Temperament and Milk Quality in Sheep and Cattle · Milk composition and clotting properties of...

Temperament and Milk Quality

in Sheep and Cattle

by

Sarula Sart

B. Sc.

This thesis is presented for the degree of

Master of Science in Agriculture

2005

The School of Animal Biology

Faculty of Natural and Agricultural Science

The University of Western Australia

Declaration

The work presented in this thesis is original work of the author, and none of the material

in this thesis has been submitted either in full, or part, for a degree at this or any other

university or institution. The experimental designs and manuscript preparation was

carried out by myself after discussion with my supervisors, Professor Graeme B. Martin,

Dr. Roberta Bencini and Dr. Dominique Blache.

Sarula Sart

July 2005

i

Table of Contents

___________________________________________________ Page

Summary v

Acknowledgements viii

General introduction 1

Chapter 1: Literature review 3

1. Introduction 3

2. The effect of temperament on animal production 4

2.1. Animal temperament and stress 4

2.1.1. Definition of temperament and stress 4

2.1.2. Measurement of animal temperament 6

2.2. Factors related to temperament 8

2.2.1. Breed 8

2.2.2. Experience and training 9

2.2.3. Age 9

2.3. The influences of temperament and stress

on animal production 10

2.3.1. The effects of temperament

in different production systems 10

2.3.1.1 Beef 10

2.3.1.2 Cows’ milk 10

2.3.1.3 Ewes’ milk 11

2.3.2. How temperament affects production 12

2.3.2.1. Behavioural reasons 12

2.3.2.2. Physiological and hormonal changes 12

2.3.2.3. Change in the immune system 14

3. Milk synthesis, milk ejection reflex and removal of milk 14

3.1. Milk synthesis 15

3.2. Control of lactation and milk ejection 17

3.2.1. Endocrine regulation of lactation 17

ii

3.2.2. The mechanism of milk ejection 19

3.2.3. Oxytocin release and milk removal

in cows and ewes 20

4. The effect of stress on milk synthesis, yield and composition 22

4.1. The factors that affect milk yield and composition 22

4.1.1. Nutrition 22

4.1.2. Season/ Lactation period 23

4.2. The inhibition of milk let-down by stress 25

4.2.1. Adrenaline 25

4.2.2. Milkers and milking techniques 26

4.2.3. Milking environment 27

4.3. The effects of stress on milk composition 28

4.3.1. How fat level is affected by stress 28

4.3.2. How protein level is affected by stress 29

5. The effects of milk composition on the processing performance

of milk for cheese 30

5.1. Process of cheesemaking 30

5.1.1. Conversion of milk into cheese 31

5.1.2. Clotting properties of milk 32

5.2. Milk composition and clotting properties of milk 34

5.2.1. Casein and fat concentrations and clotting

properties of milk 34

5.2.2. Protein level and clotting properties of milk 35

5.3. Measurement of clotting properties of milk 35

6. Conclusions 36

Chapter 2: Experiment 1: Oxytocin dose-response in calm and nervous ewes 37

Introduction 37

Materials and methods 38

Results 39

Discussion 40

iii

Chapter 3: The effects of temperament on the production and

clotting properties of milk from Merino ewes 43

Introduction 43

Experiment 2: The effects of the temperament of Merino ewes

on milk yield and composition 46

Introduction 46


Results 48

Discussion 49

Experiment 3: The effects of temperament on clotting properties

of milk from calm and nervous Merino sheep 52

Introduction 52


Results 55

Discussion 57

Chapter 4: The effects of temperament on the yield and composition

of milk from Holstein cows 60

Introduction 60

Experiment 4: Repeatability of open-field tests with a human

in Holstein cows 63

Introduction 63


Results 69

Discussion 74

Experiment 5: The relationship between the temperament and milk

quantity and quality in Holstein cows 77

Introduction 77


iv

Results 79

Discussion 82

General discussion for Chapter 4 84

General discussion 86

References 91

v

Summary

It is well known that cows produce more milk if they are comfortable at milking,

because stress from milking may cause them milk ejection problems. Temperament is

an intrinsic characteristic of the animals so may affect the level of comfort at milking,

and stress from the milking process itself may have a greater impact on animals with

nervous temperament than on those of nervous temperament. When the milking

becomes a stressor, it may affect secretion of milk ejection hormones that, in turn,

may affect milk yield and composition. There is little evidence for how animal

temperament affects milk quality in different farm animals. In this thesis, I have

examined the effects of temperament on quantity and quality of the milk from Merino

ewes and Holstein cows. I also tested whether temperament affected the processing

performance (clotting properties) of the milk from Merino ewes.

The general hypotheses tested were:

1. Calm ewes would produce more milk of better quality than nervous ewes, and,

consequently, the clotting properties would be better in the milk from calm

ewes than from nervous ewes.

2. Calm cows would produce more milk of better quality than nervous cows.

In the experiments with sheep I used animals that had been genetically selected for

“calm” or “nervous” temperament over 14 generations. In the experiments involving

cows I assigned animals to temperament groups based on their scores in a

temperament test. Temperament was measured by using an open-field test with a

human and a flock-mate.

In the first experiment I tested whether calm Merino ewes would require a smaller

dose of oxytocin for inducing milk let-down and for removing milk from their udders

than nervous Merino ewes. In addition, I tested the minimum doses of intramuscular

injections required to obtain the ejection of milk from calm and nervous ewes, and

determined whether or not different doses affected milk protein or fat. I found that

there was no difference between the calm and nervous ewes on the requirements for

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oxytocin for milk removal. A dose of 1 IU oxytocin was sufficient to achieve milk

ejection in both calm and nervous ewes. There was no clear effect of dose of oxytocin

on protein or fat concentrations of milk from ewes of either temperament group. The

dose of 1 IU had a significant effect on the fat concentration in the calm ewes, but this

was probably a chance observation.

In the second experiment, I used an intramuscular dose of 1 IU oxytocin and

compared the milk yield, milk protein and fat concentrations from the calm and

nervous Merino ewes. Calm ewes were expected to produce more milk than nervous

ewes, and to produce more protein and fat in the milk than the nervous ewes. The total

milk yield over the 18 weeks of lactation did not differ between the groups, so the

first hypothesis was not supported. This may be because the oxytocin injection

eliminated the stress of handling and milking. Stress from milking might inhibit the

oxytocin release in some individual animals, resulting in low milk yield. However,

exogenous oxytocin injection may overcome this problem. The hypothesis that calm

ewes would produce more protein than nervous ewes was strongly supported,

suggesting that genetic selection for calm temperament in Merino sheep could

improve milk quality. The casein concentration in the milk from the calm ewes was

also significantly higher than in the milk from the nervous ewes. The data from this

experiment, however, did not support the hypothesis that the fat concentration would

be affected by temperament, although it appeared to be affected by the milk output of

the ewes or by withdrawal of milk from them.

Experiment 3 was designed to examine the relationships between milk concentrations

of protein and the clotting properties of milk from calm and nervous ewes. The

hypothesis tested was that the milk produced by the calm ewes would have better

clotting properties, because of higher concentration of protein, especially casein, than

the milk produced by nervous ewes. The hypothesis was not supported. Neither rennet

clotting time nor rate of firming were decreased by the high protein or casein

concentration in milk from calm ewes. On the contrary, curd consistency was greater

for the milk from nervous ewes than for that from calm ewes. From these results it

appeared that the differences in milk protein concentrations between calm and

nervous ewes was too small to affect clotting properties. The conditions under which

the milk was clotted might have greater impact on clotting properties than the protein

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concentration of the milk: milk pH, for example, may be more important than the

slight variations in milk composition.

In experiments 4 and 5 I studied the effects of temperament of Holstein cows on milk

yield and composition, with the same hypotheses that had been tested in Merino ewes.

Cow temperament was measured twice with an open-field test, using human presence

as a ‘stressor’, comparing the responses of the second test with those of the first test.

The cows were less agitated in the second test than in the first test, with lower

numbers of steps, crossings, vocalizations, defecations and urinations. The milk

outputs over 12 weeks, and milk protein and fat concentrations were compared across

the range of different temperaments. No differences were found in the milk output,

protein or fat concentrations between the temperament classes. I concluded from these

results that the temperaments of the dairy cows from the same herd in same breed

vary too little to affect milk yield or composition.

It was concluded that genetic selection for temperament can improve milk quality

during the early stage of selection, but the impact is not significant when the animals

have been selected for many years for both milk production and ease of handling.

Hormonal patterns of different temperament animals should be studied so we can

learn how milk protein is affected by temperament. More work is also needed on the

temperament test to improve the measurement of the temperament of calm and

nervous animals that were used in this thesis.

viii

Acknowledgements

It was a great opportunity to do my Master’s Degree in Agriculture at the School of

Animal Biology at The University of Western Australia with a scholarship from the

Australian Agency for International Development (AusAID). I express my deepest thanks

to my supervisors Prof. Graeme B. Martin, Dr. Roberta Bencini and Dr. Dominique

Blache for their strong support for my work during the degree program. I also would like

to equally appreciate the support and help from AusAID scholarship officers, Ronda

Haskell and Cathy Tang.

I want to thank Dr. Philip E. Vercoe and Dr. Ian Williams who reviewed my project

proposal and commented on my thesis.

I would like to send my great appreciation to those who have contributed to this work. I

especially would like to express my gratitude to Kevin Murray and Dr. Guijun Yan for

their help on my statistical analysis; Steve Gray for his management of the experimental

animals and his support and help during the study; Margaret A. Blackberry, Christen

Hunt, John Beesley, Aprille Chadwick, Dean Thomas, Teuku Reza Ferasyi, Beth

Paganoni, Sid Saxby, Suely Lima, Sedat Yildiz, Graciela Pedrana, Jenny Cheng for their

help with milking, and other staff and postgraduate students for their help and discussion

on my programme and sharing their experience with me.

I also want to express my appreciation for Dr. Turigen Bayar, Dr. Aurigele, Dr. Dorgi,

Sarula, Xiang Yun, Tegusihua and other staff and students from Inner Mongolia

Agricultural University, and the manager and the technicians from the university research

farm, for providing facilities and support during my work there. My appreciation also

goes to Lifang Wang and other technicians from Yili Dairy Group for analysing milk

samples and help for my study in Inner Mongolia, China.

Last but not least, I would like to thank my son, Charles Sart for his everlasting love,

great company and joy for my life in Australia. I would like to say thank you to my mum

and sisters for their extraordinary love, wonderful support and encouragement during my

studies.

General Introduction

1


Modern trends in farm practice towards bigger herds and flocks leads to lack of

familiarity of the animals with their human managers. As a consequence, human

handling of the animals may lead problems such as wasted time, injuries to stockmen

and unnecessary stress to the animals (Boissy & Bouissou, 1988). Therefore,

information about the way that the environment and management affect the

responsiveness of domestic animals’ to stressors should be valuable for the

assessment of their welfare and productivity.

The responsiveness of animals to stressors is affected by their temperament, defined

as their fear response to human handling and to threatening environments (Murphy,

1999). Temperament is being paid more and more attention as we aim to improve

animal productivity as well as limit stress during husbandry practices. Understanding

and observing animal temperament is also becoming particularly important in terms

of the ethics and welfare of our industries.

Various techniques have been used to measure the temperament of experimental or

farm animals. Importantly, measures of temperament reflect the fear responses of the

test animals so it is necessary to approach any new test system, or to adjust old

systems, so that they are more accurate and agree with the philosophical concepts of

‘temperament’. A temperament test should reflect behavioural responses and reaction

of the animals to different fear-inducing factors. The open-field test with a human and

a herd-mate (a cow held in a pen at the end of the arena) has been used in the work in

this thesis to measure the temperament of experimental cows. The cows were tested

twice in an open-field test to allow comparison of their responses in the first and

second tests, and also to allow assessment of the various behaviours recorded in the

test. The aim of this study was to contribute more data from temperament tests so that

researchers and animal producers could either make comparisons with other testing

systems or develop tests used in farm management.

In addition, temperament is reported to affect both ease of handling and productivity

in most farm animals. As we shall see in the review of the literature, beef cattle of


2

poor (nervous) temperament cause management problems such as time wasting and

causing injuries to the handlers. In addition, they have lower daily gain and lower

feed conversion ratio, and produce poorer quality meat in a feed-lot than good (calm)

temperament cattle. The temperament of production animals is paid more attention in

the dairy industry because it is seen as a major factor that affects the quantity and

quality of milk and milk products. Poor temperament dairy cows have been reported

to have lower milk production and slower milking rate than good temperament cows.

For sheep, the functional parameters of the udder, such as milk yield, milk flow rate

and milk ejection latency, are strongly affected by the level of fearfulness. Also, milk

ejection latency in nervous ewes is much longer than it is in calm ewes.

In contrast to the wealth of data on milk quantity, little is known about the effect of

temperament on the composition of milk from cattle or sheep. I designed my research

project therefore to fill this gap by testing whether milk quality could be improved by

selecting good temperament cows and ewes. I also studied the clotting properties of

the milk (i.e. processing performance for making cheese) from calm and nervous

ewes. Sheep milk is rarely consumed fresh and most of it is diverted into cheese

production. Sheep milk is very suited to making cheese because of its high protein

and total solids. The yield and quality of cheese from sheep milk is affected by the

composition of the milk so, if milk composition is affected by ewe temperament, it

will eventually affect the quality of the final product.

The specific questions that I attempted to answer in this thesis are:

• Is milk quality improved by increasing the protein and fat content when Merino

sheep have been selected for temperament?

• What are the effects of these differences in milk composition on the clotting

properties of ewe milk?

• Is the temperament of dairy cows sufficiently variable in a herd that it has an

impact on the quality of the milk?

• Is the open-field temperament test including a human reliable for dairy cows?

Chapter 1 Literature Review

3

Chapter 1

Literature review

1. Introduction

The general aim of studying animal temperament is to define how animals with

differing temperaments respond to human handling and to threatening or stressful

environments. It also identifies physiological changes in the animal that are caused by

stress. A better understanding of animal temperament allows us to minimize the effect

of stressors, to increase the level of animal welfare and to provide better criteria for

genetic selection.

Selection of dairy animals for less reactivity to strange environments, and reduction in

the effects of stressors associated with environment and management, may improve

milk yield as well as its composition. Milking is a ‘contradictory procedure’ in that

handling by a milker may stress an animal but the same procedure also stimulates the

udder to send a signal to the neural system to release the hormones that are necessary

for milk let-down.

The level of fright, or stress, depends on the animal’s attitude to humans or how

nervous it is and, in turn, this degree of stress determines the amounts of hormones

released at milking. The levels of these hormones influence milk ejection, milk

removal, milk yield and milk composition and, ultimately, the quantity and quality of

cheese that is made from that milk.

In this chapter I have reviewed the current knowledge of animal temperament and the

effects of stress, the relationship between them and how temperament affects

production. In addition, the interaction between humans and animals at milking is

analysed with respect to the inhibition of milk ejection. The mechanisms of milk

synthesis, milk ejection and hormonal control of milk ejection and removal are


4

considered so as to illustrate how stress affects milk yield and composition. Finally,

the cheese-making process is reviewed to examine the effects of milk composition on

the quality of cheese obtained from the milk.

2. The effect of temperament on animal production

There are several definitions of animal temperament based on the behavioural and

physiological responses of animals that have been subjected to stressful and

threatening environments. The basic trait of temperament is the animal’s reactivity to

a stressor (Boissy, 1995) and this reactivity and the ability to cope with the stressor

varies from animal to animal. Temperament affects productivity in all farm animals

through their responses – their changes in behaviour and in their physiological and

immune systems in both the short term and the long term.

2.1. Animal temperament and stress

2.1.1. Definitions of temperament and stress

Animal temperament has been defined in many ways. The temperament of cattle has

been defined as the behavioural reaction to either human handling (Fordyce et al.,

1985, 1988a; Voisinet et al., 1997) or strange or unfamiliar environments (Kilgour,

1975; Fordyce et al., 1988b; Murphy, 1999). Temperament has also been described as

an animal’s behavioural characteristics caused by changes in the physiological,

hormonal and nervous systems that result in a special disposition compared with other

animals of the species (Kilgour, 1975). Some authors have also described

temperament as ‘emotionality’ (Hall, 1934; Kilgour, 1975; Murphy, 1999), or

personality (Gosling, 2001). It seems that this term is commonly used when

“emotion”, “feeling” or “personality” of the animals are studied. ‘Emotional activity’

should be considered for the description of temperament because the behavioural

responses of animals could be caused by their emotional state (Ramos & Mormède,


5

1998). Hall (1934) considered the emotivity of an animal as being related to the

behavioural and peripheral changes hypothesized to accompany high sympathetic

nervous system activity. Ramos & Mormède (1998) stated that emotion is associated

with behavioural/physiological changes that are generated by non-ordinary situations.

Gosling (2001) found that studying the personality of the animals would provide

opportunities to examine the biological, genetic and environmental bases of

personality, personality changes, links between personality and health and personality

perception.

'Fearfulness' is regarded as a basic trait of the temperament or personality of an

animal, and is considered to be an undesirable emotional state (Boissy, 1995). Boissy

(1995) defined ‘fearfulness’ as the general susceptibility of an animal to frightening

situations from both human handling and the environment. Stress is considered as the

response of an organism to environmental stimuli that threaten its internal equilibrium,

and such stimuli are perceived and evaluated by the ‘emotional system’ (Ramos &

Mormède (1998). Both fearfulness and stress are thus basic traits that reflect the

temperament of animals.

Domestic animals are generally divided into two temperament classes: calm (docile,

quiet) and nervous (aggressive, flighty). Animals that are calm and less stressed in the

presence of humans are said to be of ‘good temperament’ and those that are nervous

and excited while being handled are said to be of ‘bad temperament’. This explains

why calm animals respond less and remain calm when they are either close to a

human, in an unfamiliar environment, or in situations where nervous animals respond

aggressively or show agitation and anxiety (Kovalcikova & Kovalcik, 1982; Fordyce

et al., 1985, 1988b; Voisinet et al., 1997).

In summary, temperament is an animal’s behavioural responses to human handling

and unfamiliar situations and is caused by the emotional states of the animal. These

responses reflect the level of fearfulness or stress of the animal in the situation. Calm

and nervous animals have different attitudes and responses to humans and to


6

threatening environments. Calm animals respond less and adapt more readily

compared to nervous animals.

2.1.2. Measurement of animal temperament

Various systems have been used to measure the temperament of different species and

for experimental purposes. For instance, measurement of movement, agitation and

flight speed are the common tests for beef cattle while vocalization, defecation,

urination, kicking and lifting the legs are common measures for dairy cattle. Arave &

Kilgour (1982) scored the temperament of dairy cows by adding twice the number of

kicks to the number of leg-lifts during milking. However, the authors defined this as

“milking/parlour temperament”. The temperament of dairy animals should be

measured by testing systems that reflect the emotional states of the animals. Kicking

or lifting legs during milking may not represent the temperament of the animals but

only the behavioural responses for the particular situation, in this case milking. As

Ramos & Mormède (1998) indicated, observation of these behavioural responses is

meaningful only if these specific responses are associated with stress and with the

emotional state of the animals.

Of the numerous temperament tests, the two most commonly used are the open-field

test (also called the ‘arena test’) and the box test (also called the ‘box agitation test’).

Both are designed to measure the fear responses or emotional states of an animal to a

strange or unfamiliar environment. They are popular because they are easy to do and

are suitable for different species. The open-field test was originally used to measure

‘emotionality’ in small animals like rats (Hall, 1934) and later was used for dogs

(Thompson & Heron, 1954; Fuller, 1967) and pigs (Beilhardz & Cox, 1967). Kilgour

(1975) successfully used it in dairy cows. A test cow was put in a 22 m2 arena with

walls. Overhead wires divided the area into 36 squares that were used for scoring the

movement of the test animals. Ambulation, vocalization, defecation and urination

were counted. However, Kilgour (1975) found no correlation between the behavioural

responses of cows in the open-field and the subjective scores that the milkers gave the

cows. This suggests that animal temperament should be tested by a measurement


7

system rather than by personal judgement, perhaps because there would be bias and

preference in personal judgement.

The box test was designed to test the agitation of the test animal when it was

separated from the flock or herd and held in an enclosed box. Putu (1988) used this

test on sheep with a box that was 1.5 metres long, 1.5 metres wide and 1.5 metres

high with a slatted wooden floor. Murphy (1999) used the same test and recorded

movement and vocalization of the sheep for one minute. Murphy (1999) also used an

open-field test that included a human and flock mates in the test arena. A person was

placed in the arena and this made the test more applicable because it measured the

response of an animal to the environment where it was tested and also to its

interaction with humans. The theory is that both agitation and vocalization rates will

be greater when animals are separated from their mates or are in unfamiliar

surroundings (Kilgour, 1975; Kovalcikova & Kovalcik, 1982). The box test has been

successfully used to measure the temperament of sheep because it can reflect the

emotional states of test animals.

Behavioural responses in an arena are often regarded as manifestations of fear. For

example, defecation and urination are seen as responses to threatening stimuli in rats

and they result from a triggering of autonomic nervous system activity under stress,

whereas ambulation and agitation are the signs of fear in cattle and sheep (Kilgour,

1975; Murphy, 1999). Defecation during the test is said to be a response to a novel

situation and to manifestation of fear (Kilgour, 1975). However, for sheep, Murphy

(1999) concluded that the elimination of wastes and sniffing of the human in the arena

are not reliable indicators of emotivity because they were much too variable.

Vocalisation seems to be a better indicator of agitation both in cattle and sheep

(Kilgour, 1975; Murphy, 1999).

Temperament has been measured in many different ways and often without much

success because of differences in definitions between authors and variations in the

factors that are assessed in temperament tests. Animals may therefore have different

temperament scores in different tests depending on which factors are incorporated

into the test situation (Fordyce et al., 1982). Temperament tests should measure an


8

animal’s behaviour in response to fearfulness and emotivity. They should thus impose

a controlled stress on an animal, and the animal’s response to this stress can be used

as a repeatable measure of temperament. Wemelsfelder et al (2001) measured the

behaviour of the pigs with a methodology called “free choice profiling”. This

methodology gave the observers complete freedom to choose their own descriptive

terms but they achieved significant agreement in their assessments of the behavioural

expression of the pigs in different tests, and accurately attributed repeatable

expression scores to individual pigs across these tests.

Burrow (1997) suggested that the nature and magnitude of the relationships between

temperament and other productive and adaptive traits should be quantified in order to

predict the likely consequences of changes in temperament through traditional

selection procedures on herd productivity and profitability. Using a combination of

measurements or repeated tests may increase the precision of the measurement and

scoring of temperament, as well as the accuracy in estimating production outcomes.

2.2. Factors related to temperament

Some factors that are from the animal itself can be related to its temperament. Breed,

experience or training, age, weight, body condition and health (e.g. worm condition)

can all be associated with temperament. I will consider three of these here.

2.2.1. Breed

Animal temperament differs between and within species, as well as between

genotypes (Ramos & Mormède, 1998; Pollard et al., 1994). Among the beef breeds,

for example, Bos indicus crosses are considered to have more nervous temperament

than B. taurus (Hearnshaw et al., 1979; Fordyce et al., 1982). Nobody has formally

compared the temperament of beef and dairy cattle so far but, among dairy breeds,

Holsteins are considered to have a better temperament than others (Lawstuen et al.,

1988). Holsteins are also known to have a good milking speed and this is consistent


9

with the general view that good temperament cows have better milking speed and

milk yields than poor temperament cows (Lawstuen et al., 1988).

Pollard et al. (1994) worked with deer and found that hybrid calves showed a stronger

tendency to avoid humans and to be less active in the presence of a human compared

with purebred red deer calves. The hybrid calves were less approachable by a human

and their behaviour was more restricted by a human.

2.2.2. Experience and training

Regular handling and human contact, as well as familiarity with the surroundings and

with husbandry routines have beneficial effects on animal behaviour and improve co-

operation with human handling. Ivanov & Djorbineva (2002) found that the previous

experience of an animal is a major factor that influences the assessment of its

emotional traits. The results from some studies have shown that early training of

heifers was helpful for them to become familiar with the milking room, and was also

useful in their behaviour and attitude to human handling at subsequent milking

(Rushen et al., 2001). Handled heifers are less reactive than non-handled heifers in a

test with human contact. Experiences in the early life of an animal can influence its

attitude in later life (Grandin et al., 1984; Moberg & Wood, 1985) and early handling

improves the human-animal relationship thus reducing animals’ fear of humans

(Boissy & Bouissou, 1988).

2.2.3. Age

Animals become calmer as they age. This is partially because they have had more

contact with humans, more training and a wider range of experiences. Kovalcikova &

Kovalcik (1982) found that younger cows were more active and motivated than older

cows in open field tests. No difference was seen between the breeds that they tested,

the Slovak Spotted and Black Spotted, or their crosses. Other authors have reported

similar results (Dickson et al., 1970; Kilgour, 1975; Hearnshaw & Morris, 1984;

Lawstuen et al., 1988).


10

2.3. Influences of temperament and stress on animal production

Temperament and stress can have negative effects on animal production because of

the behavioural and physiological responses that are evoked. Farmers expend more

time and energy handling nervous or aggressive animals while production or product

quality from those animals may fall.

2.3.1. The effects of temperament in different production systems

2.3.1.1. Beef

Quite a lot research has been done on temperament in beef production systems.

Generally, nervous cattle have more problems in confined circumstances than calm

cattle. For example, stress-induced “dark cutting” of meat costs the beef and lamb

industry $40 million every year in Australia (Wynn, 1994). Higher temperament

scores (more nervous) also lead to higher bruise scores for the back, hips and pin bone

areas compared with lower-scored (calm) cattle (Fordyce et al., 1988b). The carcasses

of nervous cattle have about 1.5 kg per carcass more bruising and dark cut trim than

those of calm cattle (Fordyce et al., 1988b). Cattle of poor temperament also have a

lower rate of weight gain, poorer feed conversion ratios, lower body condition and

dressing percentages than those of good temperament (Petherick et al., 2002). High

score steers and cows tend to produce less tender beef than low score animals

(Fordyce et al., 1988b). Thus, being stressed at slaughter may change the pH of meat

and explain the undesirable outcomes for flavour, tenderness, quality in storage and

“dark cutting” (O’Shea et al., 1974; Fordyce et al., 1988b).

2.3.1.2. Cows’ milk

The temperament of the dairy cow influences both yield and milking speed

(Kovalcikova & Kovalcik, 1982; Lawstuen et al., 1988). Kovalcikova & Kovalcik

(1982) reported that quieter cows that responded to strange environment by a lower

motor activity were likely to have higher production than nervous cows. Lawstuen et


11

al. (1988) found that cows of good temperament tended to milk faster, resist mastitis

better and calve easier than the cows of nervous temperament. Kilgour (1975)

speculated that, as calmer cows were better producers, over time farmers will have

eliminated less productive nervous cows from the herd. Lawstuen et al. (1988)

reported that the heritability of temperament in dairy cows was 12% and heritability

of milking speed was 11% - these values are low but may nevertheless help dairy

producers in the genetic selection of their cows for production. Burrow & Corbet

(2000) confirmed that temperament was a heritable trait but still believed that

experience modifies temperament to a greater extent than genetic selection (Burrow &

Dillon, 1997; Petherick et al., 2002).

It is clear that the temperament of cows affects milk yield, milking speed and mastitis

status but it seems that there only been a single study of the effects on milk

composition. Breuer et al (1999) demonstrated the milk fat and protein were

positively correlated with the fear and behavioural responses of cows. However, the

authors did not define these behaviours in the way that temperament is defined in this

thesis. Work is needed in this area so that farmers will know whether temperament

significantly influences milk fat and protein content.

2.3.1.3. Ewes’ milk

A few researchers have worked on the effect of temperament on sheep milk

production. Functional parameters of the ewes’ udder were studied in sheep of

different temperaments by Ivanov & Djorbineva (2002) who found relationships with

milk production, milk flow rate and milk ejection latency. Calm ewes produced 23%

more milk than nervous ewes when machine milked (Ivanov & Djorbineva, 2002).

Milk ejection latency in nervous ewes was much longer (5.3 seconds) than it was in

calm ewes (1.9 seconds), suggesting that calm ewes ejected their milk much faster

than nervous ewes. In this study, temperament was tested in a milking parlour by

assessment of behaviour. However, the authors did not pursue the problem of why

nervous ewes produced less milk compared with calm ones or how to avoid

incomplete milking in nervous ewes. It is possible that hormonal changes were


12

induced in nervous ewes by the stress of being placed on the milking platform,

putting on teatcups and milking inhibit milk ejection.

As in dairy cattle, milk yield and milk ejection latency in ewes are affected by

temperament and by the level of fearfulness when the animals are machine milked.

More work is needed to determine the role of temperament on milk composition and

quality in ewes.

2.3.2. How temperament affects production

2.3.2.1. Behavioural reasons

An animal’s reaction to stress is to behave differently. There are two opposite fear-

related behavioural responses: the first is active avoidance such as movement, escape

or hiding, and the second is active defence such as attack or threat (Boissy, 1995).

Moving to the other side of the paddock in response to stressors can be regarded as

active avoidance, while aggressive and violent reactions to handlers can be described

as active defences. Behavioural changes also include the number of agitations, bleats,

defecations and urinations. These changes can be induced by the stress of, for instance,

being separated from the flock (Kilgour, 1975; Kovalcikova & Kovalcik, 1982;

Fordyce et al., 1982, 1988a, 1988b; Putu, 1988; Voisinet et al., 1997). Changes in

climate, feed or shelter, transportation or milking will make nervous animals even

more afraid and uneasy.

All these behavioural changes in nervous and sensitive animals may result in them

becoming aggressive to their mates, becoming separated from the flock, decreasing

water and food intake, losing weight, or even falling sick (Fell, 1994; Wynn, 1994;

Fell et al., 1999).

2.3.2.2. Physiological and hormonal changes

Rather than behavioural reactions, the most important effect of temperament on

production is caused by physiological, mainly hormonal, changes under short or long-


13

term stressors (Wynn, 1994; Lean, 1994; Fell et al., 1999; Giles, 1994). For example,

increased respiration rate, wide-open mouth and laboured breathing are caused by

high ambient temperatures. Animals increase their heat loss through evaporation from

the lungs and skin, or by reducing food intake when the air temperature rises above

body temperature (Giles, 1994), and they will die if they cannot overcome severe

hyperthermia (Fell, 1994). Thus, there are powerful physiological changes in an

animal’s body when it is stressed by severe climate or by other stressors in the

surroundings.

The hormones of the hypothalamus-pituitary-adrenal (HPA) axis are involved in

stress responses (Naylor et al., 1990; Moberg, 1991; Wynn, 1994; Boissy, 1995; Fell

et al., 1999). In response to fear and stress, corticotrophin-releasing hormone (CRH)

is released from the hypothalamus and reaches the pituitary gland where it stimulates

the secretion of adrenocorticotrophic hormone (ACTH). ACTH coordinates the

synthesis and release of glucocorticoids such as cortisol, from the adrenal cortex

(Moberg, 1991). Animals also release adrenalin from the adrenal medulla if they are

stressed and the blood concentrations of cortisol and adrenalin increase significantly

after milking (Negrão & Marnet 2003). However, Kilgour & Szantar-Coddington

showed that the adrenal response had little promise as an indirect criterion for

selection of lamb survival.

In addition, oxytocin release is inhibited by stress (Lightman, 1992). Negrão &

Marnet (2003) found that more adrenalin and noradrenalin were released on Day 1

than on Day 15 of lactation when the ewes were machine-milked, and that these ewes

did not show a significant release of oxytocin at the earliest milking. The fact that

cortisol release after milking is related to the duration of lactation suggests that the

animals were stressed by milking, especially during early lactation. Fearfulness of

milking will lessen in a lactation proceeds due to increased familiarity with the

situation.

Dairy animals produce milk by transferring substrates and nutrients to target tissues,

particularly the mammary glands, and both the process of transfer and the


14

responsiveness of the target cells to the supply are controlled by specific hormones,

many of which are responsive to stress. It is therefore no surprise that milk production

will be limited by stress – in particular, it would be acutely affected if the secretion of

oxytocin is inhibited.

2.3.2.3. Change in the immune system

Temperament and stress can also affect the immune system due to the hormonal

changes that they induce. Recent research by Fell et al. (1999) showed that there was

a marked correlation between the flight time and haematological parameters:

circulating cortisol, IgA and total white cell numbers were found to be higher in

aggressive cows than docile ones (Fell et al., 1999). In this study, none of 12 calm

cattle fell sick but 5 out of 12 nervous animals were hospitalized during the

experiment. These results show that physical and hormonal changes related to

changes in an animal’s immune systems can reduce their ability to avoid disease and

limit their productive performance.

Generally, animal production is influenced by temperament and stress in various ways

in farm groups and in individual animals. The examples given above show that poor

temperament can reduce production in beef and dairy cattle and in sheep.

Temperament can also influence the different products from the same animal. For

instance, temperament affects quantity, tenderness, and meat pH in beef cattle and

milk volume and milking speed in both cows and ewes. More work has been done on

the relationship between temperament and animal production in cattle than in sheep

and, in cattle, beef production has been examined in greater detail than milk

production. More research needs to be done in both dairy cattle and sheep on the

relationships between temperament and milking techniques, milk yield and milk

composition.

3. Milk synthesis, milk ejection reflex and removal of milk


15

The synthesis and secretion of milk is a complex process involving a series of

physiological and hormonal changes in the mammary gland and neural systems that

precede the onset and maintenance of lactation. A knowledge of processes that control

milk synthesis and the milk ejection reflex is essential if we are to understand the

factors involved in a successful lactation. This part of the literature review therefore

covers the synthesis of ( protein, fat and lactose) as well as the neural and hormonal

control of the milk ejection reflex and the removal of the milk.

3.1. Milk synthesis

Milk synthesis and ejection are regarded as the two phases of milk secretion. Milk

synthesis is the formation of milk by the cells of the alveolar epithelium in the

mammary gland. The histology and cytology of secretory tissue are similar in all

species even though the mammary glands may differ in size, number and shape. The

primary structure of the mammary secretory tissue is the alveolus. An alveolus is

roughly spherical and composed of one layer of epithelial cells that surround a cavity

or lumen. Each alveolus is surrounded by capillaries that provide blood for milk

synthesis (Wooding et al., 1970; Schmidt, 1971). The main components of milk,

protein, fat and lactose, are synthesized in the epithelial cells in different ways.

Larson (1965) studied the synthesis of milk protein and found that the most important

protein in the milk of ruminants is casein. The protein concentration is about 3.2% in

cows’ and about 5.7% in ewes’ milk (Harding, 1995). The principal families of

protein in cows’ milk are αs1-casein, αs2-casein, κ-casein, β-casein, α-lactalbumin, β-

lactoglobulin, serum albumin and immunoglobulins IgG1, IgG2, IgA and IgM

(Goodman et al., 1983; Farrell et al., 1987). These proteins are formed in different

ways. Ninety percent of total proteins such as the α-casein complex, β-casein, α-

lactalbumin and β-lactoglobulin are synthesized from free amino acids in the

secretory cells in the mammary gland. The remaining 10%, including the

immunoglobulins and blood serum albumin, are absorbed directly from the

bloodstream (Larson, 1965).


16

Fat, another important milk component, is composed predominantly of triglycerides.

The fat concentration is about 3.9% in cows’ milk and up to 7.1% in ewes’ milk

(Harding, 1995). The major precursors for milk fat are glucose, acetate, β-

hydroxybutyrate, the triglycerides of the chylomicra, and low-density lipoproteins

from the blood (Popják et al., 1951a; Schmidt, 1971). Fifty percent of the milk fatty

acids are derived from plasma lipids. These are mainly long-chain acids (Riis et al,

1960). Short-chain fatty acids (from C4 to C14) and some of the palmitic acids are

formed in the mammary gland through lipogenesis (Popják et al., 1951a, 1951b). The

fat in the milk of ruminants contains a higher percentage of short-chain fatty acids

than that in the milk of non-ruminants (Schmidt, 1971).

Lactose is the main sugar found in milk. It is a disaccharide composed of one

molecule of glucose and one of galactose. The primary precursor of lactose is blood

glucose. In the mammary gland, the glucose molecule is phosphorylated to form

glucose-6-phosphate, and then converted into glucose-1-phosphate. The glucose-1-

phosphate is united with uridine triphosphate (UTP) to form uridine diphosphate

glucose (UDP-glucose). UDP-glucose is then united with free glucose to form lactose

with the liberation of UDP. The last step is catalysed by the enzyme lactose

synthetase (Schmidt, 1971; Larson, 1969).

The entire process of milk synthesis and lactation is controlled by a series of

physiological processes. The structure and function of the secretory tissue and the

ducts of the mammary glands are regulated by interactions between sex steroids and

metabolic hormones. After milk is synthesized, it is secreted into the alveolar lumen

and drained away by a small duct called the intercalary duct where it is retained until

the second phase of secretion, milk ejection, itself a neuro-hormonal reflex.

3.2. 3.2. Control of lactation and milk ejection

3.2.1. Endocrine regulation of lactation


17

Hormones are involved in the development of the mammary gland and initiation and

maintenance of milk secretion. The metabolic hormones, prolactin, growth hormone

(GH), placental lactogen (PL), glucocorticoids, thyroxin and insulin are particularly

important. The sex steroids, oestrogen and progesterone are especially required for

mammary growth (Schams, 1976). The roles of these hormones on the maintenance

of milk secretion differ between species. For instance, sheep and goats require

prolactin, GH, adrenal corticoids and thyroid hormone, whereas rabbits initiate or

enhance milk secretion in the presence of prolactin alone (Forsyth, 1986).

Prolactin seems to play an important role in onset and maintenance of milk secretion

in all mammals. It is secreted by the anterior pituitary gland in response to the

suckling or milking stimulus, along with oxytocin from the posterior pituitary gland

(Meites, 1959; Koprowski & Tucker, 1973a; Gorewit et al., 1992). Prolactin is

critically important for initiation of lactation in cows (Oxender et al., 1972; Tucker,

2000). In synergism with sex steroids and thyroid hormones, prolactin and GH are

thought to play an important role in the systemic adjustment of maternal metabolism

during pregnancy and lactation, stimulating the development of the mammary gland

and the differentiation and function of mammary cells to secrete milk (Forsyth, 1986).

However, there are differences of opinion about the function of prolactin. Tucker

(2000) stated that lactogenesis (initiation of milk secretion) is the only function

clearly established to prolactin so far. Buttle et al. (1979) also suggested that placental

lactogen fulfils a role as a stimulator of differentiation of epithelial cells when

prolactin is absent or suppressed in sheep and goats.

Only a small amount of prolactin is needed for the maintenance of lactation in most

ruminants (Schams, 1976) and, although it is essential for the initiation and

enhancement of milk secretion in both ruminants and monogastrics, it cannot increase

milk secretion in some species (Cowie, 1969; Koprowski & Tucker, 1973a; Schams,

1976). Thus, prolactin concentration in blood had little correlation with milk yield in

cows (Koprowski & Tucker, 1973a). Tucker (2000) noted that prolactin does not limit

secretion of milk in cows and goats and, in ewes and mice, it only partially affects


18

lactation (Hooley et al, 1978; Forsyth, 1986). Koprowski & Tucker (1973a) also

reported that the milking stimulus to the teats of cows stimulated prolactin secretion

in early lactation but not in late lactation.

For the maintenance of lactation, GH is more important than prolactin in ruminants

(Forsyth, 1986). It has been known for a long time that administration of GH can

increase milk yield in dairy cows (Hutton, 1957). In a more recent study with dairy

ewes by Fernendez et al. (1995), injection of 160 mg of bovine somatotrophin BST

increased milk yield by 34% during Weeks 3-8 of lactation and by 53% during Weeks

11-23 of lactation. Baldi (1999) also found that treatment with BST increased milk

yield by 20-30% in dairy ewes and 14-29% in dairy goats.

GH seems to act by partitioning available energy away from tissues and toward milk

production (Forsyth 1986). Baldi (1999) supported this by demonstrating that there

was no difference between the dry matter intakes of BST-treated and control groups,

showing that BST improved the metabolic efficiency of the animals.

The concentration of glucocorticoids remains low until parturition, at which time

large amounts are secreted, perhaps in association with the stress of the birth process

(Tucker, 2000). Among the glucocorticoids, cortisol is the predominant hormone with

a major function in the mammary gland in cattle, sheep and goats. In association with

other hormones, it causes differentiation of the lobulo-alveolar system and sets up

lactation (Tucker, 2000; Peterson & Linzell, 1974; Cowie & Tindal, 1971). Thus,

injection of glucocorticoids into non-lactating cows with a well-developed lobulo-

alveolar system can induce lactation (Tucker & Meites, 1965). The stimulus of

milking releases glucocorticoids and, in contrast with the situation with prolactin, this

response is maintained throughout the lactation (Koprowski & Tucker, 1973b).

However, cortisol concentration is negatively associated with milk yield in machine-

milked ewes (Negrão & Marnet, 2003), suggesting that the animals might produce

less milk under stress because cortisol levels are increased.

3.2.2. The mechanism of milk ejection


19

In this step, milk is expelled from the alveoli and ducts towards the teat. This process

is known as the milk-ejection reflex, milk let-down or draught (Cowie et al, 1951).

Milk ejects under hormonal and neural control rather than the direct control of the

central nervous system. Most animals need a suckling or milking stimulus to promote

the secretion of the hormones that control milk ejection.

The neuroendocrine mechanism that controls milk ejection was first proposed by Ely

& Petersen (1941). A neural stimulus from the teat, due to the sucking of the young or

stimuli from milking, reaches the central nervous system and causes the posterior lobe

of the pituitary gland to release oxytocin. The oxytocin is then carried to the

mammary gland in the blood and there it causes contraction of the myoepithelial cells,

thus forcing the milk from the alveoli into the small ducts (Ely & Petersen, 1941).

Wooding at al. (1970) demonstrated the same effect of oxytocin through electron

microscopic observation of milk ejection.

Ely & Petersen (1941) stated that milk ejection was not under the direct control of the

central nervous system. However, the central nervous system influences lactation by

regulating the activity of the hypothalamo-hypophyseal axis and the output of

pituitary hormones, and by controlling the blood flow through the mammary gland,

thus regulating the supply of hormones and precursor substances to the tissue (Cowie

& Tindal, 1965).

Efferent and afferent innervations are involved in milk ejection in different ways. The

efferent innervation of the mammary gland is sympathetic. Efferent fibres innervate

the smooth muscles within or surrounding the teat meatus and this keeps the meatus

closed between milkings. Stimulation of these efferent sympathetic nerves also causes

vasoconstriction, reducing milk secretion by decreasing blood flow to the udder.

Afferent innervation arises primarily from the sensory nerve fibres in the teat and skin,

and it is involved in the initiation of the milk-ejection process (Schmidt, 1971).

In summary, the sucking stimulus is required for the ejection of milk. Sucking or

udder stimulation causes secretion of the milk let-down hormone, oxytocin, from the


20

posterior pituitary gland. The central nervous system plays a role in lactation by

regulating the hormones that control blood flow to the udder. Efferent innervation is

also involved in milk ejection as it also influences blood flow.

3.2.3. Oxytocin release and milk removal in cows and ewes

Oxytocin is the predominant hormone that controls milk ejection and removal. A

threshold oxytocin level of 3-5 pmol/l is required to induce alveolar milk for a

complete milking in dairy cows (Schams et al., 1984). The injection of oxytocin after

normal milking removes extra alveolar milk in ewes (Heap et al., 1986). Manual

stimulation before milking helps animals release oxytocin in both cows and ewes

(Mayer et al., 1984, 1991; Bruckmaier et al., 1997a). In dairy cows, milk yield and

milk flow are improved by manual stimulation while milking time is reduced (Mayer

et al., 1984). In this study, the concentration of oxytocin reached 11.1 pmol/l after 30

seconds and 16.4 pmol/l after manual stimulation of the udder for one minute. Milk

yield and milk flow were significantly higher in the group with manual stimulation

than the group without stimulation (Mayer et al., 1984). Similar results were observed

in dairy ewes by Bruckmaier et al. (1997a), who compared Ostfriesian and Lacaune

dairy sheep in terms of their oxytocin release and milking characteristics. While the

concentration of oxytocin was increased dramatically in Lacaune ewes in response to

the stimulation for two minutes and the start of milking, oxytocin concentration was

elevated only slightly in Ostfriesian breed in the same. However, milk yields were

increased by teat stimulation in both breeds in early lactation (Bruckmaier et al.,

1997a).

Oxytocin release and milk ejection patterns have been studied more closely in sheep

and the data have explained the differences between individuals and breeds.

Labussière (1988) studied Sardinian and Lacaune ewes and discovered two types of

milk flow: “one emission” and “two emissions”. Ewes with two emissions gave

cisternal milk successfully and most of the alveolar milk, while the ewes with one

emission held a certain portion of the milk and fat in the “upper part of the udder”.

Later, Mayer (1989) showed that the different types of milk flow were due to

differing releases of oxytocin in different breeds. The pooled mean oxytocin


21

concentration during milking was elevated to only 15 pg/ml in the East Friesland and

to about 30 pg/ml in the Lacaune ewes. The oxytocin release always occurred before

the second flow peak in Type 2 (two emissions) ewes. If the ewes did not release

oxytocin, a second flow peak was not initiated, thus leading to Type 1 (one emission).

Because ewes with one emission do not release oxytocin, they do not eject their

alveolar milk. This pattern is more common in ewes that have not been selected for

milking. It may be related to them being less habituated to milking and becoming

stressed during the process. Labussière (1988) proposed that the failure to release

oxytocin may be due to stress or to adrenergic functions as a response to disturbances

during milking.

In conclusion, sheep show milk flows with one or two emissions. The one-emission

pattern is more common in non-dairy animals whereas two-emission pattern is

common in dairy animals. Sheep with only one emission do not eject milk because

they do not release oxytocin when they are milked. The milk collected from the first

emission is cisternal and low in fat. Ewes with one emission have less fat in the milk,

lower total lactational outputs as well as a shorter lactation. Non-dairy sheep may

have one emission and not eject their milk because they are nervous. If that is the case,

milk from nervous sheep would differ in yield and also composition from the milk

from calm sheep.

4. The effect of stress on milk synthesis, yield and composition

Many factors affect milk yield and composition. Some are extrinsic factors, such as

environment, season, nutrition and management, while others are intrinsic factors like

genetics, age, endocrine system or structures of the mammary glands. Other than

these, the stress and fearfulness from milking may also influence the yield and quality

of milk. This part of this literature review will show how nutrition, season/lactation

and stress in the milking environment, animal-human interaction and milking

techniques affect milk yield and composition. Furthermore, I discuss how stress


22

affects milk synthesis and removal. Consequently, we will see how stress influences

milk output, and protein and fat levels in milk.

4.1.Factors that affect milk yield and composition

The quantity and quality of milk produced depends on the amount of mammary tissue

that is available to produce that milk and also on the secretory efficiency of cells in

which milk components are made. The quantity and quality of milk also depend on

hormones that develop mammary tissue or control milk ejection and removal, and on

the availability of suitable nutrients from which the tissue manufactures milk.

4.1.1. Nutrition

Nutrition is a major factor influencing milk yield and composition as the cells of the

mammary gland require a constant and optimum supply of precursors to synthesize

milk components. The amount of food and the intake of energy affect milk yield as

well as milk composition. Specifically, energy intake influences the composition and

amount of milk fat. When lactating animals have a positive energy balance, the fatty

acids in milk will change depending on whether the animal has a high carbohydrate

level or a high fat level in the diet. As energy balance decreases and becomes negative,

dietary supplies of acetate and glucose decrease. This reduces the synthesis of short-

chain fatty acids by the mammary tissue and increases mobilization of adipose tissue

and incorporation of long-chain fatty acids into milk fat (Belyea & Adams, 1990;

Palmquist et al., 1993).

Diets that alter fermentation affect fat content because the precursors required by the

mammary gland to synthesize fats are generated in the rumen (Nickerson, 1995).

Milk protein synthesis is influenced by the factors that regulate microbial growth

because the amino acids required for milk protein synthesis are derived from micro-

organisms in the rumen. Starch is also necessary to maintain microbial populations

and subsequent microbial protein synthesis (Palmquist et al., 1993; Nickerson, 1995).

For example, at low feeding rates, an increased proportion of grain improves milk

production and protein content. However, higher grain intakes (above 50% of dietary


23

DM) decrease milk fat content because of the increased intake of fermentable starch

(Palmquist et al., 1993).

Lactating animals need diets high in energy and all the substances required for milk

synthesis. Both the amount and the composition of the diet influence milk yield and

composition. A negative energy balance would decrease milk yield as well as milk fat

and protein content.

4.1.2. Season/lactation period

Nutritional variation, due to changes in the availability and quality of pasture and feed

through the year, and physiological changes during lactation are two of the most

important factors that influence milk production (Lucey & Fox, 1992; Kefford et al.,

1995; Auldist et al., 1998). Farm animals have a peak in milk yield shortly after the

start of lactation then the yield gradually decreases until the end of the lactation. Milk

fat, protein and lactose concentrations also vary as lactation progresses. The

concentrations of fat and protein tend to increase (Auldist et al., 1998) because, as

Bencini & Purvis (1990) observed, there is a negative correlation between milk output

and the protein and solid content of the milk. Similarly, Lucey & Fox (1992) found

that the concentration of milk fat and total protein were higher, whereas casein and

lactose were lower, in late-lactation than in mid-lactation milk. It should be

emphasized that cow’s milk from late lactation (after 250 days of lactation in this case)

is high in total protein, low in casein and high in whey protein. On the other hand,

Kefford et al. (1995) disagreed, having observed that casein levels were higher in

late-lactation milk. This may have been due to the diet that their cows were fed

because there was an interaction between diet and stage of lactation for casein and

whey protein (Kefford et al., 1995). However, the total outputs of fat and protein were

higher in early lactation than in late lactation as more milk was produced in early than

in late lactation.

There is an interaction between the effects of the time of year (season) and stage of

lactation on the concentrations of protein, casein and lactose. The difference between


24

the concentrations of these components in early lactation and late lactation is larger in

winter than in summer (Auldist et al., 1998).

Heat or cold stress is another seasonal factor that influences milk yield and

composition. The most suitable temperature for dairy cows to achieve maximum milk

production is 10-20 ºC and heat and cold outside of this range adversely affect milk

yield (McDowell, 1981). Percentage of fat and protein are lowest during summer

(Bruhn & Franke, 1991; Auldist et al., 1998; White et al., 2004) because heat stress

decreases feed intake by reducing the rate of passage and increasing water

consumption (McDowell, 1981). A low ratio of acetate to propionate, from rumen

fermentation, may be the reason for the low fat content. A reduced amino acid intake

in the hot months may affect the protein content of milk produced. Cold stress has

less impact on milk yield and composition than heat stress because feed intake is

increased to fulfil the animals’ need to maintain body temperature (McDowell, 1981).

In summary, the interactions between season and stage of lactation have a large

impact on milk yield and composition. Yield reaches a peak in early lactation and

then decreases gradually until lactation is complete. Milk fat and protein levels

increase as lactation progresses. Any seasonal stresses that lower energy balance, such

as poor nutrition or hot and cold weather, reduce milk production.

4.2. The inhibition of milk let-down by stress

Farmers know well that animals should not be frightened during milking because

stress inhibits milk ejection. The milking environment, including both the facilities

and the milker, may also influence milk secretion. For instance, animals may be

frightened when they are milked in strange environments or milked by a new milker

(Bruckmaier et al., 1993, 1997b). For the same reasons, it is more difficult to milk

young heifers as they are not accustomed to milking (Holmes & Wilson, 1984). This

may be explained by stress-induced secretion of adrenalin and the effects of this

hormone on the milk ejection reflex.


25

4.2.1. Adrenalin

As early as 1941, Ely & Petersen had studied changes in the milk ejection reflex when

lactating cows were startled. They found that milk ejection ceased in a lactating cow

when the cow was frightened by an exploding paper bag or by placing a cat on its

back (Ely & Petersen, 1941). Interestingly, in lactating women, emotional

disturbances such as embarrassment, pain or discomfort have been found to block

milk ejection (Newton, 1961).

Fright and stress activate the sympathetic neuro-adrenal system and cause the release

of adrenalin and this is thought to inhibit milk ejection (Ely & Petersen, 1941; Hsu &

Crump, 1989). Adrenalin is thought to act in two ways. First, adrenalin and

noradrenalin interfere in synaptic transmission in the central nervous system, at the

the supraoptic nuclei, the posterior hypothalamus and mesencephalic reticulum. This

leads to vasoconstriction in the mammary gland that prevents oxytocin from reaching

the myoepithelial cells (Ely & Petersen, 1941). Second, adrenalin is a physiological

antagonist to oxytocin (Denamur, 1965; Schmidt, 1971) so might directly inhibit milk

letdown by a direct action on mammary tissue.

There is no doubt that stress or fearfulness stimulates the secretion of adrenalin and

that adrenalin causes vasoconstriction in the udder. Whether this prevents oxytocin

from reaching the myoepithelial cells and thus effecting milk ejection is not clear

because hormones like oxytocin generally exert their effects on the basis of

concentration, not availability. It seems more likely that stress and adrenaline block

milk ejection, and influence milking characteristics and yield by blocking the action

of oxytocin.

4.2.2. Milkers and milking techniques

Milkers and milking technique influence the comfort of the animals, and thus the

milking results, as does the milking environment. The way in which animals are


26

handled and milked determines whether or not normal milk ejection occurs under

these circumstances. Research on the effects of handling on milking behaviour and

milk yield was performed by Rushen et al. (1999). The presence of an aversive

handler decreased milk yield and increased residual milk by 70%. Heart rate and

movement during milking increased when the aversive handler was present but milk

yield and residual milk did not change when a gentle handler stood by the cows

during milking (Rushen et al. 1999). This shows that different handling techniques

can affect efficiency of milking and milk yield.

Other techniques, such as machine milking, hand milking or feeding during milking

also affect milking characteristics. Cows produce more milk when milked by hand

than by machine (Uvnäs-Moberg et al., 2001). More sensory stimulation is applied by

the hands during the procedure in hand milking, and this increases the secretion of

lactational hormones and leads to a better milk ejection. Furthermore, it has been

reported that oxytocin adapts behaviour and physiology to facilitate lactation in

mammals, including cattle, besides its main function in milk let down (Uvnäs-Moberg

et al., 2001). Oxytocin promotes bonding between individuals and mother and young

in sheep (Keverne & Kendrick, 1994; Kendrick et al. 1987). If that is the case,

animals should have better behaviour, rearing abilities and temperament because of

continued oxytocin release during lactation.

4.2.3. Milking environment

The milking environment includes the place where animals are milked, the milking

facilities that are used in milking and milkers or other humans who are involved in

milking or feeding. The milker, milking facilities or other factors like loud sounds or

feeding during milking may have an impact on the removal of milk as they change the

levels of milk ejection hormones and the hormones that inhibit milk ejection.

New environments or strange noises cause anxiety that may cause problems in

milking. Cows secrete less oxytocin when they are milked in an unfamiliar room,

have more residual milk (milk left in the udder) and produce a lower milk yield


27

(Rushen et al., 2001). Milking the animals in a familiar and quiet place is more

comfortable and acceptable to them. Some farmers play music to avoid sudden sounds

that shock the animals (Bencini 2005, personal communication).

Young heifers may not have a normal milk ejection before they become familiar with

the milking routine (Holmes & Wilson, 1984). They should therefore be allowed to

become accustomed to the yard and milking shed before they calve (Holmes &

Wilson, 1984).

Feeding the animals during milking can induce oxytocin release. Oxytocin is released

by feeding in dogs and sows (Uvnäs-Moberg et al., 1985). Both oxytocin and

prolactin levels are higher in cows that are fed during milking (Svennersten et al.,

1995; Johansson et al., 1999). However, the release rate of prolactin is not as high as

it is when the cows are milked (Svennersten et al., 1990). Feeding during milking

increases milk flow, shortens milking time and increases milk yield (Johansson et al.,

1999). Johansson et al. (1999) found that milking-related release of cortisol was

almost absent when feed was provided to lactating cows. The lowered cortisol levels

when the cows were fed and milked at the same time may be an expression of an

enhanced stimulation of the anti-stress-like pattern of oxytocin-mediated effects, and

a response to the increased amount of sensory stimulation (Uvnäs-Moberg et al.,

2001).

In brief, milk characteristics and milk yield are influenced by handling and milking

techniques. Aggressive handling decreases milk yield by increasing residual milk.

Hand milking is more efficient to remove milk than machine milking as the

stimulation during hand milking can induce more oxytocin release. Unpleasant factors

before or during milking can inhibit normal milk ejection so animals should be

milked in a quiet and peaceful environment. It is important to feed the animals during

milking as this may improve oxytocin release and reduce stress at the same time.

4.3. The effects of stress on milk composition


28

Stresses from the environment and from milking may affect milk composition by

affecting the delivery of nutritional substances to the mammary glands or by inducing

hormonal changes that influence milking efficiency. The restriction of blood flow

through stress-related adrenalin release (Ely & Petersen, 1941) may lead to less

nutritional substances being delivered to the capillaries around the alveolar epithelium

where milk is synthesized. Furthermore, residual milk increases when incomplete

milking occurs causing changes in milk composition.

4.3.1. How fat level is affected by stress

Milk fat concentration rises during milking. This is best explained by the variation in

fat content as it leaves the udder (Schmidt, 1971; Labussière, 1988; Nickerson, 1995).

The level of fat in the milk depends on how much of the milk is removed from the

udder. Residual milk can contain up to 20% fat (Nickerson, 1995). The amount of

residual milk increases when oxytocin release is inhibited; for example when the

animals are milked in an unfamiliar environment (Rushen et al., 2001; Macuhová et

al., 2002), in the presence of an aggressive handler (Rushen et al., 1999) or when the

animals are milked without feeding (Johansson et al., 1999).

Non-dairy animals do not eject their alveolar milk, which increases residual milk and

decreases fat in milk that can be extracted from the mammary glands (Labussière,

1988; Mayer, 1989). In dairy cows, the first-drawn milk may only be 1-2% fat, while

at the end of milking, fat content is normally between 5 and 10% (Nickerson, 1995).

The difference is even larger in ewes that are machine milked. In ewes with no milk

ejection reflex, 75% of the fat can be retained in the udder because the milk that can

be removed is cisternal and high-fat alveolar milk is left behind (Labussière, 1988).

Similarly, even in dairy sheep, animals that are stressed during milking will have poor

milk ejection and their alveolar milk will not be removed.

4.3.2. How protein level is affected by stress

Few authors have studied the relationship between stress and protein content in milk.


29

It is possible that constricted blood flow caused by stress affects the delivery of

nutrients to the udder for the synthesis of milk protein (Schmidt, 1971). So, when

animals are stressed, fewer amino acids are available for protein production. The

second possible effect of stress on milk protein concentration may be related to

oxytocin release during milking. Recent research by Negrão & Marnet (2003) has

shown that cortisol and adrenalin levels increased significantly after milking,

suggesting that milking is a stressor. Negrão & Marnet (2003) also found that the

oxytocin level at milking was positively related to milk yield, fat and protein. They

did not explain how milk protein would be affected by oxytocin but their findings

suggest that, when the milking becomes a stressor, the levels of stress hormones are

increased and oxytocin level is decreased, resulting in low yields of milk with low

levels of fat and protein.

The third possibility of the impact of stressors on the level of protein in milk is that

stress activates the plasminogen-plasmin system (PPS), an enzymatic mechanism in

milk that breaks down casein (Silanikove et al., 2000). Plasmin is the predominant

protease in milk and it produces boiling-resistant peptides from β-casein, αs1-casein

and αs2-casein (Andrews, 1993). Plasminogen is an inactive form of plasmin (Politis

et al., 1989) and the conversion of plasminogen to plasmin is modulated by

plasminogen activator (PA) (Politis et al., 1990). PA inhibitors are produced by the

mammary epithelial cells and secreted into milk (Politis et al., 1990), preventing

plasmin activity from breaking down milk casein. When stress occurs, cortisol from

the activated hypothalamus-pituitary-adrenocortical axis causes liberation of PA from

mammary epithelial cells into the mammary cistern where it activates the

plasminogen-plasmin system and increases the break-down of caseins (Silanikove et

al., 2000). This break-down of caseins may affect the total amount of caseins, as well

as protein concentration, because casein is the major protein in milk (about 80%).

Thus, milk protein could be affected by the level of stress at milking because of

insufficient supply of amino acids to the mammary glands, decreased oxytocin level

or increasing plasmin activity. If so, nervous animals would produce less protein in

milk than calm animals.


30

5. The effects of milk composition on the processing performance of milk for

cheese

The production of high quality raw milk is important for cheese manufacturers as the

quantity and quality of cheese (Storry & Ford, 1982b; Storry et al., 1983; Bencini,

1993, 2002). High cheese yield is obtained from a high milk yield, but high cheese

yield can also be achieved from milk of high fat and casein levels (Chapman, 1981;

Lucey & Fox, 1992). This part of the literature review will examine how the

composition of milk alters the constituents of cheese and how milk protein, casein and

fat affect cheese yield and quality.

5.1. Process of cheesemaking

Cheese is one of the most popular dairy products because of its nutritional value and

long shelf life. The nutritional value of cheese is high because it is high in protein,

calcium and minerals. The shelf life of most cheeses is much longer than that of raw

milk, pasteurised milk or yoghurt.

5.1.1. Conversion of milk into cheese

Cheese is made by fermenting lactose to lactic acid and converting the solid

constituents of milk, mainly fat and casein, to curd by removing water in the form of

whey (Green & Manning, 1982; Chapman, 1981). When cheese is made, milk is

fermented and clotted into a solid curd at a certain temperature and pH by adding

‘rennet’ (the most widely used enzyme that is extracted from the fourth stomach of a

young milk-fed ruminant), also known as ‘chymosin’ (EC3.4.23.4), ‘coagulant’ and a

‘starter culture’ (Tamime, 1993; Walstra et al., 1999). This process is called

coagulation and the time taken for coagulation to occur is called ‘clotting time’ or

‘rennet coagulation time’.


31

After coagulation, the curd is cut to allow removal of the whey, a process that is

called ‘syneresis’. The last step of making cheese is to concentrate the curd and to salt

it. The different types of cheese (hard, semi-hard and soft cheeses) are determined by

the proportions of the components, fat and ‘cheese solids not fat’ (mainly casein), that

are present, and the proportion of moisture retained in the cheese (Chapman, 1981;

Tamime, 1993).

Some components of milk are indispensable for cheese making. Casein is the most

important component because it is related to coagulation of milk, an integral stage of

cheese production. Ninety percent of the casein fractions of milk protein are αs, β and

κ-casein and they are usually present in a ratio of 4:2:1 (Hill & Wake, 1969). κ-casein

is readily and quantitatively available for milk clotting when rennet is added because

of its micelle-stabilizing ability (Hill & Wake, 1969). The casein micelles are

subjected to specific proteolysis by the rennet to strip off the hydrophilic portion of κ-

casein (Dalgleish, 1979). Coagulation occurs after the addition of rennet at a time that

depends on the prevailing rate of κ-casein hydrolysis, followed by random

aggregation of casein particles to form a network (Storry & Ford, 1982a; Dalgleish,

1979; Jenness, 1979; Green & Manning, 1982).

Calcium is the other substance necessary for milk coagulation because casein

aggregates only in the presence of divalent ions (Tamime, 1993). There is evidence to

show that the coagulation of cows’ milk is improved by the addition of calcium

(Storry & Ford, 1982b) and that milk is coagulated more effectively when it contains

a higher level of either casein or calcium (Chapman & Burnnet, 1972).

Cheese making is a complex procedure that converts milk components into a solid

curd through fermentation and coagulation with the addition of rennet. Casein and

calcium play important roles in the process of converting milk into cheese.

5.1.2. Clotting properties of milk


32

The clotting properties of milk are considered to be important because they determine

the yield and quality of cheese (Storry & Ford, 1982a; Green & Manning, 1982).

These properties usually include the speed of coagulation, the firmness of the curd

and curd consistency. Clotting properties are influenced by the temperature at which

the milk is clotted, by the amount of rennet, by pH and by the levels of calcium, fat,

protein and casein in the milk.

Clotting time is decreased with reduced pH and increased temperature, and increased

by the dilution of the milk. Increasing temperature from 25 to 50°C reduces milk pH

and shortens clotting time (Storry & Ford, 1982b). Bencini (2002) also found that

clotting time was reduced when the coagulation temperature was increased from 30 to

38°C for both ewes’ and cows’ milk. However, milk does not clot when it is heated

for more than 30 minutes above 75°C (Ustunol & Brown, 1985). Balcones et al.

(1996) reported that milk pH affected the rennet clotting time and the curd firming

rate in sheep milk. The rennet clotting time was decreased when the pH of the milk

was adjusted from 6.8 to 6.2 (Balcones et al., 1996).

Besides pH and temperature, coagulation of milk also depends on the ability of casein

to form a network (Green & Manning, 1982; Howells, 1982). A linear relationship

exists between coagulation strength and the concentration of casein. In other words,

the casein level in milk strongly affects clotting properties. Rennet clotting time is

increased by diluting milk (Storry & Ford, 1982b). Coagulum strength one hour after

clotting is increased by reducing pH, by adding CaCl2 and, particularly, by increasing

total casein (Storry & Ford, 1982b).

Some clotting properties can be improved by adjustment of cheese making techniques,

but not others. Coagulation time, for example, can be improved by reducing pH, the

addition of CaCl2 or increasing the rennet concentration in mid-lactation milk.

However, these adjustments do not produce better results in early-lactation milk

(Lucey & Fox, 1992). On the other hand, other clotting properties of milk such as

slow aggregation or low gel firmness are not improved by the adjustment of pH


33

(Lucey & Fox, 1992). These characteristics may depend more on the milk

components.

The clotting properties of milk alter cheese yield and quality in two ways. First, the

speed of coagulation and its firmness are determinant in cheese yield and quality

(Storry & Ford, 1982a). Second, the coagulation of milk and the texture of the cheese

depend mainly on the manner in which the casein coagulates and on the extent of

accompanying proteolysis (Green & Manning, 1982). If there are any changes in the

conformation of casein or the structure of the proteolysis, the firmness of the gel as

well as the quality of the cheese will be affected. For example, milk with a long

clotting time and low gel firmness usually develops into a poor quality cheese as low

curd firmness decreases syneresis (Nsofor, 1989) producing a cheese of high moisture

content.

The clotting properties of milk are very important for cheese making as these

characteristics affect cheese output and quality. Whether or not a good curd is

obtained depends on pH, temperature, addition of calcium and the composition of

milk. Clotting time may be improved by the adjustment of pH, temperature, CaCl2

level and rennet concentration, but firmness of the curd and texture of the cheese are

more related to the composition of the milk.

5.2. Milk composition and clotting properties of milk

Changes in milk composition affect the clotting properties of milk and the yield and

quality of cheese. Changes in milk composition due to season have a large impact on

cheese making. Adjustments to the manufacturing process are sometimes needed to

produce an acceptable curd. Some milks are better than others for cheese making

while some, because of their composition, are not even suitable for clotting. For

instance, cows in late lactation fed on high quality feed produce more milk and more

cheese with low moisture. The complex interactions of milk protein and other

components may be the reason that cheese making is affected (Kefford et al, 1995).


34

Mastitic milk does not clot because of a high pH, a high soluble protein and a low

casein content (Green & Manning, 1982). Overall, each component of milk has its

own function in cheese making and influences the cheese in its own way.

5.2.1. Casein and fat concentrations and clotting properties of milk

Casein and fat are the two main components that determine cheese yield as they are

the main sources of the solid constituents in milk that are transferred into curd

(Chapman, 1981; Green & Manning, 1982; Storry & Ford, 1982a). Milk with more

fat and casein leads to high cheese yield (Chapman, 1981; Howells, 1982). Casein

levels in the milk also influence the quality of the cheese produced. For example, late-

lactation milk has long clotting times, slow rates of aggregation and low curd

firmness because of its high pH and low casein content (Lucey & Fox, 1992).

Fat is important for cheese making not only because it affects the cheese yield but

also because the proportion of fat is related to cheese quality (Chapman, 1981). When

the fat content is too high, it will be difficult to control moisture in cheese because

more fat is lost in the whey (Howells, 1982; Storry et al., 1983). It is generally

accepted that a casein to fat ratio of 0.7:1 is best for cheese making (Howells, 1982;

Chapman, 1981; Harding, 1995). Milk is standardised to this ratio in some cheese

factories by removing a portion of fat from whole milk or by adding a quantity of

skimmed milk or cream if the casein to fat ratio of the milk is not suitable (Chapman,

1981).

5.2.2. Protein level and clotting properties of milk

It is clear that the total protein in milk has an impact on cheese due to the relationship

between the contents of casein and protein. A few researchers have shown that there

is a positive relationship between the concentration of protein and cheese output and

quality (Pellegrini et al., 1997; Storry & Ford, 1982b). Coagulation strength was

increased with an increased protein level and calcium concentration in cow’s milk in

the studies by Storry & Ford (1982b). A linear relationship between rate of firming

and protein concentration was observed by Bencini (2002). Increased protein


35

concentration decreased the renneting time and increased curd consistency in sheep

milk. Bencini (2002) compared the clotting properties of ewes’ and cows’ milk and

concluded that sheep milk is more suitable for cheese production as it has a higher

concentration of protein and fat than does cows’ milk and, as such, has better clotting

properties (Bencini, 2002). Higher yielding, better quality cheese can be made by

increasing the protein content in the milk from which the cheese is made.

5.3. Measurement of clotting properties of milk

In most of the studies that were described in this literature review the instruments

used to measure clotting properties of milk were different. In addition, in these

publications (Dalgleish, 1980; Storry & Ford, 1982a, 1982b; Storry et al.,1983) the

clotting time was visually assessed by observing the milk in a glass vessel, and the

curd consistency was studied one sample at a time (Bencini, 1993).

Foss Electric (Denmark) released the Formagraph when it was developed in 1980s.

This instrument could process 10 samples at a time. It can provide objective

measurement of clotting time and rate of firming, and the curd consistency depends

on the machine. Later, Foss Electric (Italy) used a better design, the

Lactodynamograph. It uses a computer-driven disk instead of the paper that was used

in the Formagraph. With these machines, objective measurements of clotting

properties became available.

6. Conclusions

Stress may affect the pattern of oxytocin release, resulting in impacts on milk

production. Inhibition of oxytocin secretion affects milk removal which, in turn,

results in increasing residual milk in the alveoli. Total fat level decreases when more

alveolar milk is retained because the alveolar milk is richer in fat than the first

fraction of milk removed.


36

Fat and protein concentrations might be decreased when an animal is stressed.

Vasoconstriction caused by stress might change the fat and protein contents in milk

by reducing blood flow to the mammary glands, which may reduce the supplies of

lipids and amino acids for milk synthesis. When casein is broken down by the

plasminogen-plasmin system that has been activated by stress, total milk protein may

be decreased.

Animals with good temperament may have better milk production because they have

a better ability to cope with stress than nervous animals. However, the influence of

animal temperament on milk yield and its composition needs to be examined because

calm and nervous animals are likely to have different milking characteristics. When

the milk composition is affected by temperament, it will consequently affect the

clotting properties of the milk that, in turn, affect both cheese yield and quality.

My area of study is the effect of temperament of sheep and cattle on milk production,

composition and clotting properties. The aim of the project is to determine whether

improved milk and cheese quality can be achieved by selecting or screening for

animals of calm temperament. The general hypothesis that I am going to test in this

thesis is: ewes and cows with calm temperament have more milk with higher protein

and fat concentrations than the ewes and cows with nervous temperament.

Chapter 2 Oxytocin Dose-responses in Calm and Nervous Sheep 37

Chapter 2

Experiment 1

Oxytocin dose-responses in calm and nervous Merino ewes

Introduction

Dairy animals normally eject milk in response to oxytocin that itself is secreted in

response to the sights and sounds associated with milking or stimulation from

cleaning or washing the teats (Linzell, 1972; Holmes & Wilson, 1984). As a result,

the milk in the udder is squeezed downwards into the teats and most of it can thus be

removed from the udder. However, non-dairy animals release little or no oxytocin so

that milk ejection does not occur, and only a small proportion of milk contained in the

udder can be removed (Labussiére, 1988; Mayer, 1989). To assist in the milking of

non-dairy animals, oxytocin is injected either intravenously or intramuscularly.

Bencini (1993) found that intramuscular or intravenous injection gave similar results,

but showed that the amount of milk removed, as well as the composition of that milk,

were affected by the dose of hormone. The dose-response for volume confirmed the

reports of several other studies (Linzell, 1972; Doney et al., 1979; Bencini et al, 1992;

Bencini, 1993, 1995). Bencini (1995) concluded that the best dose for removing milk

from Merino ewes was 1 International Unit (IU), and that oxytocin injections did not

affect the fat content of the milk produced until that dose rate reached 5 IU.

There are differences between breeds and individuals in endogenous oxytocin release

at milking. Mayer (1989) found that 47% of East Friesland ewes released oxytocin

during milking while only 18% of Lacaune ewes did so. The reason for differences

between individuals within a breed was not clear. Doney et al. (1979) suggested that

the injected dose of oxytocin must be greater than endogenous doses to counteract the

action of adrenalin that the animals released due to stress. Thus, one possible

explanation of between-animal variation is differences in the responsiveness of their

adrenergic pathways to stressors from milking (Labussiére, 1988). In support of the


possibility that calm and nervous ewes from the same breed may release different

amounts of oxytocin during milking is the observation that, in cattle, the calm cows

are less stressed by human handling than the nervous cows (Petherick et al., 2002). If

the stress level is greater in nervous than in calm ewes at milking, their adrenalin

levels would be higher, so nervous ewes would need a bigger dose of oxytocin to

remove milk from their udders.

The aim of this experiment was to study responses to oxytocin in calm and nervous

Merino ewes to test the hypothesis that the calm ewes need a smaller dose of

intramuscular oxytocin than the nervous ewes for maximum milk removal. The

experiment also determined the dose of oxytocin that would be suitable for the

milking of those ewes in subsequent experiments and tested whether differing doses

of oxytocin altered the protein and fat contents in milk from calm and nervous ewes.

Materials and methods

Five calm and 5 nervous Merino ewes with single lambs were selected from a flock

that has been established to generate calm and nervous animals by genetic selection.

The “Allandale Temperament Flock” has been selected for 15 years, primarily on

their temperament, at the research farm of The University of Western Australia. The

original sheep from 1990 and 1991 were the male and female progeny of commercial

Merinos ewes of mixed ages that had been joined with commercial Merino rams of

the Australian Merino Society (AMS). Selection of the weaners at the age of 12-14

weeks was based on an assessment of temperament in an arena test and a box

agitation test. The lambs were always maintained as one flock, calm or nervous. Rams

were also selected for temperament and calm rams mated with calm ewes and nervous

rams mated with nervous ewes. Lambs born were thus the progeny of the selection

line of ewes and rams (Murphy 1999).

Experimental ewes were examined 2-5 days after lambing to ensure their udders had

no blind teats or mastitis. The ewes were hand-milked for 5 days. These ewes have

never been milked before. They were injected with 2 IU of oxytocin (Ilium Syntocin)

intramuscularly, and the udders were empted immediately after the injection. Four


hours later, the ewes were injected with either 0, 0.25, 0.5, 1 or 2 IU oxytocin

intramuscularly in a Latin square design. The ewes were fed lupin grain during

milking. The teats were sprayed with an iodine-based commercial preparation

(Alfadyne Teat Sanifise, Delaval, Australia). The ewes and their lambs grazed

together on green subterranean clover-based pastures during the experiment, and they

were separated on the mornings on which they were milked.

Milk samples were kept in a cool room at 4˚C until composition was measured. The

amount of milk produced at the second (experimental) milking was used to calculate

hourly rate of production. These were then multiplied by 24 to obtain daily milk

output. Protein, fat and lactose concentrations were measured with a “Milko Scan

133” (Foss Electric, Denmark).

Statistical analysis was carried out using an ANOVA (Latin Square) for analysis of

milk yield, fat and protein concentration. The difference between calm and nervous

ewes in milk yield were analysed by one-way ANOVA (in Randomised Blocks).

Effects were assumed to be significant when the level of probability was 5% or less (p

≤ 0.05). Results are presented as mean ± standard errors.

Results

The difference between the daily milk outputs from either calm or nervous ewes

injected with saline (0 IU oxytocin) or 0.25 IU oxytocin was nearly significant (P =

0.06; Fig. 2.1). When the oxytocin dose reached 1 IU, milk yields were increased (P =

0.002) in both groups, compared with saline-treated controls. There was no difference

in milk outputs between the doses of 1 IU and 2 IU in the calm (P = 0.58) or nervous

groups (P = 0.42).


0

500

1000

1500

2000

2500

0 0.25 0.5 1 2Oxytocin dose (IU)

Figure 2.1. Milk output (g/day) of calm and nervous ewes after injection of differing doses of oxytocin

(Means ± SE).

Fat concentration was not affected by any dose in calm or nervous ewes (P = 0.23;

Table 2.1). Protein concentration was also not affected by any of the doses in calm or

nervous ewes (P = 0.20; Table 2.1).

Table 2.1. Concentrations of fat and protein in milk (mean ± sem) from calm and nervous Merino ewes

injected with differing doses of oxytocin.

Fat concentration (%) Protein concentration (%)

Dose (IU) Calm Nervous Calm Nervous

0 4.40 ± 0.33 5.23 ± 1.13 5.40 ± 0.13 5.27 ± 0.14

0.25 5.73 ± 0.59 6.88 ± 0.82 5.30 ± 0.16 5.22 ± 0.13

0.5 4.69 ± 0.45 7.01 ± 0.44 5.41 ± 0.18 4.93 ± 0.18

1 6.98 ± 1.20 6.51 ± 0.94 5.34 ± 0.12 5.20 ± 0.15

2 6.44 ± 0.88 6.67 ± 1.02 5.30 ± 0.18 5.23 ± 0.15

Discussion

The hypothesis that calm ewes require a smaller dose of oxytocin than nervous ewes

to extract milk was not supported because the dose-response lines were the same.

These were non-dairy animals that had never been milked by humans so all of the


ewes, regardless of whether they had been selected as calm or nervous, were probably

stressed by the procedure. Endogenous oxytocin activity during milking was probably

poor in both temperament groups, so their requirements for exogenous oxytocin were

similar. This explanation is supported by observations with cattle. Cows are

frightened at their first few milkings and when they are milked in unfamiliar

surroundings (Holmes & Wilson, 1984; Rushen, 2001). For this reason, young heifers

do not have normal milk ejection until they become familiar with the milking routine

(Holmes & Wilson 1984). In this experiment the ewes were milked for only 5 days,

not long enough for them to become familiar with the milking process. Calm ewes

may habituate more quickly to the routine and may have started producing more

endogenous oxytocin, than nervous ewes, in a longer-term study. Clearly, this

experiment needs to be repeated, taking into consideration the potential for

experimental ewes to adjust to the milking routines. Alternatively, neither calm nor

nervous animals were stressed by hand-milking so oxytocin was equally able to

facilitate milk ejection in both groups. A measure of behaviour on the milking

platform or of activation of the sympathetic axis would have been a valuable indicator

of the level of stress experienced by the ewes.

The injected oxytocin increased the quantity of the milk that could be removed from

the udders. This confirms results reported by earlier researchers (Linzell, 1972; Doney

et al., 1979; Bencini et al, 1992; Bencini, 1993, 1995). Bencini (1993) also studied

oxytocin dose-responses in Merino ewes and found that 1 IU increased the amount of

milk that could be extracted from the udder, and that no extra milk could be extracted

with 5 IU and 10 IU. She suggested that 1 IU of oxytocin injected intramuscularly

would be adequate for full milk let-down in Merino ewes. In the present study, the

milk outputs did not differ between the doses 1 IU and 2 IU in either group, so a dose

of 1 IU was confirmed for use in subsequent experiments.

None of the oxytocin doses affected the fat concentration in the milk from the calm or

nervous ewes, although there were indications of an increase in fat percentage as the

doses increased. This outcome was similar to that seen by Bencini (1993, 1995), who

also studied Merinos and found that milk fat content was not affected by the bigger

dose of 5 IU but was increased with 10 IU (Bencini, 1993, 1995). Generally, this

agrees with the contention by Labussiére (1988) that milk extracted with the help of


exogenous oxytocin should have a higher fat content because fat globules accumulate

in the alveoli. The lack of a significant effect of 1-2 IU on the fat concentration, in

either calm or nervous ewes, suggests that doses in this range would avoid the

withdrawal of variable amounts of alveolar milk in the next studies.

Protein concentrations were not affected by any of the doses used in this experiment,

in either the calm or nervous groups. This observation was also consistent with

research by Bencini (1993, 1995) who also found that protein was not affected, even

with 10 IU oxytocin, in Merino ewes. Protein concentration seems not to be affected

by the fraction of the milk removed from the udder.

In conclusion, an intramuscular injection of 1 IU oxytocin is suitable for measuring

milk yield in calm and nervous non-dairy ewes. This dose does not greatly affect the

fat or protein content of the milk extracted. It may be necessary to test ewes with

different temperaments in a larger number of samplings to determine whether the

calm ewes need a smaller dose of oxytocin for maximum milk withdrawal. Finally,

the ewes should be allowed to become accustomed to milking. Under these

circumstances, differences between calm and nervous animals may become apparent.

Chapter 3 Effect of Temperament on Production in Sheep

43

Chapter 3

Experiments 2 and 3

The effects of temperament on the production and clotting properties

of milk from Merino ewes

Introduction

Improving milk yield and quality is a popular topic in the dairy industry and there has

been a lot of progress through genetic selection and improved nutrition. Nowadays,

increasing the quantity and quality of milk produced by improving the milking

environment and the management of the animals is on the agenda of both dairy

producers and animal welfare supporters. One possibility is to reduce stress at milking

and improve milk quantity and quality by selecting animals with calm temperament.

This is because calm animals are more relaxed at milking and comfort at milking can

improve milking characteristics (Lawstuen et al., 1988), thus improving milk

production.

The “temperament” or “emotivity” of an animal is defined as its fearfulness and

reactivity to strange or threatening environments, including the presence of humans

(Murphy, 1999). Kilgour (1975) described temperament as an animal’s behavioural

response to physical, hormonal and nervous system stresses. In domesticated animals,

temperament is normally described as being either ‘calm’ or ‘nervous’. Specifically,

the perceptions and attitudes of calm and nervous animals to human handling and

threatening environments are different, therefore, their responses and levels of stress

could be different. Calm animals could be less stressed and more relaxed in these

situations than nervous animals.

Animal temperament affects production because nervous animals can be stressed

more easily than calm animals and stress can reduce productivity. Nervous, flighty or

aggressive temperaments have been reported to be associated with slow growth rate,


44

low feed conversion rate and increased difficulty of handling (Fordyce et al., 1988b;

Petherick et al., 2002). In dairy cows, temperament is reported to affect milk yield,

milking speed, lactation length, and even the occurrence of mastitis (Kilgour, 1975;

Lawstuen et al., 1988). In sheep, temperament is related to the production of wool

(Behrendt, 1994), meat (Wynn, 1994) and milk (Kovalcikova & Kovalcik, 1982), and

to reproductive performance (Hinch, 1994).

The Australian Merino sheep is an extensively-grazed, wool-producing breed and has

not been selected for the best temperament for working closely with humans. They

are not comfortable with being handled at shearing, ear tagging or other husbandry

treatments (Bencini, 1993). Merino ewes are normally not milked so the process of

milking is probably an intensive stressor to them. It is difficult to get them onto

milking platforms, to accept the positioning of teatcups and to be milked. In response

to these stressors, Merino ewes would be expected to produce adrenalin and this

hormone inhibits milk ejection (Ely & Petersen, 1941). Vasoconstriction caused by

adrenalin may also reduce the blood flow that delivers nutritional substances to

mammary tissues for milk synthesis. An insufficient supply of lipids and amino acids

for fat and protein synthesis could potentially lead to lower quality milk. Incomplete

milking and increased plasmin activity could be another two factors that change fat

and protein levels in milk (Labussiére, 1988; Silanikove et al., 2000).

In addition to these stress-related aspects, milk output is limited by differences

between animals in the physiological process that control the emission of milk from

the udder. There are two types of emission in sheep: single emission and double

emission. Ewes with only one emission of milk during milking do not release

oxytocin when they are milked so they do not release their alveolar milk and all of the

milk collected from these ewes is cisternal milk. Cisternal milk has a lower

concentration of fat than alveolar milk so one-emission ewes have lower total

lactation yields, shorter lactations, and a lower fat production (Labussiére, 1988).

Mayer (1989) found the single-emission pattern to be more common in non-dairy than

in dairy breeds of sheep whereas a double-emission pattern is more common in dairy

breeds. Labussiére (1988) also suggested that the single emission pattern might be

caused by stress. If that is the case, there should be a difference between milking

characteristics in calm and nervous animals as calm animals have a better adaptability


45

to strange environment and respond better to the human handling. The research in this

chapter was designed to test whether genetically-selected calm and nervous ewes

differ in their responses to being milked and their milk ejection patterns, therefore,

resulting in different milk yield and composition. If the composition of milk is

affected by the temperament of the animal then the clotting properties of that milk

should also be affected. More cheese can be produced from better quality milk.

Higher protein, casein and fat content can improve the clotting properties of milk.

There are two experiments in this chapter:

Experiment 2 – to determine the effect of temperament on milk yield and composition

in Merino ewes.

Experiment 3 – to determine the effect of temperament on clotting properties of milk

from Merino ewes.


46

Experiment 2

Temperament, milk yield and composition in Merino ewes

Introduction

There was evidence that animal temperament can affect milk production in farm

animals. In cows, for example, poor temperament has been reported to lead to lower

milk production and to a slower milking rate (Kilgour, 1975; Lawstuen et al., 1988).

For sheep, Dimitrov Ivanov & Djorbineva (2002) reported that the functional

parameters of the udder, such as milk yield, milk flow rate and milk ejection latency,

are affected by the level of fearfulness, and that that milk ejection latency is much

longer in nervous ewes (5.3 sec) than in calm ewes (1.9 sec). Direct comparison

among these studies is not simple, however, because Dimitrov Ivanov Djorbineva

(2002) classified their sheep according to their behaviour on the platform. It is

probably more appropriate to say that they studied animals with different capacities to

habituate to the dairy environment. This is probably related to temperament but it is

not the same as temperament, so it is at best an indicator.

In contrast, little is known about the effect of temperament on the composition or

quality of sheep milk. However, when stress affects milk ejection it should also affect

milk composition because it will increase residual milk. Residual milk has a high fat

content so, when milking is incomplete, fat content is reduced (Labussière 1988). Any

interference with milk ejection by stress will reduce fat concentration in the milk that

is removed from the udder. Stress may also affect the level of protein in milk because

it activates the plasminogen-plasmin system (PPS) leading to an increased break-

down of casein (Silanikove et al., 2000) and casein normally represents

approximately 80% of milk protein (Chapman, 1981).

Using the ‘Allandale temperament flock’, a sheep flock that has been selected for 15

years to improve productivity, adaptability and ease of handling, I tested the

hypothesis that ewes of calm temperament would produce more milk of better quality

than ewes of nervous temperament. The specific hypotheses were:


47

1. Calm ewes will produce more milk than nervous ewes.

2. Milk from calm ewes contains more fat and protein than that from nervous ewes.


Experiment 2 followed the procedures used in Experiment 1 (Chapter 2). Initially, 16

calm and 16 nervous ewes were milked after they were selected from the Allandale

temperament flock. However, 2 of the calm ewes were excluded from the results

obtained as their lambs died during the experiment, leaving measurements from 14

calm and 16 nervous ewes.

In the morning, the ewes were injected intramuscularly with 1 ml normal saline

containing 1 IU of oxytocin and udders were emptied. About 4 hours later, the ewes

were again injected with 1 IU oxytocin and milked. This process was repeated each

week from Weeks 2 to 7, and then each fortnight until Week 18 after lambing, when

the lambs were weaned. The second milking was used to calculate hourly rate of

production. Hourly rates were then multiplied by 24 to obtain daily milk output.

Samples from the second milking were analysed with a “Milko Scan 133” (Foss

Electric, Denmark) to determine the concentration of fat and protein.

Statistical analysis was carried using an ANOVA for repeated measurement for milk

yield and composition. Effects were accepted as significant when the level of

probability was 5% or less. Results are presented as means ± standard errors.

Results

There were no differences between calm and nervous ewes in their daily milk yield

(Fig. 3.1 Top, P > 0.05). Total milk yield over 18 weeks was similar: 150.1 kg from

calm ewes and 161.8 kg for nervous ewes. There was also no difference in fat

concentrations in milk from the calm and nervous ewes during the 18 weeks of

lactation (Figure 3.1 Centre, P = 0.17). Over the 18 weeks of lactation, the


48

concentration of protein was higher by about 0.4% in the milk produced by calm

sheep compared with milk from nervous sheep (Figure 3.1 Bottom, P = 0.03).

5

6

7

8

9

10

Fat (

%)

calm

nervous

4.0

4.5

5.0

5.5

6.0

6.5

7.0

2 3 4 5 6 7 8 10 12 14 16 18

Lactation period (week)

Prot

ein

(%)

calm

nervous

0

500

1000

1500

2000

2500M

ilk y

ield

(g/p

er d

ay)

calm

nervous

Figure 3.1. Top: Milk yield in ‘calm’ (n = 14) and ‘nervous’ (n = 16) Merino ewes. Centre: Milk fat

in ‘calm’ (n = 14) and ‘nervous’ (n = 16) Merino ewes (Means ± SE). Bottom: Milk protein in ‘calm’

(n = 14) and ‘nervous’ (n = 16) Merino ewes (Means ± SE).


49

Discussion

The hypothesis that calm ewes produce more milk than nervous ewes was not

supported, although, in this experiment, there was considerable variation among ewes

in both groups (range 9 to 20 kg over the experimental period). Total milk yield from

calm and nervous ewes did not differ for the lactation period of 18 weeks. This

disagrees with the results from Dimitrov Ivanov & Djorbineva (2002) who milked

106 calm and 54 nervous ewes and found that the calm ewes produced 23% more

milk than the nervous ewes. There are three major differences between the two

experiments: the number of ewes, the methods of measurement of temperament, and

the use of oxytocin.

The failure of the calm ewes to produce more milk than the nervous ewes in this

experiment may have been due to the way they respond to the oxytocin injections, as

indicated in Chapter 2. All ewes were injected with 1 IU oxytocin at every milking.

This injection was needed to override the effect of exogenous adrenalin produced in

response to the stress of the milking process (Doney et al., 1979; Bencini, 1993). We

were not able to measure ‘normal’ milk production from calm and nervous animals

without oxytocin injections. The milk production from ewes with differing

temperaments would be more comparable if this problem could be avoided.

Milk did not differ in fat content between calm and nervous ewes. This was not

consistent with previous studies in which fat content was higher when a larger

proportion of milk could be removed from the udder (Labussiére, 1988; Ely &

Petersen, 1941; Bencini, 1993). More milk can be removed when the animals are not

stressed (Wellnitz & Bruckmaier, 2001), or when oxytocin is injected (Heap et al.,

1986; Bencini, 1993, 1995; Ely & Petersen, 1941). However, in both the present study

and Experiment 1 (Chapter 2), the difference between the calm and nervous ewes was

not significant. This may be caused by large variation in the samples. The fat content

of milk is strongly related to the fraction of the milk that is removed from the udder or,

by corollary, the amount of alveolar milk that is withdrawn. So, sampling could affect

the amount of milk removed from the udder and the fat content in the milk. Even

though we attempted to empty the udder properly throughout the experiment, there

may be sampling errors. For example, some milkers often had to spend a long time


50

attempting to withdraw the last fraction of milk, and this may have affected the fat

concentration in the samples.

The hypothesis that the milk of calm ewes has more protein than that of nervous ewes

was supported. There are no other reports on the effects of temperament or stress on

the protein content of milk in any farm animals. The mechanism behind this effect is

unknown. A possible explanation could be the activation of the plasmin system that

breaks down casein (Silanikove et al., 2000). Politis et al. (1989) indicated that an

increased concentration of plasmin decreases milk yield during the lactation period.

They also indicated that injections of somatotrophin could suppress plasmin

production in the mammary glands, thus allowing a greater persistence of milk

production. Lucey & Fox (1992) suggested that a low casein level in late lactation

milk may be caused by increased plasmin and plasminogen concentration. This is yet

to be investigated for sheep. Protein synthesis in milk is a complex process. More

research is needed to ascertain if the process of protein synthesis is affected by long-

term stress, or whether the protein breaks down or is combined with other

components in the milk by hormonal changes due to short-term stress at milking.

Merino ewes of calm temperament can produce better quality milk of increased

protein levels, indicating the value of selecting sheep with a calm temperament.


51

Experiment 3

Temperament and the clotting properties of milk from Merino sheep

Introduction

Higher yielding, better quality cheese can be made if the clotting of the milk is rapid

and the curd reaches a high consistency (Bencini, 2002). Faster renneting time and

greater curd consistency are linked to higher concentrations of particular milk

components, particularly casein. Milk coagulates by the specific hydrolysis of κ-

casein when rennet or chymosin is added (Green & Manning, 1982) so it is important

to have milk with a high level of casein if the goal is good quality cheese. Fat is the

other solid constituent that is concentrated into curd. The level of fat in milk affects

the output of cheese (Chapman, 1981; Green & Manning, 1982). Fat concentrations in

cows’ and goats’ milk has no effect on renneting time, but curd consistency is poor

when the fat content is too high (Storry et al., 1983).

Milk with high concentrations of protein and fat produces more cheese, so the

production of cheese is better from the same amount of sheep milk than cow’s milk

because sheep milk is richer in protein and fat (Bencini, 2002). Protein level affects

the clotting time, the curd consistency and the texture of the cheese that is produced.

However, optimal quality and yield are only achieved when the protein-to-fat ratio is

about 1 or the casein-to-fat ratio is 0.7 (Howells, 1982).

A lot of research has been done on the clotting properties of cows’ milk under various

processing conditions. The clotting characteristics of cows’ and ewes’ milk of

different composition have been compared, as have different compositions of cows’

milk due to different lactational stages or diets (Bencini, 1993, 2002; Lucey & Fox,

1992; Cavani et al., 1991). Comparison of the clotting properties of milk within a

breed of sheep is rare and no experimental work has been done to compare the

clotting properties of milk from the animals of differing temperament. Therefore,


52

clotting time, curd firming and curd consistency were measured in milk from calm

and nervous ewes obtained in the previous experiment.

The hypotheses tested in this experiment are:

1. Milk from calm ewes will have a shorter renneting time than that from

nervous ewes.

2. Milk from calm ewes will have a shorter rate of firming than that from

nervous ewes.

3. Milk from calm ewes will have a better curd consistency than that from

nervous ewes.


Milk samples were taken from the 14 calm and 16 nervous ewes in Experiment 2.

Standard rennet (Streptococcus thermophilus and Lactobacillus bulgaricus; 2 μl/ml)

was added to the ten milk samples each time with a ten-spoon dispenser.

Clotting properties of each sample were measured with a Lactodynamograph (Foss

Electric, Italy) after the composition had been measured. The Lactodynamograph

works on a similar principle to the Formagraph (Foss Electric, Denmark). Viscosity

increases when milk clots to the point that a pendulum submerged in the clotting milk

will be put in motion. Ten wells are attached to a moving thermostat-controlled plate

that oscillates at a constant rate. A pendulum that is attached to a mirror is suspended

in each well. The mirror reflects dots of lights onto a roll of a photosensitive paper,

and the paper turns at a rate of 2 mm per minute (Figure 3.4). The Lactodynamograph

uses a computer program instead of the photosensitive paper used in the Formagraph.

The mirrors project a continuous line on the paper while the milk is still liquid. The

viscosity of the milk increases when the clotting starts. When the viscosity reaches the

clotting line the pendulums begin to move and the line projected. The length of the

line between the beginning of the run (corresponding to the addition of rennet) and

the point of bifurcation of the line represents the renneting time (R, rennet clotting

time). The time taken for the two arms to reach a spread of 20 mm represents the rate


53

of firming (K20) and the spread of the arms after 30 minutes represents the

consistency of curd (A30) expressed in mm.

Pendulum

Mirror

Flashing�Light source

Thermostat-controlled oscillating plate

Photosensitive paper

Milk

Figure 3.4. Schematic representation of the Formagraph (Foss Electric, Denmark).

After the measurement of clotting properties, the samples were taken out of the

machine and the curd was broken. Whey samples were collected to measure whey

protein in a Milko Scan 133 for further determination of casein in each milk sample.

Concentration of casein is calculated thus:

Casein% = Total milk protein% – Whey protein%

An ANOVA for repeated measurement was used to analyse the casein concentration

in the milk from calm and nervous ewes and to compare the renetting time, rate of

firming and curd consistencies between calm and nervous ewes. Results were

accepted to be significant when the level of probability was 5% or less. Results are

presented as mean ± standard errors.


54

Results

The casein concentration was significantly higher (P = 0.03) in the milk from calm

ewes than from nervous ewes (Figure 3.5). The result is very similar to that seen for

protein content in Experiment 2. This is not surprising as there is a close relationship

between casein and total protein concentrations. The casein content did not drop at the

end of the experiment as it normally does at the end of a lactation period.

3.0

3.5

4.0

4.5

5.0

5.5

2 3 4 5 6 7 8 10 12 14 16 18

Lactation period (week)

calm

nervous

Figure 3.5. Milk casein in ‘calm’ (n = 14) and ‘nervous’ (n = 16) Merino ewes (Means ± SE).

The rennet clotting time of the milk from calm and nervous ewes did not differ

(Figure 3.6 Top, P = 0.17). The K20 values for the milk from calm and nervous ewes

also did not differ over the 18 weeks of lactation (Figure 3.6 Centre, P = 0.36). A30

was greater in milk from nervous ewes than from calm ewes. The difference was

significant in the first three weeks of lactation with minor differences through to

Week 18 (Figure 3.6 Bottom, P = 0.03).


55

10

11

12

13

14

15

16

17

18

R (m

in)

Calm

Nervous

1.3

1.5

1.7

1.9

2.1

2.3

2.5

K20

(min

)

Calm

Nervous

40

45

50

55

60

65

70

75

2 3 4 5 6 7 8 10 12 14 16 18

Week

A30

(mm

) CalmNervous

Figure 3.6. Processing properties for milk from ‘calm’ (n = 14) and ‘nervous’ (n = 16)

Merino ewes. Top: Renneting time (R); Centre: Rate of firming (K20). Bottom: Curd

consistency (A30). All values are mean ± SE.

Discussion


56

The higher protein content in milk from calm ewes did not improve rennet clotting

time. This differs from the results of work by both Bencini (1993, 2002) and Balcones

et al. (1996) who found that increased protein concentration decreased renneting time

in both cows’ and ewes’ milk. In the study of Bencini (1993), the range in protein

concentration was 4.9 to 6.8%, while the range in the present experiment was 4.6

(nervous) to 4.9 (calm) in early lactation, and 6.1 (nervous) to 6.8 (calm) in late

lactation. This constraint makes it difficult to detect a significant difference in

renneting time without a much greater increase in the protein concentration.

Rate of firming was also not affected by increased protein or casein concentrations.

This was consistent with the results of Bencini (1993, 2002). Rate of firming is

correlated to the concentration of casein because it represents the speed at which the

micelles become aggregated after the casein glycopeptide has been cleaved by the

rennet (Dalgleish, 1979). However, rate of firming is also very sensitive to the pH at

which the milk clots. Best aggregation takes place at a pH of 5.2-5.3 (Jenness &

Patton, 1959). It seems likely that the rate of firming is not only associated with the

concentration of the protein in the milk but also depends on the speed that the κ-

casein micelles aggregate at the optimum pH.

Curd consistency was better in the milk from nervous than from the calm ewes even

though the total casein concentration was lower. This is not consistent with the

findings of Storry et al. (1983) or Lucey & Fox (1992). Storry et al. (1983) found

strong positive correlations between the coagulum strength (expressed as the firmness

at 1 h after clotting) and total casein. However, in their experiment, goats’ milk had a

lower coagulum strength than cows’ milk despite having a higher content of casein.

They also found that coagulum strength was positively correlated with the total Ca,

Mg and Pi, but not with the fat content or casein:fat ratio. It seems that curd

consistency is not improved by high casein concentration if the other components of

the milk are not optimal for obtaining good curd consistency.

There were only small differences in milk composition between the calm and nervous

groups and these might not explain the differences seen in curd consistency. Storry &

Ford (1982b) could not find differences in coagulation strength and casein


57

concentration in milk from Jersey or Friesian cows. They suggested that any

differences between breeds in composition of permeate or casein micelles would not

have a major role in determination of coagulation strength.

The outcome of the present experiment may also have resulted from the processing

procedure. Clotting properties of ewes’ and cows’ milk are affected by pH (McMahon

et al., 1984; Balcones et al., 1996). A fall in pH is associated with faster rennet

clotting time, faster firming rate and higher curd consistency (Balcones et al., 1996;

Lucey & Fox, 1992). Unfortunately, pH was not measured in Experiment 3 so it is not

known whether this affected the outcome. This might be important because the order

in which the samples were processed may have affected the pH of the milk.

In summary, improvement in milk composition caused by calm temperament does not

seem to improve greatly the clotting properties of the milk. There are two possible

explanations. First, the differences in composition between the two milks was too

small to have a major effect on clotting properties; second, the conditions under

which the milk was clotted may have had more of an impact than the composition of

the milk and thus affected the outcome. To obtain a more definitive answer, the effect

of ewe temperament on clotting properties might need to be retested with these factors

better controlled. The pH of the milk should be adjusted to be the same for the two

groups, or pH should be measured at all stages of the process with processing of

samples organised to ensure delays in processing of each sample is random between

calm and nervous sheep.

Chapter 4 Effect of Temperament on Production in Cattle

58

Chapter 4

Experiments 4 and 5

The effects of temperament on the yield and composition of milk

from Holstein cows

Introduction

Milk quantity and quality can be improved by genetic selection, and by better

nutrition, management, facilities and milking techniques. In dairy cows, temperament

also affects milk yield, ease of milk removal, milking speed, length of lactation,

mastitis resistance and ease of calving (Kovalcikova & Kovalcik, 1982; Lawstuen et

al., 1988; Van Reenen et al., 2002). Cows with good temperament have better

reproductive characteristics than those with poor temperament (Kovalcikova &

Kovalcik, 1982; Lawstuen et al., 1988).

Historically, temperament selection has probably been considered as a factor for ease

of handling (Kilgour, 1975) but the primary selection criteria for dairy cows are high

milk production and high milk fat content (Lawstuen et al., 1988). It is perhaps time

to reconsider cow temperament as a criterion for genetic selection. Dairy cows with

poor temperament have two types of problem at milking. First, milk yield and milking

speed are related to the temperament as nervous cows have milk let-down problems.

It is well known that a cow should not be frightened or stressed before or during

milking because milk ejection will be compromised. Whether or not the cows are

frightened at milking also depend on the genetic and individual variation among the

animals in their ability to cope with stress. Calm cows can more easily cope with

stress than nervous cows, so cows with good temperament have better milk

production and milk flow (milking speed). Milk yield was seemed to be lowest on

farms where the cows were highly fearful of humans (Hemsworth & Barnett, 2000).


59

Second, cows with poor temperament are usually more difficult to handle. Cows that

are more fearful of humans are more likely to injure the milkers (Hemsworth et al.,

1989). It is also more time-consuming to handle poor temperament cows due to their

avoidance responses to milkers, refusal to move into milking parlours, and

depositioning of teat-cups. Clearly, it is advantageous to select dairy cows for calm

temperament or to train the cows to be calm in order to minimize stress levels in daily

management. A broader aim is to improve the welfare of the animals as well as their

productivity.

Dairy and non-dairy animals differ in temperament and respond differently to

milking. Dairy cows have been selected to be high producers of milk and to work

with people concerned in their day-to-day handling. Therefore, they are not stressed at

milking as much as non-dairy animals are. Indeed, dairy cows can be stimulated to

release oxytocin and let down their milk at milking time simply by seeing the milking

facilities, hearing the sounds of the feeder or sensing the stimuli associated with

preparing the udders and positioning the teat-cups. In contrast, non-dairy animals do

not release oxytocin at milking time so effective milk ejection does not occur.

Consequently, non-dairy animals are not milked out completely and their milk

composition and quality are compromised.

There is little evidence about the differences between individual animals at milking. It

has been reported that there are differences between dairy sheep and non-dairy sheep

with respect to patterns of oxytocin release. There is also variation in oxytocin release

among individuals (Mayer, 1989), perhaps because of differences in their level of

stress and their temperament. Some researchers have attempted to test the fearfulness

of dairy animals by measuring heart rates and stress hormone levels before, during

and after milking (Hopster et al., 2002; Van Reneen et al., 2002). However, it is hard

to tell what the cause of fear is in these situations because the animals might have

been stressed by milking or by the measurement procedures.

It was concluded from Experiment 2 (Chapter 3) that Merino ewes with calm

temperament produced better quality milk, having more protein, than Merino ewes of

nervous temperament. This prompted result led me to investigate whether the same

would apply to dairy cows.


60

I had two major interests. One was to test the temperament of the cows using an open-

field test and study the relationship between the temperament of the cows and their

milk production. The reason to use an open-field for the temperament test was

because most of the authors scored the temperament of the cows according to their

observations during milking or handling in their studies on the relationship between

the temperament and milk production (Dickson et al., 1970; Kilgour, 1975; Purcell et

al. 1988; Lawstuen et al., 1988). My second interest was to test the same hypothesis

that was tested on Merino ewes on Holstein cows to determine how much variation in

milk quality there is among cows from one herd. In Experiment 2 (Chapter 3), I used

a flock in which ewes had been genetically selected for 14 generations to be calm or

to be nervous. I was also interested to study the correlation between the temperament

of the cows and the milk production that was obtained under normal milking

performances. This differs from the work of other researchers who studied milking

characteristics of cows under specific stressors such as an unfamiliar environment,

feed withdrawal or the presence of an aggressive handler (Holmes & Wilson, 1984;

Bruckmaier et al., 1993, 1997b; Rushen et al., 1999). Thus, there are two experiments

in this chapter:

Experiment 4: Repeatability of open-field tests with a human in Holstein cows;

Experiment 5: The relationship between temperament and milk quantity and quality in

Holstein cows.


61

Experiment 4

Repeatability of open-field tests with a human in Holstein cows

Introduction

The temperament of farm animals has been defined and measured in differing ways

by different authors. Some define temperament as behavioural responses to the

presence of a human or a challenging environment, while others define it as

emotivity. Kilgour (1975) defined temperament as the behavioural characteristics

resulting from the individual’s physiological, hormonal and nervous organization, that

contribute to the unique disposition of one animal in contrast to other members of the

species. In dairy cows, temperament has sometimes been regarded as a behavioural

characteristic or a ‘personality’, and at other times as the response of cows to humans

or human handling during milking. Generally, temperament is an animal’s response to

humans and surroundings. These responses reflect the animal’s fear level and that is

related to physiological and hormonal changes due to stress.

The techniques for measuring the temperament of diary cows also vary. The

temperament has been measured in a testing system, by recording behaviour during

milking, or even by scoring the milker’s judgement of the cows’ interactions with

their handlers during daily management. For example, Kilgour (1975) was the first to

use an open-field test for dairy cows and measured their temperament by counting the

number of squares they entered (ambulation score), the numbers of defecations and

urinations (elimination score) and the numbers of vocalizations (vocalization score).

Later, he and his colleagues calculated the temperament score of dairy cows by

adding the number of kicks (multiplied by two) and the number leg-lifts (Arave &

Kilgour, 1982). Other researchers had suggested that leg-lifting and kicking were

responses of dairy cows’ to their fear of humans (Hemsworth et al., 1989; Breuer et

al., 2000; Van Reenen et al., 2002). However, there was also a contradictory report in


62

which both very tame and fearful animals were less likely to step or kick during

milking (Bremner, 1997).

In addition, there are different opinions about whether or not humans are the most

fear-inducing factor to cattle. Munksgaard & Jensen (1996) suggested that housing in

a barren environment increases the motivation of cattle to explore and become fearful.

Others argued that human handling is stressful and that responses to this reflect

temperament in cattle (Boissy, 1995; Rushen et al., 2001; Van Reenen et al., 2002).

Open-field testing in which there was a human standing in the arena has been used

with sheep, but not with dairy cows. If we are to determine how much temperament or

fear of humans affects milking, we need to establish a reliable test of the fear response

of cows to humans.

The factors that are tested in an open-field test vary as well. Most authors counted the

movements of the cows and the number of vocalizations, defecations and urinations.

Others examined kicking behaviour of calves or the response of cows to a moving

ball in the arena. Sniffing is said to be an unreliable sign of fear for sheep in an open-

field test (Murphy, 1999). However, there is lack of evidence as to whether sniffing

behaviour is manifested by dairy cows in an open-field test. Freezing behaviour is

considered to be sign of great fear (Murphy, 1999).

Another factor is that test cows become familiar with their surroundings and their

situation as the test time is extended. Cows become less responsive and less fearful

when tested for the second time or when they stay longer in a test arena (Kovalcikova

& Kovalcik, 1982; Dellmeier et al., 1985; Kilgour, 1975). In the study by Kilgour

(1975), cows showed a greater decrease in both ambulation and vocalization scores on

the second and third days of tests than they did on the first day. He explained that the

arena was more familiar to the cows in the second day and the third day because of

their earlier experiences (Kilgour, 1975).

Therefore, in Experiment 4 I used a new approach, namely that of an open-field test

including a human. Each cow was tested twice so the experiment also tested the

hypothesis that the cows become familiar with the arena and thus respond less in the

second test than in the first.


63

The other questions that I attempted to answer in this experiment were:

1. Does an open-field test including a human demonstrate the temperament of the

dairy cows?

2. What are the manifestations of fear in an open-field test for dairy cows?


Thirty seven Holstein cows of 2-9 years of age from the farm of the Inner Mongolia

Agriculture University, Inner Mongolia, China were tested for temperament twice

(one week apart) with an open-field test including a human that was not known to the

cows. Temperament was scored for each cow by the numbers of steps, crossings,

defecations, urinations and sniffing behaviours.

Test procedure

A test arena was built with wood and metal posts between the milking shed and the

exercise area of the cows. The arena was approximately 27 m2 (7.9 metres long × 3.6

metres wide) with walls 1.8 metres high covered by plastic (Figure 4.1).

Pen with1 cow

0.8 m

1.8 m 2.2 m 2.2 m 1.7 m

Exit

Zone 5 Zone 4 Zone 3 Zone 2

Zone1

7.9 m

3.6 m

Entry

Human

Figure 4.1. Diagram of the open-field arena used to measure temperament in dairy cows.

There were two gates on opposite sides of the arena, one for entering and one for

leaving the arena. The entry gate was at the end of a chute out of the milking shed and

the exit gate gave access to the exercise yard. The ground was cleaned and was

divided into 5 zones marked with dry chalk. These zones were numbered from 1 to 5


64

starting from the point where the human stood. Another cow was held behind the test

human.

The test cow was ushered quietly into the arena when the person in the arena was

ready. Timing started as the cow entered. The observer in Zone 1 stood still and

counted the numbers of the steps and crossings of the zones. A crossing was counted

when both the forefeet of the cow crossed the line on the ground. The zone where the

testing cows came nearest to the human in the arena was recorded, and the numbers of

defecations and urinations were also counted. Another observer outside of the arena

counted the vocalizations. Other behavioural characteristics, such as sniffing the fence

or the human, “freezing” behaviour (a cow is very frightened and stands still) and

pushing the fence in an attempt to escape, were also recorded. Each cow remained in

the arena for 5 minutes. The arena floor was cleaned after each test.

Temperament scoring

Temperament of the cows in different groups was described as below:

Calm – Slow movement, few steps and zone crossings, none or very few

vocalizations, defecations, urinations or sniffing.

Medium – Medium speed or movement, some steps and zone crossings; a few

vocalizations, one defecation, urination, or sniffing behaviour.

Nervous – Frequent or rapid movements, many steps and zone crossings; some

vocalizations, defecations and urinations, and frequent sniffing of the human or

the fence.

The experimental cows were divided into three groups according to their temperament

scores in order to compare milk production and composition between groups.

Temperament score was assessed according to the number of steps and crossings,

vocalizations, defecations, urinations and sniffing behaviours of the test cows in the

first test. Cows were given a score of 1, 2 or 3 according to one of three ranges in the

number of steps, crossings and vocalizations, and according to two ranges in the

number of defecations, urinations and sniffing behaviours. The ranges of test data

corresponding to each score were evenly divided according to the total range of steps


65

taken (18 to 115) or crossings made (2 to 27), allowing classification of cows as calm,

medium, or nervous.

Score 1 2 3

Number of crossings 2-9 10-18 19-27

Number of steps 18-50 51-82 83-115

Number of vocalizations 0-1 2-4 5<

Number of defecations 0-1 2

Number of urinations 0 1

Sniffing behaviour no sniffing sniffing

Crossings, steps and vocalizations are regarded as the clearest signs of fear, so cows

with the most frequent expression of such behaviours were given a score of 3. The

temperament score was then calculated by summing the counts of these behaviours

for the individual cow. The total score ranged from 6 to 13 (only 1 cow was scored

13). The cows were classified, as shown in Table 4.1, as “Calm” when the total score

was 6-7 (mean total score 6.63 ± 0.18), “Medium” when the total score was 8-10

(8.47 ± 0.12), or “Nervous” when the total score exceeded 10 (10.90 ± 0.35). The

mean ages of these groups were 6.5 ± 0.6 (“Calm”), 4.7 ± 0.4 (“Medium”) and 4.5 ±

0.7 years (“Nervous”).


66

Table 4.1. Calculation of temperament scores and assignment of cows to temperament classes. Cow no.

Age (y)

Cross score

Step score

Vocalise score

Defecate score

Urinate score

Sniff score

Total score

Temperament class

96-22 8 1 1 1 1 1 1 6 Calm 97-3 7 1 1 1 1 1 1 6 Calm 98-5 6 1 1 1 1 1 1 6 Calm 00-4 4 1 2 1 1 1 1 7 Calm 95-4 9 1 1 1 2 1 1 7 Calm 97-6 7 1 1 1 1 1 2 7 Calm 98-8 6 1 2 1 1 1 1 7 Calm 99-7 5 1 1 1 1 1 2 7 Calm

01-16 3 1 2 1 2 1 1 8 Medium 01-8 3 2 2 1 1 1 1 8 Medium 02-3 2 2 1 1 2 1 1 8 Medium

98-11 6 1 1 1 2 2 1 8 Medium 98-12 6 1 1 1 2 2 1 8 Medium 98-4 6 1 1 2 2 1 1 8 Medium 98-7 6 1 1 2 2 1 1 8 Medium

99-10 5 2 1 1 2 1 1 8 Medium 99-13 5 1 2 1 1 2 1 8 Medium 99-5 5 1 2 1 1 1 2 8 Medium 00-7 4 2 2 1 1 2 1 9 Medium 00-8 4 2 1 1 2 2 1 9 Medium 01-2 3 2 2 1 2 1 1 9 Medium 01-4 3 2 2 1 1 1 2 9 Medium 01-6 3 2 2 1 2 1 1 9 Medium

97-12 7 1 2 1 2 2 1 9 Medium 97-14 7 2 2 1 1 1 2 9 Medium 98-2 6 2 2 1 2 1 1 9 Medium

99-15 5 2 2 1 1 1 2 9 Medium 02-9 2 2 2 1 2 1 2 10 Nervous

97-15 7 1 2 1 2 2 2 10 Nervous 98-10 6 2 2 3 1 1 1 10 Nervous 99-14 5 2 2 2 1 1 2 10 Nervous 99-2 5 2 1 3 2 1 1 10 Nervous 00-5 4 3 3 1 1 1 2 11 Nervous

96-17 8 3 3 1 1 2 1 11 Nervous 01-12 3 3 3 1 2 1 2 12 Nervous 01-3 3 2 3 3 1 1 2 12 Nervous

02-13 2 3 3 3 2 1 1 13 Nervous

Statistical analysis

Student’s T-test was used to compare the number of steps and crossings in the first

and second tests in the arena. Results were assumed to be significant when the level

of probability was 5% or less. Regression analysis was used to determine the strength

of the relationship between the numbers of steps and crossings in the tests. Results are

presented as means ± standard errors.


67

Results

The numbers of the steps and crossings were strongly linearly related within the first

open-field test (Figure 4.2). The numbers of the steps and crossings were also strongly

related in the second open-field test, but the cows made fewer steps and crossings in

the second test so that the observations tended to be clustered around the bottom part

of the line (Figure 4.2).

y = 3.7668x + 16.623R2 = 0.8039

0

20

40

60

80

100

120

140

Step

s

y = 4.8957x + 10.052

R2 = 0.8599

0

20

40

60

80

100

120

140

160

Crossings

Step

s

0 5 10 15 20 25 30

Figure 4.2. The relationship between the numbers of steps and crossings in the first open-field test

(top) and in the second open-field test (bottom).

The cows made significantly fewer steps (P = 0.02) and crossings (P = 0.001) in the

second test than in the first test. The mean steps of temperaments classes made in the

second test were 28.9 ± 10.4 (“Calm”), 50 ± 7.3 (“Medium”) and 68.9 ± 9.8

(“Nervous”), and the mean crossings were 3.8 ± 1.9 (“Calm”), 8.4 ± 1.5 (“Medium”)

and 11.7 ± 1.5 (“Nervous”) (Table 4.2). Most of the cows remained in Zones 2 (0.8

metres away from the human) or 3 (1.7 meters away from the human). In the first test,


68

only 3 cows entered Zone 1 (where the human was), and none remained only in Zone

4 (3.9 meters away from the human). In the second test, 3 cows moved into and

remained in Zone 4 and thus made very few steps and crossings.

Table 4.2. The behaviour of cows (steps, crossings, zones nearest to human) in the 2 open-field tests.

Steps Crossings Nearest zone Cow no. Temperament First Second First Second First Second

00-4 Calm 55 33 9 2 3 3 95-4 Calm 49 25 6 2 2 4 96-22 Calm 18 19 3 3 2 2 97-3 Calm 26 5 6 1 3 3 97-6 Calm 47 91 9 16 2 2 98-5 Calm 30 9 5 1 3 4 98-8 Calm 51 13 7 2 3 3 99-7 Calm 24 36 2 3 3 3

Mean ± sem 38 ± 6 29 ± 10 6 ± 1 4 ± 2 00-7 Medium 66 49 18 11 2 2 00-8 Medium 45 137 11 29 2 1 01-16 Medium 54 55 7 10 2 2 01-2 Medium 65 81 14 11 2 2 01-4 Medium 59 59 11 5 1 3 01-6 Medium 54 60 11 11 1 2 01-8 Medium 72 58 16 12 2 2 02-3 Medium 45 55 11 9 2 2 97-12 Medium 58 7 9 1 3 4 97-14 Medium 82 51 18 10 1 2 98-11 Medium 41 11 8 2 2 3 98-12 Medium 43 9 7 3 3 3 98-2 Medium 80 62 13 7 2 2 98-4 Medium 43 25 7 4 3 3 98-7 Medium 34 60 6 8 2 3 99-10 Medium 46 24 14 4 3 3 99-13 Medium 56 69 8 9 2 2 99-15 Medium 63 32 15 5 3 3 99-5 Medium 54 46 9 8 2 2

Mean ± sem 56 ± 3 50 ± 7 11 ± 1 8 ± 2 00-5 Nervous 115 44 27 8 2 3 01-12 Nervous 97 77 21 14 2 2 01-3 Nervous 101 139 16 22 2 1 02-13 Nervous 111 88 25 13 2 2 02-9 Nervous 71 58 16 11 2 1 96-17 Nervous 111 81 20 13 3 3 97-15 Nervous 62 51 9 8 2 3 98-10 Nervous 74 48 13 14 2 2 99-14 Nervous 79 62 11 8 2 3 99-2 Nervous 50 41 11 6 2 2

Mean ± sem 87 ± 8 69 ± 10 17 ± 2 12 ± 2 Total 2231 1870 429 306 Mean ± sem 60 ± 4.03 51 ± 5.29 12 ± 0.96 8 ± 1.00


69

The number of vocalizations fell by 30% between the first and second tests (Table

4.3). Ten cows vocalized in the first test and 9 cows in the second test. Among these,

5 cows vocalized in both tests. Among these cows, one cow vocalized 15 times in the

second test while did 11 the first test, the rest of the cows vocalized the same or fewer

times in the second test than the first test.

Both the numbers of cows that defecated and urinated, and the numbers of those

behaviours, decreased in the second test compared to the first test (Table 4.3). The

number of cows that defecated fell from 18 (49%) to 10 (27%), the number that

urinated from 8 (22%) to 2 (5%). No relationship was found between the number of

crossings and number of sniffing behaviours (P = 0.13).

Other behaviours, such as attempting to escape from the arena, were observed in a

few cows in both tests. One cow attempted to escape from the arena by pushing the

fence in both tests. However, she was otherwise classified as a ‘calm’ cow with low

total numbers of movements, vocalizations defecation and urination. Another cow

that attempted to escape was ‘nervous’. Two cows attempted to escape the arena in

the second test. One cow jumped out of the arena after 2 minutes and 44 seconds of

the second test.


70

Table 4.3. Vocalizations, defecations, urinations and sniffing behaviours by test cows in the two open-

field tests.

Vocalization Defecation Urination Sniffing Cow no. Temperament First Second First Second First Second First Second

00-4 Calm 1 1 0 0 0 0 0 0 95-4 Calm 0 0 1 0 0 0 0 0 96-22 Calm 0 0 0 0 0 0 0 0 97-3 Calm 0 0 0 0 0 0 0 0 97-6 Calm 0 0 0 0 0 0 1 0 98-5 Calm 0 0 0 1 0 0 0 0 98-8 Calm 0 0 0 0 0 0 0 0 99-7 Calm 0 0 0 0 0 0 1 0 00-7 Medium 0 3 0 0 1 0 0 1 00-8 Medium 1 0 2 0 1 0 0 0 01-16 Medium 0 0 1 0 0 0 0 0 01-2 Medium 0 0 1 0 0 0 0 0 01-4 Medium 0 0 0 1 0 0 1 1 01-6 Medium 0 0 1 0 0 0 0 0 01-8 Medium 0 1 0 0 0 0 0 1 02-3 Medium 0 0 1 0 0 0 0 1 97-12 Medium 0 0 1 0 1 0 0 0 97-14 Medium 0 0 0 1 0 0 1 1 98-11 Medium 0 1 1 0 1 0 0 0 98-12 Medium 1 0 1 1 1 1 0 0 98-2 Medium 0 1 1 0 0 0 0 0 98-4 Medium 4 0 1 1 0 0 0 0 98-7 Medium 4 0 1 0 0 0 0 0 99-10 Medium 0 0 2 0 0 0 0 0 99-13 Medium 0 0 0 1 1 0 0 0 99-15 Medium 0 0 0 1 0 0 1 1 99-5 Medium 0 0 0 0 0 0 1 0 00-5 Nervous 0 0 0 1 0 0 1 1 01-12 Nervous 0 0 1 0 0 0 1 1 01-3 Nervous 11 2 0 0 0 0 1 1 02-13 Nervous 11 15 1 0 0 0 0 1 02-9 Nervous 0 0 1 0 0 0 1 0 96-17 Nervous 0 0 0 0 1 0 0 1 97-15 Nervous 0 0 1 0 1 0 1 0 98-10 Nervous 5 0 0 1 0 1 0 0 99-14 Nervous 2 1 0 1 0 0 1 1 99-2 Nervous 9 9 1 0 0 0 0 0

Total 49 34 20 10 8 2 12 12

Mean±sem 1.3 ± 0.50 0.9 ± 0.48 0.5 ± 0.10 0.3 ± 0.08 0.2 ± 0.07 0.1 ± 0.04 0.3 ± 0.08 0.3 ± 0.08


71

Discussion

The ambulatory or motor activity of a test cow in an open-field test is related to

excitability or agitation and can thus be used as an objective assessment of fear or

temperament (Kilgour, 1975; Kovalcikova & Kovalcik, 1982; Boissy & Bouissou,

1995). However, the animals will probably habituate to the test situation

(Kovalcikova & Kovalcik, 1982; Kilgour, 1975). The hypothesis that the cows would

respond less in the second test than in the first test was strongly supported because the

cows made fewer steps and crossings in the arena in the second test. This observation

agrees with that of Kilgour (1975) and is consistent with the reported decrease in

agitation, both in cows and calves, when the animals are held for increasingly longer

periods in a test arena (Kovalcikova & Kovalcik, 1982; Dellmeier et al., 1985). Thus,

the animals adapt readily to any stress induced by the test conditions but the test

results are consistent between tests because the number of steps and crossings made in

the second test was fewest in the calm group and most in the nervous group.

Nevertheless, there was a strong relationship between the numbers of steps and the

numbers of crossings in both tests. This is reassuring if not surprising – only a few

steps were required to move from one zone to the other, so there is a strong

relationship between these values because most cows moved into different zones

during the test. Only a small number of cows remained in one zone during the entire

5-minute period of observation.

Most cows were aware of, and avoided, the human presence in the arena. Most of the

cows remained in Zones 2 or 3 in both tests, and did not enter into Zone 1 where the

human was, so their natural attraction toward the herd-mate held behind the human

was inhibited. However, none of the cows remained only at the far end of the arena,

Zone 4, away from the human. It seems that the cows would take a few steps into

Zone 3 before recognising the presence of the human in front of them in the holding

pen, suggesting that some cows only moved into and remained in Zone 4 in the

second test because they had become familiar with the arena after the first test.

In sheep, Murphy (1999) also observed that nervous animals actively avoided the

human, even in the presence of flock mates. However, several cows classified as

“nervous” approached the human in the present test, suggesting a poor relationship


72

between flight distance (the minimum distance of the test animal from the human) and

the classification of temperament in these animals. Murphey et al. (1981) suggested

that there were breed differences in flight distance – for example, dairy cows have

shorter flight distances than beef cattle. In the present experiment, test cows seemed

to come near the human only after it appeared to be safe or harmless to do so,

although some cows approached closely to investigate by sniffing.

The number of vocalizations was reduced in the second test compared to the first test,

and the number of the cows that vocalized also decreased slightly. These observations

were similar to the results reported by Kilgour (1975). Vocalization and locomotion

were said to be another important behavioural sign of low levels of fear (Boissy &

Bouissou, 1995). The data presented here indicate that the level of fearfulness in the

cows was reduced in their second appearance in the arena.

Kilgour (1975) indicated that defecation was an automatic response to fear. He

explained that, sometimes, dung was dropped in an arena to establish territorial rights

or to give a strange area an odour that was familiar to the subject, and that this action

was an attempt to reduce fear. The decreasing numbers of cows that both defecated

and urinated and the numbers of those behaviours, between the first and second tests,

indicates that these behaviours are a good manifestation of fear or stress in an open-

field test.

The number of sniffing events was the same in the first and second tests and was not

related to the number of crossings made by the cows. However, most of the cows that

exhibited sniffing behaviour were of “nervous” or “medium” temperament. This

suggests that sniffing is a sign of stress in cattle. However, this conclusion is not

consistent with the interpretation of behaviour exhibited by sheep in an open-field test

where sniffing was suggested to be an unreliable indicator of emotivity (Murphy,

1999).

It appeared from this experiment that other behaviour, such as attempting to escape

from the arena, was not a significant sign of fear because this was displayed by

“calm”, “medium” and “nervous’ cows. It appeared that these cows remembered the


73

previous test and that they could not wait to be released. They exhibited little

agitation except for their attempt to get out of the arena.

The observation that none of the cows showed freezing behaviour, except for one cow

that stood still for about 1 minute but responded normally in the rest of the test,

indicates that most of the cows were not very frightened. The reason why one of the

cows jumped out of the test arena during the second test was not clear. She was a

“medium” temperament cow according to her behavioural response in the first test.

This cow was not in oestrus at that time. This observation might suggest that other

factors such as handling of the animals before the test could affect behaviour during

the test.

In brief, an open-field test including a human is a suitable way of measuring the

temperament of dairy cows because it induces and tests the level of fear that the cows

display in the test situation. Repeating the test can reduce fear, but the second test still

can reflect the different levels of fear response in cows of different temperament by

showing the level of behavioural reaction.


74

Experiment 5

The relationship between temperament and milk quantity and

quality in Holstein cows

Introduction

The results from previous investigations have shown that machine milking can cause

stress and reductions in milk yield (Israilzhanov, 1980; Bruckmaier et al., 1993;

Tancin et al., 1995; Marnet & Negrão, 2000). The underlying mechanism of this

phenomenon was thought to be reduced milk ejection caused by stress-induced

inhibition of oxytocin release (Bruckmaier et al., 1993). Subsequent studies found

that stressors from milking did not modify oxytocin release into the blood circulation

but, rather, reduced the binding of oxytocin to its receptors in the mammary gland

(Bruckmaier et al., 1997b). In other studies high plasma concentrations of cortisol,

high heart rates, bad-behavioural reactions and reduced oxytocin release were all

linked with an increase in the size of the fraction of residual milk. Thus milk yield

was decreased when the cows were milked in an unfamiliar surroundings or in the

presence of a handler who handled the cows aggressively before milking (Bruckmaier

et al., 1993, 1997b; Rushen et al., 1999).

Oxytocin release is also associated with milk yield, fat and protein concentration in

machine milked ewes (Negrão & Marnet, 2003). In addition to oxytocin, Negrão &

Marnet (2003) measured the circulating concentrations of cortisol, adrenalin and

noradrenalin during the first milking in primiparous ewes and found that cortisol

values were negatively related to milk yield. There was no correlation between values

for adrenalin or noradrenalin and milk yield, fat or protein, but there was positive

correlation between oxytocin release and milk yield, and fat and protein

concentrations in the milk. The effect of oxytocin on protein content was not

explained. Tancin et al. (1995) reported that high levels of cortisol and noradrenalin

were associated with milking stress and disturbance of milk ejection in Holstein cows.

Overall, these reports support the conclusion that milking, if it becomes a stressor,


75

will reduce milk ejection by reducing the secretion or action of oxytocin and,

consequently, there will be reductions in milk output and probably the fat and protein

content of the milk.

The temperament of dairy cows has been reported to affect both milk yield and

milking speed (Kovalcikova & Kovalcik, 1982; Lawstuen et al., 1988). Cows with

good temperament are bigger producers and have a faster milk flow than cows with

bad temperament (Lawstuen et al., 1988). Little is known about whether variations in

the temperament of cows in the same herd can affect concentrations of fat and protein

in the milk. Theoretically, cows could be stressed during milking because of human-

animal interactions and the animals’ intrinsic fear of human handlers (Hopster et al.,

2002). Moreover, nervous cows could be more stressed by milking because they have

less ability to cope with stress. This suggests that, compared to calm herd-mates,

nervous cows might release less oxytocin or be more susceptible to blockade the

action of oxytocin, and thus eject milk less effectively, thus producing less milk with

less protein and fat.

Experiment 5 was designed to study the relationships between temperament and milk

yield and composition in Holstein cows during the normal process of machine-

milking. The hypothesis that calm cows produce more milk of better quality than the

nervous cows was tested.


Experiment 5 followed on from Experiment 4. The cows were machine-milked twice

per day after the temperament tests. Regardless of temperament, all cows were fed

hay, corn silage and protein concentrates during the experiment.

Cows were ushered into the milking shed immediately after feeding and were not fed

during milking. Once per week for 12 weeks, the total milk outputs for that day’s two

milkings were recorded by milk meters and the milk was sampled. The milk samples

were analysed with a Bentley 150 (Infrared Milk Analyser) to determine protein, fat

and lactose content.


76

Of the cows studied in Experiment 4, only 23 (5 “calm”, 11 “medium” and 7

“nervous”, 2-8 years old) were used for the analysis of milk yield and composition.

For the remaining animals, too many values were missing. Daily milk yield and fat

and protein percentages in the morning milk were used for analysis because some of

those cows were not milked in the afternoons. Morning milk outputs were regarded as

the daily yield for the cows that were not milked in the afternoons. The data used for

analysis were adjusted to take into account the day of lactation in order to enable

better comparisons.

ANOVA with a general linear mixed model (SAS® Software Version 8.2, Copyright

© 1999-2001, SAS institute Inc., Cary, NC, USA) was used to analyse changes in

milk yield, protein and fat with time for each of the temperament classes. The main

hypotheses examined whether the fixed factor temperament (calm, medium, nervous)

and the covariate ‘days in lactation’ together with the temperament by the days in

lactation interaction have any effect on each of milk yield, fat and protein

concentration. The random factor cow within temperament was included in the model.

Regression analysis was used to determine the correlation between the behavioural

factors and milk yield, milk fat and protein. Effects were assumed to be significant

when the level of probability was 5% or less. All data are presented as mean ±

standard error.

Results

The interaction between the temperament and days in lactation was found not to be

statistically significant for milk yield, fat or protein (Figure 4.4 indicates slopes very

similar). Subsequently, main effects of days in lactation and temperament were

examined. There were no significant differences between the temperament and the

milk yield, fat or protein concentrations (P = 0.95 for milk yield, P = 0.94 for fat

concentration or P = 0.58 for protein concentration). However, days in lactation had

effects on milk yield, fat and protein concentration.

Figure 4.4 could not be included in the digital version of this thesis for technical reasons. Please refer to the physical copy of the thesis, held in the University Library.


78

There was no relationship between the number of steps in the test arena and the

average milk yield for the 12-week period, for the 8 cows that were in the same part

of their lactation period (100-170 days). The same applied to the fat content and the

protein content in the milk produced from these cows (Figure 4.5).

y = 0.0129x + 13.911

R2 = 0.0249

10

12

14

16

18

20

Milk

yie

ld K

g/da

y

y = -0.0001x + 3.624

R2 = 0.0001

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

Fat (

%)

y = 0.0005x + 2.8614

R2 = 0.0279

2.6

2.8

3.0

3.2

0 20 40 60 80 100 120

Numbers of steps

Prot

ein

(%)

Figure 4.5. Relationships between temperament (number of steps in the open-field test) and milk

production (top) and milk content of fat (centre) and protein (bottom) in dairy cows.


79

Discussion

The hypothesis that calm cows would produce more milk than nervous cows was not

supported but this may have been because the animals were generally calm and the

range in the values of the behavioural measures was too narrow to allow the

hypothesis to be tested. This differs from the observations of Kovalcikova & Kovalcik

(1982) who reported that calm temperament cows that responded less to a strange

environment were likely to have high milk production. In their study, the cows were

tested for temperament in a 10-square-metre room with 9 equal squares, similar to the

open-field test used in the present study. Their cows were Slovak Spotted and Black

Spotted and their crosses, whereas the cows in the present study were Holsteins.

Temperament and response to novel environments could differ between these breeds.

Age might be another important factor affecting both temperament and milk yield. In

the report from Kovalcikova & Kovalcik (1982), the relationship between production

and behaviour was significant in young cows but not in older cows. The age range of

the cows in present experiment was 2-8 years, a big range that may affect variability

in temperament scores as well as milk production. Finally, by the time we had

selected cows in comparable stages of lactation, the numbers of animals was quite

low, making it difficult to detect relationships between production and behaviour.

Lawstuen et al. (1988) reported that high production Holstein cows appeared to have

good temperament, but the temperament scores were assessed by the herd-owners

according to their daily management and observations. Similar systems of assessment

were used by Dickson et al. (1970) and Purcell et al. (1988) who scored the

temperament of cows according to their behaviours at milking, such as movements in

the milking parlours or moving of legs or kicks during milking. However, neither

found any relationships between production and temperament in Holstein cows.

Dickson et al. (1970) found no correlation between temperament and milk yield or

stage of lactation. Despite the different methods of measuring behaviour, these

observations agree with those of the present study, again supporting the argument

that, in general, Holsteins show a very narrow range of temperament behaviours,

perhaps because of their long history of selection as dairy animals.


80

Kilgour (1975) studied the behaviour of Jersey cows in an open-field test. Again, he

could not find any correlations between the behaviour of the cows in the arena and the

temperament ratings that were scored by the milkers. He suggested that the “flighty”

cows tended to be bigger producers whereas the “quiet type” cows covered a range of

milk productions. However, milk production of the cows was not measured in the

experiment in which he tested the cows for temperament in the open-field.

Kovalcikova & Kovalcik (1982) found that the numbers of vocalizations and

defecations was not related to milk production. They measured the behaviour of cows

in an arena test and their milk production and found no relationship between the steps

and milk yield, milk fat or protein (Kovalcikova & Kovalcik, 1982). The numbers of

steps, vocalizations and defecations that cows make in an open-field are normally

efficient indications of fear (Kilgour, 1975), and the cows produce less milk when

they are frightened (Rushen et al., 1999). Therefore, the results from the present study

suggest that the cows did not take their fear responses from the test into the milking

situation. They may be comfortable at milking even though they were stressed in the

open-field test, so their milk production was not correlated to their behaviour scores

in the open-field test.

Other factors that could have affected milk production by the cows in the present

experiment were lactation period and age. It is well known that lactation period

influences both quantity and quality of milk (Nickerson, 1995) and cow age affects

both temperament and milk yield (Dickson et al., 1970; Kilgour, 1975; Hearnshaw &

Morris, 1984; Lawstuen et al., 1988; Wilmink, 1987). When I arrived at the research

farm of the Inner Mongolia Agriculture University, I found that the cows were in all

different stages of lactation. The effect of the lactation appeared to be significant

although cows that were before 250 days of lactation were used for analysis for milk

production. Data were collected for a short period compared to the lactation period.

Variation in both lactation period and age might hide any effect of temperament on

milk production. Clearly, the experiment should have been conducted with cows with

similar lactation periods and similar ages.


81

General discussion for Chapter 4

The open-field test with a human is suitable for testing temperament in dairy cows

and can provide repeatable data. The results from the open-field tests also suggested

that a combined temperament score that uses all test factors is feasible. Boisy &

Bouissou (1988) stated that modern management leads to a lack of familiarization of

animals with humans and that this may cause problems such as wasted time, injuries

to the stockman during husbandry and unnecessary stress for the animals. Therefore,

it is necessary to include human-animal interactions in temperament tests. The human

in the arena was a stranger to the test cows in Experiment 4 and this might have

induced fear in the cows resulting in an increase in behavioural activities such as

agitation, vocalization and defecation.

Some of the behaviours appeared to be better indicators of fear than the others, and

most of the findings in the open-field test were consistent with the descriptions from

other authors. Ambulation and agitation are generally said to be fear responses in

cattle (Kilgour, 1975; Munksgaard & Jensen, 1996) and in sheep (Murphy, 1999).

Defecation during the test has been regarded as a fear manifestation in cattle (Kilgour,

1975) and vocalisation has been regarded as a good indicator of agitation both in

cattle and in sheep (Kilgour, 1975; Murphy, 1999). From the observations in

Experiment 4, one could draw the conclusion that ambulation (steps and crossings),

vocalization, defecation and urination were all useful indicators of fear in strange

environments or test situations because the numbers of these behaviours decreased

from the first to the second test.

Sniffing and flight distance are likely to be weak indicators of fear in tests. The

sniffing activity of the test cows did not weaken in the second test and the number of

cows that sniffed around did not decrease, even though most of the cows that had

sniffing activity were otherwise classified as “nervous”. These results agree with

observations from other authors. Kilgour (1975) classified sniffing as an automatic

reaction of cattle in an open-field test and Boissy & Bouissou (1995) suggested that

sniffing a novel object was a sign of low fear in cattle. Flight distance was shown to

relate to factors such as breed and past experiences of the cows with humans

(Murphey et al., 1981; Purcell et al., 1988).


82

Temperament scores vary according to test situations. Some tests of temperament are

more likely to reflect the intrinsic fearfulness of an animal, and others reflect

situation-specific fear resulting from the environmental condition at the time of

testing and whether or not an animal has experienced those situations (Petherick et al.,

2002). In Experiment 4, the temperament scores of the cows were estimated by

combining the number of steps, crossings, vocalizations, defecations, urinations and

sniffing. Among these behaviours, movements and vocalization were considered to be

more efficient indicators than the others. The scores appeared to be good indicators of

fear in the novel test situation for these cows.

The finding that temperament was not significantly correlated with milk production

may be because the experimental cows were Holsteins that had been selected for dairy

productivity for hundreds of years. In that process of selection, nervous cows would

have been systematically eliminated from the herd because they are hard to handle or

to milk. Thus, all the cows in Experiment 5 were reasonably calm and the ranges of

temperament scores between the groups were small. The same technical issue was

noted by Dickson et al. (1970) who concluded that there are mostly calm cows in

Holstein dairy herds because temperament is a heritable trait (Dickson et al. 1970;

Lawstuen et al., 1988). We therefore cannot conclude from the present study that cow

temperament within a herd does not affect milk yield, or the protein or fat content in

the milk produced. Rather, we need to find more precise measures of temperament to

differentiate between the animals, or perhaps conclude that there is too little to be

gained by further work in this area. Further, the differences in outcomes between the

experiments with cows and sheep in this thesis can also be explained by genetic

selection. The clear difference between the milk from calm and nervous ewes, not

seen in cows, was detected because the sheep had been selected to be either calm or

nervous.

Chapter 5 General Discussion

83

General discussion

The results in this thesis have shown that the total milk protein and casein

concentration could be improved by selecting Merino sheep for calm temperament.

The “Allandale Temperament Flock” is a good demonstration that calm temperament

can be developed by genetic selection, and production is improved at the same time.

Murphy (1999) demonstrated that calm ewes had better maternal behaviour and

rearing ability than the nervous ewes, and the results in Chapter 3 show that calm

ewes produced better quality milk than nervous ewes. Selecting animals with calm

temperament can also be helpful for easy handling in daily management.

By contrast, Holstein cows did not show any differences in the milk yield among the

temperament groups, so the hypothesis that calm temperament cows would produce

more milk than the nervous temperament cows was not supported. This is probably

not surprising because Holstein cows have been selected for dairy purposes for

hundreds of years so cows would have been selected for calm temperament with or

without direct intention. The heritability of temperament in dairy cows is 12%

(Lawstuen, 1988) so, when the good temperament cows were kept and bad

temperament cows were eliminated, calm cows would accumulate in the dairy herds

by inheriting this temperament from generation to generation. The experimental sheep,

on the other hand, had been selected for extremes of calm or nervous temperament so

were more likely to reveal the benefits of such selection, particularly with small group

sizes.

Dairy and non-dairy animals have different milking characteristics. Dairy animals are

easily stimulated by factors such as the sounds and sights related to milking, so milk

ejection takes place readily, whereas non-dairy animals eject milk poorly. The results

from the Merino ewes and Holstein cows may also differ because of these aspects of

lactation physiology. Nevertheless, dairy producers should keep in mind that better

quality milk can be obtained by genetic selection for calm temperament. Animal

producers can use the information presented in this thesis when they are considering


84

non-dairy sheep for genetic selection. Animal breeders should consider animal

temperament as a selection criterion that will help improve production.

The findings from this work also indicate that the temperament of dairy cows from

the same herd will not affect the milk composition under normal milking conditions.

Most of the authors who reported inhibition of milk ejection, due to stress-related

oxytocin inhibition or blockage, studied milk ejection in primiparous cows (Van

Reenen et al., 2002; Hopster et al., 2002) or ewes (Negrão & Marnet, 2003), in an

unfamiliar milking room (Rushen et al., 2001), or under other stressful conditions

such as the presence an aversive handler (Rushen et al., 1999). The first few milkings

for new heifers or ewes, and being milked in an unfamiliar room, are stressful for the

animals. The experimental cows in the present work were milked by the normal

milkers from the farm, using the same milking routines, two days after their

temperament test in the previous experiment. Those cows were arguably so familiar

with the milking situation that they were not stressed. They may have been stressed

straight after the temperament tests, but they quickly got back to normal so there was

no correlation between behavioural tests and milk production, within a herd, as seen

also by Purcell et al. (1988). However, the authors did observe a weak between-herd

correlation of parlour score with milk production. Surprisingly, cows with high

parlour scores (restless) had higher milk production than the cows with low parlour

scores (Purcell et al., 1988). This might be because temperament is generally tested

under fear-eliciting conditions. The cows may not bring the fear of the test situation

into their normal milking situation, except for the first couple of milkings after the test.

Milking behaviours of the cows or the ewes were not statistically analysed in this

work. However, from general observation during the experiments, it can be

concluded that the animals can be adapted for milking by training. For example,

milking was not likely a problem for the dairy cows because most of the cows had

been milked for a long time when the experiment started. There were 10 first calvers,

but the latest calver had been milked for 80 days when the experiment started.

Clearly, the Holstein is a dairy breed and is well-known for having a good

temperament.

The situation differs with the Merino ewes in the study. They had never been milked


85

or trained to work with humans. The process of milking would be a stressor to these

animals, especially at the beginning, and the level of stress would be even greater for

the nervous ewes than the calm ewes. This is supported by the dramatic fall in milk

yield and fat concentration in nervous ewes in Weeks 2 and 3. On the other hand,

milk yield and fat concentration were both stable in calm ewes throughout the

experiment. However, after a couple weeks of training, most of the ewes were co-

operative when being ushered onto the platform and during milking. This observation

is similar to that of Bencini (1993), who showed that both Merino and Awassi ×

Merino ewes learnt the milking routine quickly.

An ability to learn and to habituate to new environments or managements is regarded

as a desirable characteristic for animals (Kovalcikova & Kovalcik, 1982). Boissy &

Bouissou (1995) indicated that the assessment of fearfulness in farm animals could be

used to predict their subsequent ability to adjust to changes and their sensitivity to the

development of stress and pathologies. Animals with calm temperament are more

likely to be able to cope with stress and adjust to changes, indicating the value of

selecting farm animals for calm temperament, as well as the value of proper training.

Cattle and sheep are flock animals so that they get nervous when they are separated

and held in an unfamiliar arena or box. Temperament tests therefore measure an

animal’s fear in this situation. The open-field test that was used in Experiment 4

(Chapter 3) was based on the design in the study of Murphy (1999) who used an

open-field test with a human and flock-mates in the arena while she tested the

temperament of Merino sheep. The philosophy behind it was to test the response of

an animal to a situation where there was conflict between attraction towards a flock-

mate and fear of the human barrier between them. Murphy (1999) suggested that, in

the presence of a human, the mobility and frequency of vocalization of sheep tended

to increase. Similarly, in Experiment 4, most of the cows did not come near to their

herd-mate because of the human in the arena.

It has to be noted that definitions of temperament and its measurement differ from one

study to another. Most authors defined the temperament of dairy cows by their

behaviours during daily husbandry or by their behaviours during milking, and they


86

called the latter ‘milking temperament/behaviour’ or ‘parlour score/behaviour’

(Dickson et al., 1970; Purcell et al., 1988). Some of them attempted to include the

human factor in milking such as the attitude and reaction of the cows to the milkers.

However, it was very hard to avoid biases on temperament scores in these studies

because scoring was done by the milkers or herd owners. Scoring was affected by the

preferences of the owners, so cows with high milk production may have received high

scores. In this thesis, the temperament of the cows was tested and scored by a stranger

to the cows, and the scores were only based on the behavioural characteristics during

the test. This test, therefore, could avoid bias and personal preference.

The open-field test including a human, as used in my work, can add one more option

to the existing numerous temperament tests. The results from the test indicated that

an open-field with a human was reliable for testing the temperament of dairy cows

because it could test the fear levels of the cows. Scientists and animal producers may

have to compare temperament tests used to choose the best one for their experimental

or management purposes. Different or repeated tests, different test options and

combined scoring may be helpful because Fordyce et al. (1982) suggested that an

animal’s temperament depends on the situation in which it is observed so that

individual or breed may score high in one test or situation but low in another.

The finding that the calm ewes produced more casein than the nervous ewes is useful

information for the diary industry. It is also useful for selecting sheep for dairy

potential. Sheep milk is very suitable for making cheese because of its high

concentrations of protein, casein and fat. Australia has a huge population of sheep

from which sheep should be selected for dairy purposes. Calm temperament should

be one of the selection criteria. Selecting Merino ewes for temperament could be an

easy and economical way to improve milk protein and casein.

In conclusion, protein and casein concentrations of milk can be improved by genetic

selection for calm temperament in Merino ewes. However, the clotting properties of

the milk were not improved by increased protein and casein concentrations in sheep

milk. For dairy cows, an open-field test including a human and a flock-mate may be

useful for measuring the fear response and, therefore, the temperament of cows. The

lack of a relationship between temperament scores and milk production in the


87

Holstein cows suggests that familiar milking routines may not be a stressor because

these animals already have a good temperament and their fear level is very low under

normal milking conditions. Further work is needed to study the hormones involved in

protein synthesis and milk removal to determine to reasons why milk protein is

affected by temperament or stress from milking.

References

88

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