the reduction of phagocytic activity of the -phages as well as of the granulocytes which are considered as the first line of defense of the bovine mammary gland against infections. This unstable physiological condition might lead to the incidence of mastitis facilitated by postpartal immunosupression (Kandefer-Szerszen et a/.

,

1992; Klucinski et

al.,

1988). The stress-situation and damages of tissues connected with the birth process, especially accrued in the soft tissue of the birth ways.

2.4 Subclinical Mastitis

2.4.1 Prevslence and Charadetfstics of Subclinical MasWs

Mastitis from the Greek mastos-breast and itis is a general term used to refer to any inflammation of the mammary gland The previous studies indicated that subclinical mastitis in dairy cattle is thought to be a multicomplex disease.

infectious subclinical mastitis the Streptococcus agalaciiae, Staphyilocioccus aueus and Escherichia mli are well known as the main causal agents (Wibawan et al., 1999). Bacteria could enter the mammary glands and multiply sufficiently to trigger the inflammatory response. Vasodilatation occurred and results in increased blood flow to the mammary glands. Inflammatory products such as prostaglandins, leucotrienes, pmteases, and oxygen radicals increased the vascular permeability. Leukocytes, initially polymorphonuclear neutrophils (PMN) leaved the blood vessels and diapedicaly enter the surrounding tissue, after which macrophages predominate.

Subclinical mastitis has remained the most economically important problem in dairy cattle in Indonesia with high prevalence ranging fmm 85 to 90%

(Sudarwanto, 1995; Wibawan et a1.,1999). The information about the pathogene- sis of subclinical mastitis is very limited. In subclinical mastitis, the milk appears to be normal. Bacteria usually, but not always, can be isolated in milk. Milk yield is depressed, and composition may

be

altered. No pathological changes of mammary gland could be observed but the histopathological examination showed that the inflammation of the mammary gland had occurred. Inflammation of mammary gland caused by colonisation as well as by invading pathogens is common among lactating dairy cows and is a major cause of economic losses.

contributes to the loss of income in the following ways. 70% from lost of milk production, 14% from death and premature culling, 8% miUc discarded at treatment and 8% for drugs and veterinary (Bailey, 1996).

S. aureus and S. agalactiae as the main causal agents of infectious subclinical mastitis often start with an acute phase and generally become chronic and subclinical. In contrast, E. coli caused an extensive damage of mammary gland tissue. This opposite characteristics are likely to results from the difference of pathogenic mechanisms used by those bacteria. By S. agaladiae and S, aureus the adhesion is thought to be the most important and critical step of infection and followed by the colonisation of bacteria on the surface of the mammary epithelial cells wbawan et al., 1999). The differences in the pathogenesis and clinical reaction of causal agents offer the opportunity to contrast the inflammatory responses and to study the early host responses to the respective causal agents by measuring the inflammatory markers such as haptoglobin, C-reactive protein (CRP) or other inflammatory mediators which might be used for the early screening test of subclinical mastitis

in

dairy cattle (Sandholm et a/., 1995). In case of acute mastitis, the clinical symptomes are

obvious and laboratory tests are not required to detect inflammation. Detection of subclinical mastitis is a challenging problem. Subclinical mastitis reduces milk production, milk composition and quality of milk and also maintains infection foci in the herd (reservoir). The extent to which various compositional changes occur depends on the inflammatory response. This depends on the virulence factor of the pathogen and the amount of affected tissue within the mammary gland. Inflammation should be analysed by monitoring various inflammatory mediators.

Ca, Mg concentration. Disturbance of the milk synthesis caused the decreasing

of casein-,

p

ladoglobulin-, a lactalbumine-, lactose concentration and fat in milk.

The high concentration of Na and CI in the milk caused a salty taste and alkalic

property of milk. The milk becomes heat unstable and was not completely

coagulated by rennin and caused failure in cheese ripening. According to Munm

in Hamann and Rund (1994) mastitis effect on milk and milk product are : (1)

fresh milk changes very easy in a rancid taste and the protein are heat unstable,

(2) pasteurised milk has an unfavourable flavour and with an unstable quality and (3) effect on making cheese are: a reduction in starter activation and

delayed coagulation time, breaking firmness decrease, lost of fat and casein in

milk at last there is a low profit and (4) problem in processing butter would

influenced the taste with an oxidation taste, present a weak aroma, prolonged

rippening butter time and inhibition of diacetyl product.

2.4.2 Diagnosis of Subclinical Mastitis

2.4.2.1 Somatic Cell Count (SCC)

Somatic cell counts (SCC) from a day's milk is the best indicator of the

extent to which the gland is involved in fighting a mastitis infection. Milk contains

cells which derive from the cow, these are somatic cells. The number of somatic

cells is immediately high after calving, but it drops rapidly during the first week of

lactation and increases again towards end-lactation. Therefore, the number of

cells depends on two factors: 1). a real increase in their quantity as a

consequence of udder initation and attraction to the inflammatory site or 2). The

dilution factor, i.e. the daily milk yield shows an inverse relation with the somatic

cell count Somatic cells are present in the milk in high number only when and

(Sandholm et a/., 1995). The quality of raw milk produced in Germany is

guaranteed by a milk quality control scheme conducted in accordance with

European Committee standards. In addition, because of its effect on the price

paid for milk, the somatic cell count in bulk milk is an indicator of udder health.

Udder health can only be maintained by strict adherence to accepted control

measures during milking, hygiene and prophylactic treatment (Fehlings and

Deneke, 2000). The cells found in milk consist of about 75% leukocytes and

about 25% epithelial cells. Normally SCC are 60-70% epithelial cell, 10%

lymphocyte and 2030% leukocytes (Sinell and Neuschulz, 1965). Leukocyte

numbers increase in response to bacterial infection, tissue injury and stress.

Increasing somatic cell numbers is a result of an increase in the number of

leukocytes.

The somatic cell count of milk serves as an indirect method to measure the

level of irritation in the mammary gland. There are 3 methods of evaluating SCC:

(1) Bulk Tank SCC (BTSCC), (2) Weighted SCC (VVTSCC) and Somatic Cell

Count Score (SCCS). The BTSCC is usually based on single sample of milk

from the bulk tank. The WTSCC is calculated on samples from individual cows

and is weighted or adjusted based

on

each cow's level of milk production. The

WTSCC for all milking cows in a herd should approximate the BTSCC. The

SCCS is based on the logarithmic conversion of the actual SCC to a linear score

as shown in Table 1.

The loss in milk production between scores 2 and 3 is estimated at 1.5

Ibs/cow/day based on an actual SCC difference of about 50,000 cells. Cows with a SCCS of 0-3 a= generally considered uninfected while cows with SCCS of 7

-

[image:28.550.78.520.163.503.2]

Table I . Relationship between somatic cell count-score, somatic

ceH count

range

and daily production loss of

dsmy

cows

(Shook

and

Saeman in Smith et a/. 2000)

The value of SCCS is associated with the ladation period, milk yield, the dens@ of cows

in a herd and the season. The first

lactation have a Iower SCCS with a h i r percentage in

the

0-3 category compared to older cows. This is not directty associated with the

age

but

mused by increased rate of

udder

infections. Unmfected

cows,

regardless of age generally have low SCC (Ebehart et a/.,

1979). High producing cows as well as high density herds trends to have a

higher SCC compared to low producing cows

or

lower dens@ herds. A high SCCS are generally observed during

the

summer time, in the months of July,

Herds with butk tank SCC above

200,000

wilt have varying degrees

of

subclinical mastitis present.

In the Table 2, data from the National Mastitis Council (1987)

show

that 6%

of the quarters in a herd could be expected to be infected in a herd with a bulk tank SCC of 200,000. At 500,000 SCC, 16% of tho quarters may be infected

with a 6% reduction in milk produdion. Ttds information lead

to

the conclusion that the monitoring of udder heal# is very important

in

order to control and

reduce the level of mastitis in a herd. Somatic

d l

count measurement provide

the

opportunity

to monitor and evaluate udder health status.

Tabk 2. Estimated infedion prevalence

and

t m s in milk produdion associated

with elevated bulk tank somatic

ceH

counts

* Pmduction loss cabMed as a percent d production expected at 200,000 cell1 ml. National M a s t i Council (1 981).

Somatic celt count under 400,000

dldmt

are typical

of

herds that have good management practices, but no partiwlar emphasis

on

mastitis control. In

contrast, somatic cell caunts greater than 500,000 cellslml indicates that one

third

of

the mammary gtands are infected

and the

lost of milk due to subclinical

2.4.22 California Mastitis Test ( C m

and

Mliscmin

Mast&% Test (WIUT)

The CMT test is a simple method to estimate the DNA

content

of milk. This test bases on an anionic detergent, Na-iauryt sulphate (Dodecyl Sulfate Sodium),

which dissolves eel membranes

and

nuclei. Consequently DNA is released and

it forms a transient gel with

the

detergent The more DNA in the milk

sample,

the higher the

viscosity of

the get (Sandholm et

aL,

1995). The v a w ~ o n

of

scoring of CMT, depend

upon

the skill of the person reading the result and the method

used to conduct the test. Uniformity in techniques are necessary if results are to

be comparable. The use of the CMT on the entire herd

at

monthly intervals can

be

extremely useful as

an

aid in detecting herd mastitis problems. individual and

total quarter infedions

can

be determined and, with proper records,

the

tevel of herd mastitis can be monitored. The relation between CW-Score and the

average somatic cell count is presented in Table 3

Table 3. Determination of CMT-Score associated with an average somatic cell count.

The advantages of

the

CMT are: (I) fairty accurate in measuring somatic

ceH

concentratin in mitk, meiating well with ather

test,

(2) it is sensitive and

can

be used for sampling

quafters,

bucket

and

buk tank milk samples, (3)

foreign material does not interfere with the

test,

(4) simple and a little equipment CMT-Scored

Negative Trace

2

3

Average somatic cell

count

(cells/ml)

300,OOO

m,m

~,~

2,700,000

[image:30.564.73.514.470.640.2]

is needed, (5) environmentat temperature changes have only a lime effed on the CMT as Long as the milk has been refrigemfed and is not over two days OM and

(5) herd mastitis levels can be estimated from bulk €ank A CMT

d

2 or 3 in tank milk indicates a probable percent

of

infected cows.

The Wisconsin Mastitis Test (WMT) is an other

method,

which frequently is used as a screening test for subclinical mastitis. The principle

af

the WAM is the same as that of the CMT except that the amount of gel formed is measured in a tube calibrated in miltimeters (mm) instead of being visually scmed. The

advantageous of WMT is conducted under precise procedures and standard

temperatwe conditions. A more accurate and

precise

method of measuring

somatic cetls in milk is

the

ekctronically m - d

somatic

cell counts

used

by Dairy Herd Improvement Association (DHIA).

Somatic cell counts is expressed in thousands of cells per milliliter, sometimes referred to as raw score, and converted

to

a Enear score ranging

TaMe 4. A comparison of methods used in subdinical mastitis

test

and their correlation with somatic cell

counts

(Eastridge and HoMet, 1992).

2.4.23 IPE1

Pest

The determinatk#1

of

mastitis irt the btd

can

be

done

by using diagnostical reagent. Recently, fieid subdical mastitis tests which are available

such

as CMT are rarely done

due

to the

price

and the avaitabifi of the reagents.

Another reagent called IPB-1

reagent,

is devetoped for a field test that is

relatively easy to use, inexpensive, and consttucted of ingredients readily are

available in domestic market. The reliability value of IP8-I is better than CMT or

WST, the

sensitivrty

of IPB-I is 0.99

and

CMT 0.92, the spectficity 0.92 with 0.36

and

the

prect'~dive value 0.95 with 0.97 respectively. The prdictive value still

2.4.2.4 Lac- dehydrogenase

Lactate dehydrogenase (LDH) is an enzyme widely distributed among the

various species of the animal kingdom and ubiquitous in the tissue of man and

other vertebrates. This enzyme catalyses the oxidation of lactate to pyruvate and

the reverse reaction, the reduction of pyruvate to lactate. LDH plays an important

role in the intermediary metabolism of various tissues. For the catalytic activities

of LDH the presence of a coenzyme nicotine amide adenine denucleotide (NAD)

is essential.

The LDH activity in milk might be used as an indicator to determine the

early stage of inflammation in the mammary glands and is recommended for the

screening technique (Hambitzer and Sommer, 1987). The value of this enzyme

in the milk is not more than 85 UA and when this value exceeded, mastitis is

present (Andersson, 1991). Accoding to Sommer et a/. (1986), LDH activity in

milk is higher in samples containing pathogenic bacteria (S. aureus) compared to

the non infectious mastitis milk. A high positive correlation was found between

cell count and LDH activity in milk containing pathogenic bacteria. The LDH

activlty increased from about 130 to 260 Un as cell count increased from

250,000 to 1.5 millionlml. No comparable results were shown by the samples

containing non-pathogenic bacteria, where the activity of LDH remained fairly

constant about 100 UII as cell count increased from about 250,000 to 1.25

millionlml.

2.4.2.5 Acute P h s e Response and Subclinical Mastitis

The maintenance of a physiologic homeostasis during ups and downs of

daily living is assured by a number of physiological mechanism. Tissue injury

and infection which represent a threat to the integrity of the organism, and

eqicilibrium. The local response of tissue

to

such

injury

or infection is acute inflammation. It's major clinical maniksWons resuit from changes in vascular caliber and flow, increased vascular permeability and attraction of leukocytes. Durtng the first few days €&tawing insult, a vast number

of

systemic and

metabolic changes occur, that is the acute response. The acute response should

be understood as a value in helping to pemtit survival during

the

period immediately foflowing iniuty and in achieving the same goals

as

We local

inflammatory response does, containment or destruction of infedious agents,

removal of damaged tissue and repair of the affected organ.

In mammals, tissue damage or inflammation at a localized site lead to

systemic changes known as the acute phase response. The acute phase reaction is an earty body defense mechanism tfiggered in response to cellular

injury manifested through a variety of adverse conditions,

such

as infection,

inflammation and advanced matignancies. This reaction provides substantial

immediate protection before humoraCmedited immunity becomes effective.

Among the varied physiological atteratian, which together produce

this

_response,

is a

change

in the circulating levels of a number

of

liver derived proteins, collectively known

as acute

phase reactant p m f e h (APRP) (Koj, $974; Saini et

a/.,

1998). Acute phae-pmtetns are serum proteins, whose concentrations

increase during

the

acute phase response to inflammation

or

infedion. The

respanse occurs in alt animals, but it is s ~ i m t t y different in various species (Eckersall et a/., 1988). APRP in man

and

animals indude C-reactive protein

(CRP), serum amyloid A (SAA), haptogtobi (Hp), cenrioplasmin, orosomucoid, al-antitrypsin, a2-moglobuIirtl comptement and coagulation proteins (Peppys

100 kDa find in blood and fomred in the Cver

of

mammals (Knura, 2000). The

assay

of

serum Hp in bovine serum or plasma is

now

a routine biochemical test

for veterinary diagnostic in Eurppe and USA. This is due to

the

ability

of

Hp to bind hemoglobin. It binds free hemoglobin, thus p r e s e ~ n g

the

body, which is

liberated from damaged erythmcytes.

SAA

and Hp concentration were

associated only with acute and sub

awte

lesions but not for animals with chronic

infhmmation (EckersaH, 1995). The possibility

of

using Hp for the

detection

of mastitis has been reported by Comer et a!. (I-), which

showed that

the

change in haptoglobin level from zero to 70 mg HpflOO ml by cows with clinical

mastitis. Until now, there is no information available about the using of H p

biological activity for early detection of subclinical mastitis.

2.4.2.6 Ekctrical Conductivity in Milk

A precise diagnose

of

udder health status is

the

base for

effedive

mastitis control. Cell count and electricai conductivity are used to indicate inflammatory

changes in the bovine

mammary gland.

The basis

of

mastitis diagnosis consists

of

the

combination of the detection of

the

inflammatory changes, but for economic reasons very often the measurement of

the

number

of

somatic cells

and the ekctrkai conductwit)c in milk performed withwt parallel bacteriological

examination (Hamann and Gyod, 1999). Accocdfng to Linzetl and Peaker (1 975), canductivlty is expressed as the molarity of a sodium chloride solution of the same electrical conductivity as

the

milk sample. Physiological studies have

shown

that in minfected glands

the

major ions responsible for milk conductivity pass into milk onky through h secretory cells and that the relatively high potassium and low sodium concentrations are mainted by

the

integrity and active

metabolism

of

the cells. The larger ducts where much

of

the

milk is stored

factors which interfere with cell metabolism will tend to alter the ionic

composition and more severe damage to the ducts or the secretory epithelium

. will have a more drastic effect because the milk will be allowed to partially

equilibrate with extracellular fluid which is rich in sodium and chloride but low in

potassium and these ions will rapidly move across the damaged epithelium. Two

relevant facts are well established. Firstly, milk fat concentration rises steadily

during milking and secondly conductivity decreases as the fat concentration is

increased.

2.5 Homeopathy as an Alternative Therapy

The main aim of homeopathic treatment is to maintain the physiological

homeostasis, which is based on natural healing. The use of homeopathic drugs

in "organic livestock fanning'' has been recommended by Europe Union.

Homeopathy is an alternative treatment, which is effective against bacterial as

well as viral infections. Contrary, antibiotic treatment had no effect on viral

infections, only avoiding the bacterial secondary infections and caused residue problems (Wheeler, 1978). Their effect follows a physiological principle, have no

negative side effect and are used mostly in small dose at LOIN concentrations

without side effects. They give no additional toxic burden for the liver and

kidneys and cause no residue, which is very important for food producing

animals. They cause no additional burden to the environment and are also

ecologically friendly.

Homeopathy can be defined as a therapy, which activates the natural cure and defense potential of the organism (Gebhardt, 1977). Homeopathy is a

system of medicat therapeutics for treating people and animals on the basis of

the principle "Similia similibus curentur" which means 'Yreats likes with likes". The

range of clinical symptoms in a healthy individual, can be used, prepared as

homeopathic remedy, to cure individuals showing similar symptoms. The precise

mechanism of action of a homeopathic on the molecular level is not yet fully

understood.

The homeopathic drugs are prepared from natural substances derived

from plants, animals, mineral, inorganic salts and organic substances.

Preparations we made on either a centisimal (1C) or decimal scale (ID).

Repeated dilution and succussion result in higher potencies, releasing more

energy in the process. Therefore, homeopathy is a system of medicine, which

concern itself with energy and not with material doses of a drug (Hamann, 1992).

Based on the potencies grade homeopathic drugs could be divided into 3

criteria: Lower potencies (D1-D6), the drugs should be used to treat chronic

cases with or without pathological changes being present, middle potencies (D7-

D30), and higher potencies (>D30) which are more energized should be

employed in acute infections (McLeod, 1991). Prepamtion of minerals and

metals are made with the 'Yrituration" method. More refined the fragment size of

the drugs caused a better "dynamic curative power"1efficacy (K6hler' 1999).

Pathological tissues are more sensitive to the drugs. Using small amount

of drugs enhances the viability of cells, but in high dose of drugs it caused cell

necrosis (Wheeler, 1978).

2.6 Advantageous of Using Combination Form of Homeopathic Drugs

Manifestation of a disease is not a consequence of a single disturbance

of an organ system but involve a complex of disturbances of those systems. This

leads to the preparation of the homeopathic combination drugs which are mostly

prepared in order to achieve a broad spectrum of drug's efficacy. Other

unexperienced in homeopathy, (2) they can be used according conventional

indications, and (3) there is only a minimal danger of accentuating the

symptoms.

2.7 Control of Subclinical Mastitis by Homeopathic Treatment

[image:38.555.74.512.307.712.2]

An overview of the use of a homeopathic treatment in controlling subclinical mastitis in Europe has been summarized in Table 5.

Table 5. An overview of the use homeopathic drugs in mastitis control.

Cases

Subclinical m a t i i s

Subclinical mastiis

Subclinical m a t i i s

Subclinical Mastiis

Subclinical m a t i i s

Num. of COWS 580 15 26 13 104 Drugs used Belladona, Lachesis comp Traumeel Phytolacca Phytolacca 30c, Thuja, Sticta , Ratanhia 30 c,

Sulphur 30 c, Thymus 30 c, Zingiber 30c

Nosodes

P h ytolacca decandra 200c Phosphorus 200c Conium rnaculatum 200c

Echinacea D2, Mercurius D6,

PhYMa- D10, Phellandrium D12, Silicea D6,

Lachesis D8 causa

S. aureus

Spontane -0usly infect4 Aplica- tion single or combina tion oral and topically Intrama- mary Results depressed sublinical mastitis incidence no significant effect was observed reject the new infection depressed sublinical mastiis incidence prophylaxis effect, decrea- se of LDH enzyme, SCC decrea- sed Reference Doren- kamp (1 992)

Egan (1 995)

Meaney

(1 995)

Searcy et

a/. (1 995)

Anders- son et a/.

Cases Chronic matiiis Mastitis Mastiis Mastiiis Mastiiis Clinical mast it is

Acute M a t i i s

Acute mastitis Acute mastitis Acute catarrhal mastiiis Num. of COWS 18 41 30 280 13 50 50 21 8 causa staph, strep, wryneba- cter

E. cdi,

Klebsiek Pseudomo

strep- tococcus,-

E. coli

S. aweus

Drugs used

Phytolacca D l

Caulophyllum 30c

mastitis noso- des (S uberis,

S. dysgalactiae

S. aureus and

E. coli Carduus wmp and Coenzy- me wmp Nosode staph, strep, Engystol Belladona, Echinacea Phytolacca decandra 200c Phosphorus 200c Conium maculatum 200c Aconitum, Phytolacca, Bryonia, Lachesis, Mercurius solubilis Awnitum D4 Phytolacca D l Bryonia D4 Lachesis D8 Lachesis D8 wmP Traumeel, Phytolacca, Belladonna Aplica- tion per os wmbina tion general at dry period per oral oral or subcutan single or wmbina tion single or wmbina tion Results

do not redu- ce milk quan- tiiy, do not prevent mastitis occurrence depressed the mastitis incidence depressed the m a t i i s incidence decreased somatic cell depressed the mastitis incidence increased milk yield, milk fat depressed incidence homeopathic prep. succes for gram ne- gative and for gram positive

treatment of E. coli

achieved depressed the infection totally bovine recover after treatment Reference Schutte A. (1 994)

Day (1986) Day (1 986) Sphor (1 989) May and Reinhart (1 993)

Seamy et 81. (1 995)

Sonne- wald (1 986)

Merck et a/. (1 989)

Merck et a/. (t989) showed that homeopathic treatment could improve the

health status of cows which before suffered from acute mastitis, especially

caused by the E. coli infection. Contrary to this, reported Meaney (1992) by

using homeopathic nosodes showed that when homeopathic remedies were

routinely applied to dairy MWS during lactating and non-lactating periods there

was no effect on SCC or intra mammary infection. Searcy et a/. (1995) reported

that homeopathic treatment using Phytolacca decandra 200c (50%), Phosphorus 200c (30%) and Conium maculatum 200c (20%) in a clinical trial depressed

subclinical mastitis incidence to 4.5 times compared to the control group. This confirmed previous observations of the benefit the homeopathic method can

provide in mastitis control in animal population. Dorenkamp (1992) showed that by using the homeopathic combination (Belladona-Homaccord ad us. vet.,

Lachesis compositum ad us. vet. and Traumeel ad us. vet) could be used to

control subclinical mastitis. A significant effect to the healthy of mammary gland

of cows suffered from S. aureus infection is shown by the treatment of Lachesis D8 (Andersson et a/., 1996). Homeopathic treatment could be applied for the

acute and sub acute mastitis, but might be not sufficient for chronic mastitis due to the irreversible damage of the parenchyma tksue of the mammary

(Dorenkamp, 2000). Furthermore, reported Klocke et ai. (2000) homeopathic

treatment could increase the healing rate of mastitis.

2.8 Homeopathica Substances

According to Internationale Gessellschaft fiir Biologische Medizin e.V.

(1998) and Tierarzneimittel 5 auflage (1994) the pharmacologische effect of

2.8. I Coenzyme compositum (Coenzyme comp. ad us.vet)

(Heel,

Baden-

Baden)

Coenzyme comp. stimulates of blocked enzymatic systems in degenerative

diseases as well as in defective enzyme functions, has a biological

activities as an intermediate catalisator, influenced to the intracellular

respiration and as a modulator of respiration enzymes.

Coenzyme compositum (Coenzyme compositum ad us.vet) consist of :

a. Coenzym A (a carrier of activated organic in citric acid cyclic, glicolysis and

lipid metabolism)

b. Acidurn ascorbicum (Ascorbic acid, vit C)(Co-factors of fermentation,

reduction oxidation system, collagen synthesis and antioxidant).

c. Natrium riboflavinum phosphoricum (Riboflavin-5'-phosphat, vit

82)

(Co-

factors of fermentation, reduction-oxidation system, flavoproteide).

d. Thiaminum hydrochloricum (Thiaminhydrochlorid, Vit B1) (Cofactor of fermen- tation, oxidative decarboxylation)

e. Pyridoxinum hydrochloricum (Pyridoxinchlorid, Vit B6)( fermentation co-factor, transaminase, dehydratase, desulfhidrase, decarboxylase)

f. Nicotinamidum (Nicotinamid) (Cofador for fermentation dehydratasen)

g. Acidum cis-aconiticum (Metabolite of TCA and redox system

h. Acidum citriwm (Citric acid) (Metabolite of TCA)

i. Acidum fumariwm (Fumarin acid) (Metabolite of TCA and redox system

exhaustion responsible)

j. Acidum a-ketoglutaricum (2-Oxoglutarat acid )(Metabolite from TCA and redox

system. Muscle relaxation, lipoid disturbance and steroid products

k. Acidum malicum (Metabolite of TCA and redox-system. Neutralize the acid

I. Acidum succinicum (Bemsteinsaure)(Metabolite from TCA is exceed. Disturbance of blood synthesis)

m. Barium oxalsuccinicum (Bariumoxalsuccinat)(Metabolite from TCA and redox -system related to mesenchym structures. Regular endocrine disturbance). n. Natrium pynrvicum (Natriumpyruvat)(Metabolite from TCA and sistem redox

system. Decreasing cell activities).

o. Cysteinum (Cystein)(SH gmupp contained factor of TCA redox system, toxicity and failure of therapy.

p. Adenosinum triphosphoricum (ATP) (Support the activities of mitochondria)

q. Nadidum (Nicotinamid-adenindinucleotid, NAD) (respiration enzyme).

r. Natrium diethyloxale aceticum (Natriumoxalazetat) (Metabolite from TCA and redox, exceed).

s. Acidum alpha-liponicum (Coenzyme made acid and dekarboxylase).

2.8.2 Traumeel (TRAUMEEL ad u s . W ) (Heel, Baden- Baden)

Traumeel consist of various substances which have a respective biological activities such as (1) the stimulate of somatic cells, (2) stimulate the inflammation (3) activation of phagocytosis (4) regulation of blood vessels, contraction and (5) decrease the gland secretion.

Traumeel consist of:

a. Arnica montana (stimulate the synthesis of somatic cells, muscle and tendon, stop bleeding). Stimulate the inflammation.

b. Calendula (stimulate the inflammation effect especially for skin, pharingitis, mucous, phlegmon, panaritium, ulcera, dermatitis.

c. Hamamelis (Relaxation of blood vessels, passive congestion).

e. Aristolochia clematitis. Locomotion inflammation, activated phagocytes

f. Atropa belladonna Decreasing gland secretion. increasing pulse frequency,

pain in extremity swollen joint, sweating, paralysis, inflammation symptom

especially lungs, mammae. Convukion digestive tract nerve and urinary tract,

dilatation pupil.

g. Bells perennis. Muscle irritation, paralysis, joint irritation, swollen, sensitive

h. Charnomilla Muscle irritation, spasm from digest tract, uterus, tract urinary,

inflammation blood vessel.

i. Echinacea angustifolia. Leukocyte increasing, agitated, lymph vessels

dilatation and exhaustive

j. Echinacea purpurea. Stimulate phagocyte T and B lymphocyte

k. Hypericum perforatum. Neurosis, skin inflammation

I. Millefolium. Bleeding caused by leaking vessels, hipotensi.

m. Symphytum officinale. Irritation until relaxation tonus muscles.

n. Hepar sulfuris. Hyper catarhalis secretion, sensitive to climate and draught

o. Mercurius solubilis Hahnemanni (mercury (11)-amido nitrate)

Oral cavrty inflammation, diarrhea, dysuria, lymph gland hyperactive.

2.8.3

Mucosa cornpositurn (Mucosa cornpositurn ad us.vet)(Heel Baden- Baden)

Mucosa compositum ad us.vet. responsible for healing unhealthy tissues

and stimulate dysfunction organ and repaired damaged mucosal tissues.

Mucosa compositum consist of:

a. Potential Organ preparat.

b. Mucosa nasalis,Mucosa pulmonis, Mucosa oris, Mucosa oesophagi, Mucosa

ductus choledochi, Mucosa jejuni, Mucosa ilei, Mucosa coli, Mucosa recti, Mucosa oculi, Pancreas, Tunica rnucosa vesicae urinariae,

c. Intermediate catalysator (activated the respiration cells, and block enzyme production)

d. Natriurn diethyloxalaceticum (Na oxalasetat)

3. MATERIAL AND METHODS

3.1 MATERIALS

3.1. I Milk and blood samples

.

3.1.2 Instruments

Bottle samples, Lactodensimeter Soxhlet, Gerber tube, waterbath, scales,

centrifuge, measure glass, pH-meter, Kjeldahl pitcher, burette, microscope,

test shell (for the IPB-1 test), incubator, thermometer, cooling box,

spectrophotometer, hematocrit capillaries, Neubauer, blood counter,

venoject, syringe.

3.1.3 Chemicals

3.1.3.1 Used for milk analysis

H2S04, amyl alcohol, &SO4, CuS04.5H20, NaOH, borat acid, HCI, methyl red indicator, buffer solution, plate count agar, peptone water, methylen

blue loeffler stain, ether alcohol, alcohol, reagents IPB-1 and Lactate

dehydrogenase kit.

3.1.3.2 Used for blood analysis

aspartate arninotransferase enzyme, Lactate dehydrogenase-, urea- and

cholesteml kit, Hayem reagent, Turk reagent, Giemsa staining, heparin.

3.1.3.3 Homeopathic drugs

Coenzyme compositum', Mucosa compositum', Lachesis compositumQ,

~raumeep, Carduus compositum' (Fa.Heel, Baden-Baden, Germany).

3.2 ANIMAL CONTINGENT

Thirty three subclinical rnastitis lactating Holstein Friesian cows were

cows therapy B and 11 cows therapy C. The study was done in a blinded form. The experiment commenced in December 1999, in pregnant condition of 3-6

months, continued until all the cows give birth 8 weeks post partum. The dairy cows are in aged between in 2-7 years.

Cows are kept in a loose barn, fed at 09.00 o'clock and milking is done at

14.00 and 02.00 o'clock by machine a-Laval. All cows were teat-desinfected

routinely after milking.

Data about milk yield, pactus time, artificial insemination

and

number of lactation are collected in a monthly annual recording.

3.3 METHODS

3.3.7

Determination of Health

Status

of Cows Concerning to Subclinical

Mastitis

Animals are screened for subclinical mastitis using IPB-1 (3.3.1 .a) and somatic cell count according to Breed method (3.3.1 .b).

a. IPBI -Test

Milk sample in equal volume with IPBI-reagent are mixed on a test shell and rotated for 15 sec. The positive reaction is shown by coagulation appearance of the test suspension. In case of a negative result the remains homogenous. The positive reaction are dassified into 3 categories:

Positive 1: it appears a light coagulation, the suspension is not homogenous. Positive 2: a distinct clotting and coagulation appears.

b. Somatic cell count

A volume of 0.01 ml milk is smeared in one square cm on an object glass

and stained with Methylen Blue Loeffler. The somatic cell is count under light

microscope 100 x 10 (Breed method).

Cows with somatic cell count (SCC) more than 400,000lml are classified as

suffering from subclinical mastitis. When the animals showed negative reactions

with IPB-1 and have SCC below 400,0001ml they are categorised as healthy.

3.3.2 Milk Qualify Analysis

For this, milk samples are collected periodically at 14.00 o'clock (evening

milking) from cows which have a normal lactation period and blood samples are

taken after the cows have been milked. Samples are collected and transported

to the laboratory in a cooling box (<lo0 C) for 1-2 hours. The analysis of milk

quality is finished at the same day.

a. pH Value.

The pH value of respective milk are determined with a pH- meter (pH 523

WTW tijv Bayem).

b. Milk Electrical Conductivity

Using a special hand apparatus, milk checker (Esai Co. Ltd, Tokyo, Japan,

precision coefficient of variation < 0,7%). Poured directly 5 ml of milk sample in the cup of this apparatus and the value will be measured automatically as mS

(millimeter Siemens). Normal the milk electrical conductivity are below 5.6 mS.

c. Milk Composition Analysis

The milk analysis is done 3 hours after milking at 25' C.

1) Milk Fat (F%)

Milk fat content is determined according to Gerber.

Amount

of

10.0 ml sulfuric acid 92% p.a. (Merck, Germany) is filled in the

alcohol (Merck, Germany). Homogenized for 10 min until the dark purple color

appeared, after that centrifuged (1,200 rpm, 3 min.). Finally, the suspension in

butyrometer is incubated in a waterbath (65' C, 5 min.). The fat content is

determined as % value.

2) Protein analysis according Kjddahl

Homogenized milk sample (0.4 g) and added 3.0 ml concentrated sulfuric acid

(98%) and 0.5 g selenium reagent mixture. Potassium sulfate (K2SO4) and

Copper-(11)-sulfate (CuS04.5H20)

used

as a catalyst, heated until the clear suspension with light yellow color occurred. The suspension is heated again for

1.5 hour and let to cool at room temperature. Added 30 ml NaOH (30%)

,

the

ammonium will be free and catch with 5.0 ml boric acid 4%(H3B03 ). This free

ammonium can be measured by acid titration (HCI 0.025N). The pure milk

protein is the value of N X 6.38 and the result of N is in percent (%) in gram N

proteinfi00 g milk sample.

d. Lactate dehydrogenase Activity in Milk

To 5 ml milk in a test tube and added with 5.0 ml concentrated ammonium sulfate followed by centrifuged (Hettich EBA 3s) for 3,000 rpm, 10

min (Tbpel, 1987). After the centrifugation the test tube is kept in the refrigerator

for 1 hour. Then milk serum is taken for the determination of LDH activities using

a diagnostioKit (Merck, Germany).

3.3.3 Blood Analysis

Blood analysis are done using standard methods (Kerr, 1989), including :

a. Total erythrocytes counts

b. Leukocytes total count

c. Hemoglobin concentration

d. Pack Cell Volume (PCV)

3.3.4 Clinical Chemistry

This were done to determine the relationship between clinical chemistry by

healthy cows as well as subclinical mastitis cows treated with respective

treatments. Serum are collected from respective cows and used for further

investigation, including:

a. Aspartate Aminotransferase enzyme activity (Enzylinem biohnerieux)

b. Lactate dehydrogenase (LDH-enzyme) activity ( ~ c o l i n e ~ Merck)

c. Blood cholesterol (Ecolinem Merck)

d. Blood Urea (bio~erieux?

All tests are done according to the respective manufactured.

3.4 DATA COLLECTION

Primary data are collected from laboratory observations