Temperament and Milk Quality in Sheep and Cattle

Transcription

1 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

2 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

3 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 Animal temperament and stress Definition of temperament and stress Measurement of animal temperament Factors related to temperament Breed Experience and training Age The influences of temperament and stress on animal production The effects of temperament in different production systems Beef Cows milk Ewes milk How temperament affects production Behavioural reasons Physiological and hormonal changes Change in the immune system Milk synthesis, milk ejection reflex and removal of milk Milk synthesis Control of lactation and milk ejection Endocrine regulation of lactation 17

4 ii The mechanism of milk ejection Oxytocin release and milk removal in cows and ewes The effect of stress on milk synthesis, yield and composition The factors that affect milk yield and composition Nutrition Season/ Lactation period The inhibition of milk let-down by stress Adrenaline Milkers and milking techniques Milking environment The effects of stress on milk composition How fat level is affected by stress How protein level is affected by stress The effects of milk composition on the processing performance of milk for cheese Process of cheesemaking Conversion of milk into cheese Clotting properties of milk Milk composition and clotting properties of milk Casein and fat concentrations and clotting properties of milk Protein level and clotting properties of milk Measurement of clotting properties of milk 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

5 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 Materials and methods 47 Results 48 Discussion 49 Experiment 3: The effects of temperament on clotting properties of milk from calm and nervous Merino sheep 52 Introduction 52 Materials and methods 53 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 Materials and methods 65 Results 69 Discussion 74 Experiment 5: The relationship between the temperament and milk quantity and quality in Holstein cows 77 Introduction 77 Materials and methods 78

6 iv Results 79 Discussion 82 General discussion for Chapter 4 84 General discussion 86 References 91

7 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

8 vi 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

9 vii 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.

10 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.

11 General Introduction 1 General Introduction 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

12 General Introduction 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?

13 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

14 Chapter 1 Literature Review 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 Animal temperament and stress 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,

15 Chapter 1 Literature Review ). 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

16 Chapter 1 Literature Review 6 threatening environments. Calm animals respond less and adapt more readily compared to nervous animals 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 m 2 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

17 Chapter 1 Literature Review 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

18 Chapter 1 Literature Review 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 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 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

19 Chapter 1 Literature Review 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 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 cooperation 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) 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).

20 Chapter 1 Literature Review 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 The effects of temperament in different production systems 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) 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

21 Chapter 1 Literature Review 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 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

22 Chapter 1 Literature Review 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 How temperament affects production Behavioural reasons An animal s reaction to stress is to behave differently. There are two opposite fearrelated 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) 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-

23 Chapter 1 Literature Review 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

24 Chapter 1 Literature Review 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 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

25 Chapter 1 Literature Review 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 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 α s1 -casein, α s2 -casein, κ-casein, β-casein, α-lactalbumin, β- lactoglobulin, serum albumin and immunoglobulins IgG 1, IgG 2, 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).

26 Chapter 1 Literature Review 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 C 4 to C 14 ) 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 Control of lactation and milk ejection Endocrine regulation of lactation

27 Chapter 1 Literature Review 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

28 Chapter 1 Literature Review 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 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 lobuloalveolar 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 machinemilked ewes (Negrão & Marnet, 2003), suggesting that the animals might produce less milk under stress because cortisol levels are increased The mechanism of milk ejection

29 Chapter 1 Literature Review 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