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Dairy Products and Farming Techniques for the Sheep Milking Industry

A report for the Rural Industries Research and Development Corporation by Roberta Bencini

September 2005

RIRDC Publication No 05/142 RIRDC Project No UWA 66A

ISBN 1 74151 205 0 ISSN 1440-6845

Dairy Products and Farming Techniques for the Sheep Milking Industry Publication No. 05/142 Project No.UWA 66A

The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable industries. The information should not be relied upon for the purpose of a particular matter. Specialist and/or appropriate legal advice should be obtained before any action or decision is taken on the basis of any material in this document. The Commonwealth of Australia, Rural Industries Research and Development Corporation, the authors or contributors do not assume liability of any kind whatsoever resulting from any person's use or reliance upon the content of this document.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details (Roberta Bencini) (School of Animal Biology Faculty of Natural and Agricultural Sciences The University of Western Australia)

Phone: 08 64882521 Fax: 08 6488 1040 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected]. Website: http://www.rirdc.gov.au

Published in September 2005 Printed on environmentally friendly paper by Canprint

Foreword

The sheep milking industry is still in its infancy even though Australia imports sheep milk products and farm gate returns for sheep milk are high (Bencini and Dawe, 1998). The lack of productive dairy sheep has been a hindrance to the establishment of the sheep milking industry (Bencini, 1999) and the industry faces the problem of identifying productive genotypes of sheep that will make sheep milking economically viable.

This project investigated the milk production, milk composition and processing performance of the milk from sheep that have extra copies of the ovine Growth Hormone gene in collaboration with CSIRO. Growth Hormone is commonly injected in dairy cattle in the USA to increase milk production, but its use to enhance milk production is not approved in our country. As the GH transgenic sheep produce the extra GH naturally, it should be feasible to develop commercial applications. Since GH gene is passed on to the progeny of these sheep the GH transgenic sheep have the potential to bring a permanent genetic gain to the Australian sheep milking flock. Animal welfare problems and changes in the legislation meant that CSIRO had to destroy the transgenic sheep before the investigation was completed. As an alternative, milk production and composition of the East Friesian sheep that was imported in Australia was recently studied. Although crosses of East Friesians with local breeds were milked in previous projects (Bencini and Aboola, 2002), the dairy potential of the pure bred under Western Australian conditions had not been studied previously.

Another problem faced by the Australian sheep milking industry is the development of dairy products suitable for Australian consumers and of markets for these products. To address this problem a new blue cheese that is milder than traditional sheep and cow’s milk blue cheeses was developed and has been very successful within the local community.

Finally, sheep milk could be marketed more profitably if it could be claimed that it has health benefits for the consumer. To substantiate these claims, the content of Conjugate Linoleic Acid (CLA) that has been found to have anti-carcinogenic and anti-atherosclerosis properties in laboratory animals was investigated. Methods to increase the content of favourable compounds in the milk by feeding supplements high in unsaturated fatty acids such as fishmeal and sunflower oil were investigated.

This project was funded from RIRDC Core Funds, which are provided by the Australian Government.

This report, a new addition to RIRDC’s diverse range of over 1500 research publications, forms part of our New Animal Products R&D program, which aims to facilitate the establishment of a viable sheep milking industry in Australia.

Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/eshop

Peter O’Brien Managing Director Rural Industries Research and Development Corporation

iii

Acknowledgments

The financial support of Rural Industries Research & Development Corporation is gratefully acknowledged.

This project has been extremely hard work. I have to thank all the people that gave their time, often overworked and underpaid, to help us in the completion of this project:

UWA Staff Peter Cowl, Shenton Park Field Station Animal Facilities Manager Svjetlana Mijatovic, General Assistant and PhD Student The milkers: Georgett Banchero, Jo Bannister, Jenny Cheng, Brian Chambers, Karen Debski, Gabriel Garcia, Piotr Jaster, Di Mayberry. David Peake, 4th Year Student Peter Hutton, Casual Milker and 4th Year Student Kevin Murray, Research Associate, Mathematics Department for help with statistical analyses Craig and Maxwell Macfarlane for support and help during the project Anna, Vanessa and Ennio Malu for friendly support and help in the sale of our dairy products

CSIRO Collaborators Dr Norm R Adams, Senior Principal Research Scientist Jan R Briegel, Senior Technician Soressa Kitessa, Senior Research Scientist Andrew Williams, Technician

Industry partners YHH Holdings: Graham Daws, Director John Blair, Stock Manager Casa Dairy Products: Cliff Tarry, Owner and Director Jane and Bruce Wilde, Jarrah-Lee Springs East Friesian Sheep Stud, Nannup (WA) CHR Hansen for donating the starter cultures for our Dairy Products Laboratory

RIRDC Dr Peter McInnes, Manager, New Animal Products Program, for his continuing support and encouragement.

Contents

Foreword ...... iii Acknowledgments...... iv Executive Summary...... vi 1. Introduction ...... 1 2. Objectives ...... 3 3. Methodology...... 4 3.1 Location and animals...... 4 3.2 Management of the animals...... 4 3.3 Measurements of production and composition of milk...... 5 3.4 Product development and production of sheep dairy products ...... 6 3.5 Statistical analyses...... 6 4 Experimental...... 7 5. General Discussion and implications...... 39 6. Recommendations...... 40 7. Communications Strategy...... 41 8. References ...... 42

Executive Summary

This project addressed some of the problems faced by the emerging sheep milking industry in Australia. Two of these were identified as (Bencini, 1999): 1) The lack of productive genotypes of dairy sheep 2) A lack of sheep milk products suitable for Australian consumers.

Sheep milk could be marketed more profitably if it could be claimed that it has health benefits for the consumer. Therefore we also investigated 3) Methods to increase the content of favourable compounds in the milk.

Thanks to funding from the RIRDC, we conducted research on these main areas of concern.

1) The lack of productive genotypes of dairy sheep The local breeds of sheep produce less than 100 litres of milk per lactation, a level of production that is not economically viable in the average sheep milking enterprise (Bencini and Dawe, 1998). The Awassi and the East Friesian sheep have been imported recently and they are reported to be the highest producers of milk in the world (Epstein, 1985; Anifantakis, 1986), so they have the potential to make sheep milking profitable. However, in both cases, only small numbers of animals were actually imported, and they are expensive to buy or difficult to find. A completely new genotype has also been developed using transgenic technology. CSIRO produced sheep that have extra copies of the ovine Growth Hormone gene. Growth Hormone (GH) is commonly injected in dairy cattle in the USA to increase milk production, but its use to enhance milk production is not approved in Australia. As the GH transgenic sheep produce the extra GH naturally, it should be feasible to develop commercial applications. The GH gene is passed on to the progeny of these sheep allowing farmers to introduce this gene into their flocks, producing permanent genetic gain, similarly to the introduction of the Booroola F (Fertility) gene. Therefore the GH transgenic sheep have the potential to bring a permanent genetic gain to the Australian sheep milking flock. Our project aimed at evaluating the milk production, milk composition and processing performance of the milk of the GH transgenic sheep. Welfare problems with the transgenic sheep and changes in the legislation underpinned the decision of CSIRO to destroy the GH sheep before we could finish our studies. Despite this, we were able to milk some transgenic sheep and found that they produce twice as much milk than the non-transgenic controls. The milk had normal composition and normal processing properties suggesting that these sheep had an enormous dairy potential. Although all transgenic sheep have been destroyed, a last minute plea from UWA and from RIRDC ensured that some semen from the rams was frozen before their destruction. The semen is currently stored at the Veterinary School of Murdoch University and we have not given up hope than one day we will be able to resurrect the transgenics and complete our studies. The new legislation and consumers’ attitude to genetically modified organisms means research in this area would be extremely expensive and is out of our reach with current funding levels.

Initially we had planned to cross the GH sheep, which were ordinary fine wool Merinos, with East Friesian dairy sheep. The idea was that if GH Merinos produced twice as much milk as the controls their crosses with a dairy breed may produce even more and provide the sheep milking industry with a very desirable genotype. Since this was not possible due to the premature destruction of the GH sheep, in the last year of the project we decided to test the dairy potential of the East Friesian sheep. Our previous project had already shown the crosses of the East Friesian with local sheep produced more milk than the local breeds. Testing the East Friesian crosses was motivated by the fact that pure East Friesian are expensive and hard to come by, and it was assumed that sheep dairy farmers would milk their vi crosses with local breeds. The dairy potential of the purebred East Friesian had not been tested previously under Western Australian conditions. Thanks to a generous offer from Jane and Bruce Wilde from Jarrah Lea Springs (Nannup) we were able to borrow 20 7/8 East Friesian sheep. We milked them for a whole lactation and measured the production and composition of their milk.

Our results indicated that the milk production of the East Friesian sheep was similar to that of the Awassi sheep. However, they had longer and more persistent lactations making them an ideal breed to produce sheep milk out of season or for a year round supply of milk.

We also continued to milk our flock of dairy sheep and confirmed that the East Friesian x Awassi genotype has the highest milk production of all the crosses we investigated.

2) A lack of sheep milk products suitable for Australian consumers To establish a viable sheep milking industry it is important to develop typical Australian sheep dairy products that are suitable for and readily accepted by Australian consumers. Famous imported cheeses such as the Roquefort, Fetta and Pecorino are often very strong in taste and may not be readily accepted by Australian consumers (Bencini, 1999).

In our previous projects we developed a range of sheep dairy products but we had never attempted to develop a blue vein cheese. In this project we developed a mould-ripened blue cheese that has a relatively short maturation time and has been well received by the local community.

3) Methods to increase the content of favourable compounds in the milk Sheep milk could be marketed more profitably if it could be claimed that it has health benefits for the consumer. In recent times a number of unsaturated fatty acids has been reported to produce health benefits. Omega-3 fatty acids present in fishmeal are some of such fatty acids. We conducted research that showed that by feeding fishmeal to sheep the omega-3 fatty acids are transferred into the milk (Kitessa et al., 2003). Therefore sheep milk farmers could use such supplement to produce health enhanced sheep milk that has increased concentrations of omega-3 fatty acids.

Conjugated Linoleic Acids (CLA) are also a group of unsaturated fatty acid isomers recently identified as anticarcinogens, potent antioxidants, modulators in the immune system, anti- atherosclerosis agents and body weight protectors (Jenkins and Lundy, 2001). Dietary CLA inhibits the growth of a number of human cancer cell lines and suppresses the development of chemically induced tumours in laboratory animals (Parodi, 1999). Among domestic ruminants, sheep milk has the highest concentration of fat and is the richest source of CLA (Banni and Martin, 1998). Some studies have shown that genetic factors (breed and/or species) can affect the levels of CLA in animal products (Lawless et al., 1999; Knight et al., 2003; Kelsey et al., 2003). We studied differences in CLA content in the milk of different breeds of sheep over eight weeks of lactation to investigate potential differences in CLA concentrations between different breeds of dairy sheep. We found that breed differences were little, but that fat tail sheep (either Awassi or their crosses with local breeds) had a more constant concentration of CLA throughout lactation, possibly due to the mobilization of body reserves stored in the fat tail. This work demonstrated that beneficial compounds such as CLA are present high concentrations in the milk of sheep their concentrations are maintained throughout lactation in the fat tail breeds. Ruminants produce CLA naturally but they do not produce concentrations of CLA that are high enough to have a significant health benefit (Knight et al., 2003). Kelly et al. (1998) and Kay et al. (2002) demonstrated that increasing the concentration of linoleic acid in the diet increases the amount of CLA produced in the milk of dairy cows. Kelly et al. (1998)

vii suggested that the objective in research should be to increase the CLA content of milk fat rather than the absolute amount secreted in milk. Therefore we investigated feeding strategies to increase the concentration of CLA in the milk of sheep. In a first experiment we fed sunflower oil, which is rich in linoleic acid, a precursor of CLA. The addition of sunflower oil to the diet increased the level of cis-9, trans-11CLA in the milk fat from a basal level of 10.6 and 9.4 mg/g of milk fat to 20.1 mg/g of milk fat and 23.9 mg/g of milk fat in Merino and Awassi sheep respectively. Our work has demonstrated that it is possible to adopt feeding strategies that increase the concentration of beneficial compounds in the milk. This opens the way for the production of designer sheep milk similar to designer milks suggested within the traditional cow dairy industry as methods to increase the marketability of cow’s milk.

viii

1. Introduction

Australia has a large ethnic population of European origin and imports some $8 million worth of sheep milk products every year (Dawe and Langford, 1987). About 8,000 tons per year of sheep milk products could find a market in Australia and to match this demand 250,000 ewes would have to be milked in 100-150 sheep dairies (Dawe, 1990). Compared to cow's milk, sheep milk produces cheeses of higher yield, whiter colour, richer taste and better nutritional quality (Kalantzopoulos, 1999). Therefore, the development of sheep milk cheeses suitable for Australian consumers would increase the variety and quality of cheeses consumed in the diverse Australian market.

The existence of potential local market for sheep milk products has been confirmed by research funded by the RIRDC (Bencini, 1999), but despite this the Australian sheep milking industry is still small. This may be due in part to the fact that returns from the sale of lambs have recently soared due to the animal industries crisis experienced in the rest of the world.

At The University of Western Australia, we have been researching the three main problems faced by the fledgling sheep milking industry, as outlined below.

Firstly, until recently Australia did not have specialised breeds of dairy sheep. Local breeds of sheep produce less than 100 litres of milk per lactation, a level of production that is not profitable (Bencini and Dawe, 1998). Milk production is a trait only expressed by ewes, and genetic improvement for milk production can only be achieved through progeny testing schemes similar to those used in dairy cattle and overseas dairy sheep. This idea was pursued by the RIRDC, with a tender awarded to Emeritus Professor D.R. Lindsay to investigate the feasibility of such schemes for the Australian sheep and goat milking industries (Lindsay and Skerritt, 2003).

The low productivity of the local breeds could be addressed immediately by importing specialised dairy breeds of sheep from overseas. This was the aim of the recent importation of Awassi and East Friesian sheep, two breeds that have the highest production of milk in the world (Epstein, 1985; Anifantakis, 1986a). These two breeds have the potential to increase yields and make sheep milking economically viable. In Previous projects we tested the dairy potential of the crosses of these imported breeds with local breeds (Bencini, 1999; Bencini and Agboola, 2002). The present project investigated 7/8 East Friesian ewes to determine their dairy potential under Western Australian conditions.

Another way to address this problem could be the use of transgenic animals. CSIRO developed a genotype of transgenic sheep that has an extra copy of the sheep Growth Hormone (GH) gene. GH is injected in dairy cows to increase milk production but its use is not permitted in Australia. The GH transgenic sheep produce the GH naturally and we investigated the possibility that the high levels of GH would result in increased milk production. While we showed that this was indeed the case in GH transgenic sheep when compared to the control fine wool Merinos, we were unable to further our studies due to a decision by CSIRO to destroy the transgenic sheep.

The second problem faced by the Australian sheep milking industry is represented by the need to develop new typical Australian sheep milk products. Imported overseas cheeses such as the famous Roquefort, Pecorino and Fetta are characterized by a strong taste that may not be suitable for Australian consumers. As we had not previously attempted to develop a blue cheese, the present project aimed at developing a mild blue style cheese that would be readily accepted by Australian consumers. 1

Lastly, we recognise that sheep dairy products could be marketed more effectively if they were shown to produce health benefits for consumers. We conducted experimental work to measure the presence of Conjugated Linoleic Acid (CLA), which have been reported to be anticarcinogens, potent antioxidants, modulators in the immune system, anti-atherosclerosis agents and body weight protectors (Jenkins and Lundy, 2001).

We also studied possible methods to increase favourable compound such as omega-3 fatty acids and CLA in sheep milk by feeding supplements to the sheep.

2. Objectives

The main objective of this project was to aid the development of an Australian sheep milking industry by: • Assessing the dairy potential of new genotypes of sheep such as the GH transgenic sheep and East Friesian sheep • Investigating sheep milk for its potential health benefits and nutrition strategies to increase the concentration of desirable unsaturated fatty acids (omega -3 and CLA) • Developing a new sheep milk blue cheese suitable for Australian consumers.

3. Methodology

3.1 Location and animals

Most of the experimental work for this project was conducted at The University of Western Australia’s Shenton Park Research Station, Floreat, where the dairy sheep were kept in paddocks. Laboratory work was conducted in the Dairy Products Laboratory in the School of Animal Biology at The University of Western Australia.

Australian Merino Society (AMS) Merinos were sourced from Allandale (Wundowie, WA), the research farm of The University of Western Australia and were used as controls in comparative experiments as they are the most common breed of sheep in Australia.

The remainder of the dairy ewes were supplied by our industry partners, YHH Holdings and Jarrah Lea Springs and were East Friesian x Awassi cross ewes, Awassi x Merino 7/8 and 15/16 cross ewes and East Friesian x Poll Dorset 7/8 cross ewes. These sheep came from Yathroo Farm, (Dandaragan, WA) and from Jarrah Lea Springs (Nannup, WA).

The work with the GH transgenic sheep was conducted at the CSIRO Research Station in Bakers Hill (WA) where the transgenic sheep were kept under strict quarantine confinement.

3.2 Management of the animals

Housing and nutrition - During the experimental periods, animals were kept in communal paddocks where they grazed irrigated pasture composed predominantly of Kikuyu (Pennisetum clandestinum) and subterranean clover (Trifolium subterraneum) and had meadow hay available ad libitum. They received up to 1 kg of lupins/head (392 g/kgDM protein, 21 MJ/kgDM energy) daily and they were also given approximately 200g of a mixture of 70% oaten chaff, 30% lupins, and 0.5% hi-cal and salt as a contentment food at each milking. When experiments were completed and/or the lactation was concluded the sheep were returned to their respective properties of origin.

Lambs that were born to the experimental ewes were housed in an outdoor pen when separated from their mothers and were offered creep feed represented by a mixture of 60% oaten chaff, 30% lucerne hay and 10% cracked lupins.

All experimental protocols were approved by The University of Western Australia’s Animal Ethics Committee, and conducted according to the recommendations of the Australian National Health and Medical Research Council for the care and handling of animals in experiments. Milking – The dairy sheep were milked on a 12 bay rapid exit milking parlour built by Prattley (Temuka, NZ, Figure 3.2.1). When possible, the sheep were fed on the platform a few weeks before lambing, so that they learnt that by walking onto the platform they received food. The sheep were milked twice a day except for the first and last months of lactation when the milking frequency was reduced to once a day. They were milked with an Alfa Laval milking machine (now DeLaval International AB, Tumba, Sweden) that had a pulsation rate of 120/min and vacuum pressure of - 40 kPa. At the end of each milking the teats were disinfected with an iodine based commercial preparation (Alfadyne Teat Sanitiser, Australia).

a b

cd Figure 3.2.1. The milking platform at Shenton Park Research Station, The University of Western Australia, is a rapid exit parlour that allows a rapid throughput of ewes per hour. a) Sheep entering the parlour. b) The author milking the sheep. c) Sheep leaving the stalls. d) Sheep leaving the dairy (photographs by Craig Macfarlane).

3.3 Measurements of production and composition of milk

Measurements of milk production and composition occurred throughout each lactation, either weekly or fortnightly. Milk production was measured with Tru Test milk meters (Tru Test Distributors, Auckland, New Zealand). These testers have a valve that diverts a proportion of the milk produced (in our case 58 g/kg produced) into a plastic jar. The jar is then weighed and the weight is multiplied by a constant to allow calculation of the daily milk output. Samples of milk were collected from the Tru Test jars, stored at 1-4oC and analysed with a Milko Scan 133 (Foss Electric, Denmark) calibrated for sheep milk. This is a single-beam infrared instrument, which measures the infra red absorption at wavelengths characteristic of the components to be analysed. When testing for fat concentration with the A filter, it measures the absorption at 5.73 µ by the carbonyl group of the ester linkage. If the B filter is used it measures the absorption at 3.5µ by the -CH2 groups. When testing for protein concentration it measures the absorption at 6.46 µ by the amine II groups of the peptide bond and for lactose testing the absorption at 9.6 µ by the hydroxyl group. Total solids are automatically calculated by the instrument by adding protein, fat, lactose and a constant mineral bias of 0.79%.

3.4 Product development and production of sheep dairy products

The development of a new blue cheese was undertaken in the Dairy Products Laboratory of The University of Western Australia. At the same time, we continued to produce sheep milk dairy products such as yoghurt, camembert cheese and Labnah (cream cheese). The following equipment was used for this project:

• Cheese vat - a stainless steel-jacketed cheese vat of 30L capacity built at the Combined Workshop at The University of Western Australia. The vat has a variable speed paddle to mix the milk and temperature adjustment to maintain the milk at the desired temperature. • Incubator - a thermostated incubator of approximately 80L capacity was used to incubate dairy products such as yoghurt and cheese. • Disc Bowl Centrifuge - An Armfield disc bowl centrifuge was used for the separation of milk fat. It was used to produce batches of low fat yoghurt and cream. • Batch pasteuriser - a laboratory batch pasteuriser of 30L capacity was built at the workshop at The University of Western Australia. This was used to pasteurise the milk prior to processing at a temperature of 72°C for 20 seconds. • Milko Scan 133 – described above, was used for analysis of milk composition before the milk was transformed into dairy products. • Lattodinamografo or lactodynamograph (Foss Electric, Italy) - This instrument was used to measure the clotting properties of the milk. These were the renneting time, r, or time it took for the milk to clot, the rate of curd formation, k20, which is the time it took for the clotting milk to reach a standard consistency of 20mm and the consistency of the curd, A30, which is the distance between the two arms of the lactodynamograph output (Bencini, 2002).

Other equipment used for the project included pH meter, balances, refrigerator (4°C) and freezer (-20°C).

For each experimental batch of dairy products the following measurements were recorded: • Composition of the milk (protein, fat, lactose, total solids) • Initial pH of the milk • Amount of milk processed • Processing procedures (e.g. amount of rennet added, type of starter culture used, time and temperature in cheese vat, etc.) • Yield of dairy products from each litre of milk • Composition of dairy products derived from the milk (protein, fat, moisture, ash)

Protein in cheese was determined with The Kjeldhal method. Fat was determined by hexane: propanol (3:2) extraction. Water and ash were determined by freeze-drying and furnace incineration at 550°C respectively. Sodium and Calcium were also determined by the atomic absorption method.

3.5 Statistical analyses

All results are presented as means ± their standard errors. Unless otherwise indicated, the statistical analysis of the milk production and composition was done by Least Squares Analysis of Variance (SuperANOVA, Abacus Concepts, 1991) and effects were assumed to be significant when the level of probability was 5% or less. Analysis of covariance was conducted on lactation data to take into account the effect of stage of lactation.

4 Experimental

Our experimental work addressed the lack of productive dairy sheep as well as the development of new sheep milk dairy products. We continued to evaluate the dairy potential of the East Friesian crosses with the Awassi and the Merino breeds as in previous projects as well as undertaking work on the GH transgenic sheep and on the 7/8 East Friesian sheep.

We conducted as series of experiments to measure and possibly increase the concentrations of favourable unsaturated fatty acids in the milk and we developed a new blue cheese.

4.1.1 Dairy Production of Awassi, East Friesian x Merino, East Friesian x Awassi and East Friesian ewes

The lack of productive breeds of dairy sheep has been a hindrance to the establishment of a sheep milking industry in Australia (Bencini and Dawe, 1998). The recent importation of Awassi and East Friesian sheep may change this situation. So far we have assessed the dairy potential of the Awassi sheep and we have shown that the Awassi sheep and their crosses with the East Friesian sheep produce more milk and have longer lactations than local sheep (Bencini, 1999; Bencini and Agboola, 2002). However, improving the genetic basis of the sheep milking flock remains an important objective for the Australian sheep milking industry (Lindsay and Skerritt, 2003). Therefore testing new genotypes of sheep such as the East Friesian is of paramount importance for the industry. The East Friesian sheep has been reported by some to be delicate and prone to pneumonia (Mills, 1989) or to be a non-flocking animal that prefers to be kept on its own (Kervina et al., 1981). In most countries where it has been imported it ended up being back crossed to local breeds originating the Assaf in Israel, the Frisonarta in Greece, the FLS in France, and the Friserra in Portugal (Flamant and Barillet, 1982). However, crosses with the Awassi have shown no adaptation to the harsh environment of Israel (Eyal and Goot, 1969; Mills, 1989) and Sardinian farmers complain about similar adaptation problems with the East Friesian x Sarda crosses (personal observation). Flamant and Morand-Fehr (1982) wrote that attempts to introduce the East Friesian sheep in the Mediterranean region failed as the breed was not adaptable to harsh conditions and to flock management. Treacher (1987) also reported that the importation of the East Friesian had little effect on local breeds and now the genetic improvement of the local breeds is attracting more attention since they appear to be better suited to their environments.

In Australia sheep dairy farmers have reported the occurrence of pneumonia in Tasmania (D. Rae, Pers. Comm.) and even in Western Australia (B. Wilde, Pers. Comm.).

Given these reports, in the last year of the project we gladly accepted the offer of milking a flock of 7/8 East Friesian ewes to study their dairy potential under Western Australian conditions. Here we report on the lactations of our flock of dairy sheep for the years 2002, 2003 and 2004.

4.1.2 Materials and methods

2002 lactation

In 2002, we milked a flock of flock of 12 Awassi, 17 East Friesian x Awassi, 16 East Friesian x Merino, 7 Awassi x Merino and 12 Merino ewes. The ewes lambed between mid May and mid July and were milked twice a day except for the first and last month of the lactation when they were milked only once a day. Milk production and composition were assessed at weekly intervals, except towards the end of the lactation when we adopted testing at two weekly 7 intervals. Production and composition data were analysed by one way ANCOVA (SuperANOVA, Abacus Concepts, 1991), where the main effect was the genotype and the covariate was the week of measurement, which is a reflection of the stage of lactation. Fisher’s protected LSD tests were adopted to compare the differences in milk production and composition between genotypes.

2003 lactation

In 2003, we milked a flock of flock of 26 Awassi, 14 East Friesian x Awassi, 5 Awassi x Merino, 15 East Friesian x Merino and 14 Merino ewes. The ewes lambed between 16 April 2003 and 13 June 2003 and were milked twice a day except for the first and last month of lactation, when milking was only once a day. Milk production and composition were assessed at fortnightly intervals for the whole lactation period. Production and composition data were analysed by one way ANCOVA (SuperANOVA, Abacus Concepts, 1991), again using the genotype as the main effect and the stage of lactation as the covariate. Fisher’s protected LSD tests were adopted to compare the differences in milk production and composition between genotypes.

2004 lactation

In 2004, we milked a flock of flock of 23 Awassi, 20 East Friesian, 13 East Friesian x Awassi, 5 Awassi x Merino, 14 East Friesian x Merino and 16 Merino ewes. The ewes lambed between 23 March 2004 and 28 June 2004. This spread of lambing was due to the fact that the East Friesian ewes from Nannup started lambing as soon as they arrived despite being pregnancy diagnosed by a vet as due to lamb at the same time of our sheep. This meant that we could not start milking them as soon as we wished and missed the beginning of their lactation. The sheep were milked twice a day except for the month of June, when milking frequency was reduced to once a day to allow the nursing of lambs and for the month of December when the frequency of milking was also reduced to once a day due to low productions. Milk production and composition were assessed at fortnightly intervals and interpolated for the purpose of drawing the lactation curve. Production and composition data were analysed by one way ANCOVA (SuperANOVA, Abacus Concepts, 1991), again using the genotype as the main effect and the stage of lactation as the covariate. Fisher’s protected LSD tests were adopted to compare the differences in milk production and composition between genotypes.

a) 1800 A separate two way ANCOVA was also performed to examine the effect of 1600 multiple births (single versus twin or 1400 triplet). To examine if breeds differed in 1200 the lengths of their lactations the 1000 variable days-in-milk was also analised Milk produced (g/day) as a dependent variable by one way 800

ANOVA. 600 4.1.3 Results 400 200

2002 lactation 0

b) Figure 4.1.1 shows the typical 1400 lactation curves for the breeds that 1000 we milked in 2002. Throughout their lactation, the East Friesian x Awassi 800 ewes produced more milk than the 600

Awassi, East Friesian x Merino, 400 Awassi x Merino and Merino ewes (p<0.05, Table 4.1.1). 200

0 The concentration of milk fat c) differed between genotypes as the 1000 Awassi sheep had higher 800 concentrations of fat in the milk than the other breeds, and the Merino and 600

East Friesian x Merino sheep had 400 lower concentrations of fat in their milk (p<0.05, Table 4.1.1). 200 0 The concentration of milk protein d) also differed between genotypes as 1000 the East Friesian x Awassi sheep had 800 lower concentrations of protein in the 600 milk than the Merino and the Awassi, East Friesian x Merino and 400 Awassi x Merino sheep had higher 200 concentrations of protein in their 0 milk (p<0.05, Table 4.1.1).

800 e) The concentration of lactose was less variable that that of fat and protein, 600 and it was slightly higher in the milk 400 of the East Friesian x Awassi sheep 200 and East Friesian x Merino ewes 0 (p<0.05, Table 4.1.1). 3029282726252423222120191817161514131211109876543210

Weeks of lactation Figure 4.1.1. Milk productions (g/day) of a) East Friesian x Awassi, b) Awassi, c) East Friesian x Merino, d) Awassi x Merino and e) Merino ewes in 2002.

Table 4.1.1. Production of milk (g/day) and concentrations of protein and fat (%) from East Friesian x Awassi, Awassi, East Friesian x Merino, Awassi x Merino and Merino sheep over the course of the lactation in 2002. Data are Means ± SE. Different letters indicate a significant difference (p<0.05) between means.

Genotype (number of sheep) Milk yield (g/day) Protein (%) Fat (%) Lactose (%) East Friesian x Awassi (17) 1225 ± 40.7 (a) 5.25 ± 0.04 (a) 6.19 ± 0.11 (a) 4.88 ± 0.03 (a) Awassi (12) 735 ± 34.2 (b) 5.66 ± 0.05 (b) 6.86 ± 0.17 (b) 4.71 ± 0.03 (b) East Friesian x Merino (16) 561 ± 21.6 (c) 5.53 ± 0.05 (b) 5.46 ± 0.13 (c) 4.84 ± 0.03 (a) Awassi x Merino (7) 545 ± 33.6 (c) 5.65 ± 0.08 (b) 6.35 ± 0.21 (a) 4.69 ± 0.05 (b) Merino (12) 336 ± 17.2 (d) 5.39 ± 0.06 (c) 5.32 ± 0.20 (c) 4.78 ± 0.03 (b)

2003 lactation

The East Friesian x Awassi ewes produced almost twice as much as the Awassi ewes and almost three times as much milk as the merino ewes (Table 4.1.2).

Table 4.1.2. Production of milk (g/day) and concentrations of protein and fat (%) from East Friesian x Awassi, Awassi, East Friesian x Merino, Awassi x Merino and Merino sheep over the course of the lactation in 2003. Data are Means ± SE. Different letters indicate a significant difference (p<0.05) between means.

Genotype (number of sheep) Milk yield (g/day) Protein (%) Fat (%) Lactose (%) East Friesian x Awassi (14) 954 ± 42.6 (a) 5.94 ± 0.05 (a) 6.95 ± 0.09 (a) 4.75 ± 0.03 (a) Awassi (26) 535 ± 21.4 (b) 6.18 ± 0.04 (b) 7.21 ± 0.11 (b) 4.57 ± 0.04 (b) East Friesian x Merino (15) 575 ± 25.2 (b) 6.08 ± 0.07 (b) 6.89 ± 0.14 (a) 4.62 ± 0.04 (c) Awassi x Merino (5) 519 ± 50.3 (b) 6.44 ± 0.09 (c) 7.43 ± 0.19 (b) 4.44 ± 0.06 (b) Merino (14) 360 ± 22.0 (c) 6.22 ± 0.07 (b) 6.93 ± 0.16 (a) 4.61 ± 0.06 (c)

The Awassi, East Friesian x Merino and Merino genotypes had significantly greater concentrations of protein in their milk and the East Friesian x Awassi had the lowest (p<0.05; Table 4.1.2). The concentration of fat was significantly higher in the milk from the Awassi and Awassi x Merino sheep than in the milk from the other breeds (p<0.05; Table 4.1.2). Lactose concentration was greater in the milk from the East Friesian x Awassi and lowest in the milk freom the Awassi x Merino ewes (p<0.05; Table 4.1.2).

2004 lactation

Figure 4.1.2 shows the lactation curves for the genotypes milked in 2004. The East Friesian x Awassi ewes produced again more milk than all the other breeds, including the East Friesian. These were remarkably similar to the Awassi in terms of milk production. (p<0.05, Table 4.1.3).

The concentration of fat was higher in the milk of the Awassi and Awassi x Merino ewes and lower in the other breeds (p<0.05, Table 4.1.3).

The concentration of milk protein also differed between genotypes as the East Friesian x Awassi sheep had lower concentrations of protein in the milk than the other breeds (p<0.05, Table 4.1.3).

The concentration of lactose was less variable that that of fat and protein, and it was slightly higher in the milk of the East Friesian x Awassi and East Friesian sheep (p=0.003, Table 4.1.3).

Table 4.1.3. Production of milk (g/day) and concentrations of protein and fat (%) from East Friesian x Awassi, Awassi, East Friesian, East Friesian x Merino, Awassi x Merino and Merino sheep over the course of the lactation in 2004. Data are Means ± SE. Different letters indicate a significant difference (p<0.05) between means.

Genotype (number of Milk yield (g/day) Protein (%) Fat (%) Lactose (%) sheep) East Friesian x Awassi (13) 777 ± 36.7 (a) 5.94 ± 0.05 (a) 7.18 ± 0.09 (a) 4.64 ± 0.03 (a) East Friesian (20) 499 ± 17.8 (b) 6.12 ± 0.05 (b) 7.00 ± 0.08 (a) 4.65 ± 0.02 (a) Awassi (23) 478 ± 23.9 (b) 6.26 ± 0.06 (b,c) 7.77 ± 0.11 (b) 4.43 ± 0.04 (b) Awassi x Merino (5) 463 ± 54.3 (b,c) 6.35 ± 0.13 (b,c) 8.11 ± 0.25 (b) 4.44 ± 0.07 (b) East Friesian x Merino (14) 371 ± 17.4 (c,d) 6.40 ± 0.09 (c) 7.03 ± 0.14 (a) 4.39 ± 0.06 (b) Merino (16) 327 ± 23.6 (d) 6.29 ± 0.70 (b,c) 7.29 ± 0.17 (a) 4.39 ± 0.04 (b)

The two way ANCOVA revealed a significant effect of birth type, with ewes that gave birth to multiple lambs producing significantly more milk (p<0.05).

The longest lactations were observed in the East Friesian sheep (197 ±12.6 days, a) followed by the East Friesian x Awassi (182 ± 6.2 days; a,b), East Friesian x Merino (157 ± 12.0 days; b,c), Awassi (131 ± 8.6 days; c,d), Awassi x Merino (120 ± 20.6 days; c,d) and Merino (108 ± 6.6 days; d).

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Figure 4.1.2. Milk productions (g/day) of a) East Friesian x Awassi, b) Awassi, c) East Friesian, d) East Friesian x Merino and e) Merino ewes in 2004. 12

4.1.4 Discussion

In general the results presented here are similar to those reported in an earlier report and confirm that the East Friesian x Awassi cross genotype is indeed superior for dairy production (Bencini and Agboola, 2002). This superior performance may be partly due to hybrid vigour, which, in turn, may be the reason why these cross ewes are also larger than the other breeds (their average weight was 71.5 ±3.16 kg, while the Awassi were 58.8 ± 1.35 kg, the East Friesian x Merino were 62.0 ± 1.35 kg and the Merino were 60.2 ± 2.10 kg).

The differences in the concentration of fat and protein in the milk between the different genotypes could be ascribed to the reported negative relationship between milk quantity and quality (Flamant and Morand-Fehr, 1982; Barillet et al. 1986). Lactose is the osmotic component of milk and by drawing water across the mammary gland’s epithelium it determines milk volume (Saacke and Heald, 1974). Not surprisingly, therefore, its concentration was greater in the more productive breeds, the East Friesian x Awassi and East Friesian. Lactose does not play a major role in the production of dairy products so differences between genotypes should not be of concern. The differences in protein and fat concentrations were quite small and the milk of these sheep, even those that have significantly lower protein concentrations in their milk, is still far superior to cow’s milk for making cheese.

The 20 East Friesian sheep milked in 2004 lambed earlier that the other genotypes. This meant that we missed the peak of lactation for a significant percentage of the ewes, which is supported by the large standard errors due to low numbers between the 5th and 9th week of lactation. Throughout lactation, their milk production was similar to that of the Awassi sheep, but for the Awassi we were able to record milk production much earlier and for the whole lactation. The East Friesian sheep have remained in our care and will be milked again in 2005. This time they will lamb together with the rest of the flock and we will be able to assess their dairy performance and compare it with that of the other genotypes. Significantly, by milking these sheep quite late in their lactation we found that they had remarkably good persistencies of lactation when compared to the other genotypes. This is important for sheep milking enterprises that need a constant or year round supply of milk because it would allow producing milk in summer simply by having two flocks lambing at different times. The length of lactation for the East Friesian sheep was similar to that of the very productive East Friesian x Awassi ewes. It will be important to continue to milk the east Friesian in 2005 to be able to compare their persistency of lactation to that of the other breeds.

Three of the lambs born to the East Friesian sheep did contract pneumonia and die, supporting the personal communications from Australian sheep dairy farmers. We are now in contact with the Spooner Research Station in Wisconsin, where similar incidences have been tackled in the past and we are expecting reports on their experiences (I. Berger Pers. Comm.).

Not surprisingly ewes that gave birth to multiple lambs produced more milk. This can be ascribed to both the effect of placental lactogen during pregnancy promoting a greater development of mammary tissue in multiple bearing ewes (Rattray et al.,1974; Davis et al., 1980), as well as the effect of the suckling stimulus from multiple lambs (Bencini and Pulina, 1997). If sheep dairy farmers use a share milking method similar to ours they would obtain more milk from ewes that give birth to multiple lambs.

4.2.1 GH transgenic ewes produce more milk and have longer lactations than non transgenic sheep

Scientists from the CSIRO Division of Animal Production at Prospect NSW developed sheep transgenic for Growth Hormone (GH). These Merino sheep have extra copies of the GH gene and produce greater amounts of Growth Hormone. Their growth rate is about 10% greater than that of control sheep, and they are leaner (N. Adams, Pers. Comm.).

GH increases milk production in dairy cows (Peel and Bauman, 1987; Bauman, 1999). Bovine SomatoTropin (bST) injections are normally used in dairies in the USA to increase milk production (Zinn and Bravo-Ureta, 1996) and in Australia to obtain lean pork (Pethick and Dunshea, 1996), but their use in cattle or sheep is not yet approved in Australia (Bencini and Pulina, 1997).

No negative effects have been reported on the composition of the milk or the health of dairy cattle and the injected bST is not translocated into the milk, which is safe for human consumption (Bauman, 1999). In sheep, GH increases milk production, but the protein concentration of the milk was reported to be negatively affected by some researchers (Fleet et al., 1986; 1988; Holcombe et al., 1988; Pell et al., 1989; Sandles et al., 1988; Stelwagen et al., 1993). Others reported no significant changes in the composition of the milk (Dell’Orto et al., 1996; D’Urso et al., 1998) and Baldi (1999) suggested that these contrasting reports may be due to the use of sheep in different stages of lactation or to different breeds of sheep. Therefore it is foreseeable that the GH sheep will have an increased milk production as a result of their higher production of GH, but we do not know how the extra circulating GH will affect the composition of their milk. Indeed these sheep represented a unique opportunity to study the effects of GH throughout lactation, which was not possible in previous studies.

4.2.2 Materials and methods

Five fine wool Merino transgenic GH ewes and 6 fine wool Merino (control) ewes were milked between weeks 4 and 21 of their lactation with the oxytocin technique (Bencini, 1995). With this method the sheep nurse their own lambs and they do not have to be milked twice a day to maintain lactation. On test days, the ewes were separated from their lambs and milked out manually after inducing a milk ejection (let-down) with an intramuscular injection of 1 I.U. of oxytocin (Ilium Syntocin, Troy Laboratories Pty Ltd, Australia). After 4 hours the sheep were injected again with 1I.U. of oxytocin and are milked out. The milk produced at this second milking was weighed to estimate daily milk output. Samples of milk were also collected and analysed for fat, protein, lactose and total solids. A 10 mL aliquote from each milk sample was also clotted with 10µL of rennet diluted 1 in 20 and the protein concentration of the resulting whey was measured with the Milko Scan. Casein content was estimated as the difference between total protein and whey protein.

Data for milk production and composition were analysed by one-way analysis of covariance (SuperANOVA, Abacus Concepts, 1991), using as a covariate the number of days since birth (days in lactation).

4.2.3 Results

The average daily production of milk from the transgenic sheep was consistently greater than that of the control, non transgenic Merino ewes (Fig 4.2.1, p<0.05). This was despite the fact that one of them had only one functional side of the udder. If data from this animal are 14 removed then the GH ewes produced twice as much as the control ewes, 2.0±0.14 kg/day versus 1.1±0.07 kg/day. During the last two measurements of milk production some of the ewes (both control and GH) had very low lactose concentrations in their milk, below 1%. This normally happens after weaning, when lactose is decomposed into glucose and galactose, which are reabsorbed by the mammary gland epithelium. This normally drives milk volume down and lactation terminates abruptly. However, this did not happen with the GH sheep and during the following week their milk production increased (Fig 4.2.1). Two weeks later the GH sheep were still producing some milk, while the controls had dried up completely (Fig 4.2.1).

The transgenic sheep had a lower concentration of protein and more specifically casein in their milk but the concentration of fat, lactose and total solids did not differ significantly between the transgenic sheep and the controls (Table 4.2.1).

Table 4.2.1. Concentration of milk components (%±SE) in the milk of transgenic GH sheep and control Merino sheep. Different letters indicate a significant difference (p<0.05) between means.

Genotype Fat Protein Lactose Casein Total Solids (number of sheep) Transgenic (5) 7.88±0.329 5.30±0.228 (a) 5.05±0.122 3.12±0.095 (a) 18.80±0.368 (a) (a) (a) Control (6) 7.34±0.186 5.76±0.131 (b) 5.19 ±0.044 4.10±0.110 18.95±0.231 (a) (a) (a) (b)

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Figure 4.2.1. Daily production of milk (kg/day) from transgenic (open circles, n=5) and control sheep (solid circles, n=6) over the course of the lactation.

4.2.4 Discussion

The hypothesis that the transgenic sheep would produce more milk due to their higher levels of circulating GH was clearly supported by the results. Despite the low number of sheep that we were able to access and milk the transgenic sheep produced almost twice as much as the control ewes.

The lower concentration of protein, particularly casein in the milk may have resulted in inferior processing performance of the milk. Unfortunately the regulations prevented us from processing any of the milk and our Lactodynamograph was out of order at the time of the experiment so that we were unable to measure the clotting properties of the milk. Our result agrees with those of Fleet et al. (1986; 1988), Holcombe et al. (1988), Pell et al. (1989), Sandles et al. (1988), Stelwagen et al. (1993) who reported that the concentration protein in the milk was negatively affected in animals injected with bST.

During the last two measurements of milk production some of the control and transgenic ewes had low lactose concentrations in their milk, below 1%, suggesting that they had weaned their lambs as low lactose in milk is observed after weaning, when lactose is decomposed into glucose and galactose, which are reabsorbed by the mammary gland epithelium. As lactose is the only molecule in milk that is osmotically active (Saacke and Heald, 1974), this normally drives milk volume down and lactation terminates abruptly. Surprisingly, this did not happen with the GH sheep and during the following week their milk production increased (Fig 4.2.1). This finding suggests that GH may play an important role in the mechanisms underlying the end of lactation.

The decision to dispose of the transgenic sheep was dictated by animal welfare problems that the animals developed as they grew. The high circulating level of GH meant that the sheep were acromegalic and that their bones and hooves continued to grow in adult animals. Hooves had to be trimmed twice as frequently as those of the control sheep. Some animals appeared disproportionate and limped quite badly.

The recent changes in the legislation on genetically modified organisms also had a major role to play. CSIRO would have faced enormous bills to rebuild all of their fences to have their premises licensed for the conduct of transgenic work. Our original plan to produce approximately 50 transgenic East Friesian cross sheep and compare them with as many controls would have meant that under the new legislation we may have been obliged to destroy and incinerate not only the transgenic sheep, but also their dams and sires, as well as their offspring and any other sheep that may have come in contact with them.

Frozen semen from transgenic rams is currently held at the Veterinary Science School of Murdoch University. It may be possible one day to resurrect these amazing sheep and continue to study their lactation physiology. However, under the current legislation, public opinion and funding regime this may remain just a dream.

4.3.1 Changes in content of Conjugated Linoleic Acid (CLA) in milk during lactation in different breeds of dairy sheep

Over the last decade there has been rapidly mounting interest in the metabolism and biological activities of Conjugated Linoleic Acids (CLA), which are positional and geometric isomers of linoleic acid (18:2). There are elegant review articles on the general biological activity (Parodi,

1994; 1999; Pariza et al., 2001) and specific roles of CLAs in milk fat reduction (Bauman and Griinari, 2003; Griinari and Bauman, 2003). The most abundant isomer found in ruminant products is the cis-9, trans-11-octadecadienoic acid (18:2 c9, t11 CLA), which is also called rumenic acid because of its abundance in ruminant products (Parodi 1999). It is produced in a hydrogenation pathway that occurs in the rumen (Hughes et al., 1982). This is also the isomer of CLA shown to be potent suppressor of tumour growth in human cancer cells and chemically induced tumours in laboratory animals (Ip et al., 1991; Lavillonniere and Bougnoux, 1999; Pariza et al., 1999; Wahle and Heys, 2002).

Most studies investigating variation in CLA levels in meat and milk have focused on dietary sources of variation, as dietary fat is the supplier of preformed CLA or its precursors (Chouinard et al., 2001). Some studies have shown that genetic factors (breed and/or species) can affect the levels of CLA in animal products (Lawless et al., 1999; Knight et al., 2003; Kelsey et al., 2003). Lawless et al. (1999) and Kelsey et al. (2003) found significant differences in the CLA content of milk from different breeds of dairy cows. However, the observations were based respectively on two and one sampling points during lactation. The present experimental work was undertaken by a 4th year student, David Peake, who studied differences in the CLA content of different breeds of sheep milked over eight weeks of lactation to investigate CLA concentrations in different breeds of dairy sheep throughout lactation. Hence, the objectives of the study were to determine 1) if there were differences in the CLA concentration in the milk of different breeds of dairy sheep, and 2) how such differences, if any, changed as days in lactation progressed.

This work has been submitted to the Australian Journal of Experimental Agriculture (Kitessa et al., 2005).

4.3.2 Materials and methods

Five different breeds of sheep from our dairy sheep herd were kept under the same conditions to compare their lactation performance. These were: Awassi (A), Merino (M), A x M, East Friesian (EF) x A and EF x M (Table 4.2.1). The Awassi is a fat tail sheep that has been selected in the Middle East under very harsh conditions. The fat tail acts as a camel’s hump, allowing the animals to survive and produce during feed shortages by mobilising the fat reserves accumulated in the tail during periods of food abundance (Epstein, 1985). The Awassi x Merino crosses also have fat tails, while the other breeds have a thin tail, which is a dominant character for the East Friesian breed (Mills, 1989).

Table 4.2.1. Number of sheep and number of observations per breed during the three months of the experimental period.

Awassi Merino A x M EF x A EF x M

Number of sheep 17 16 8 19 17 Data points: July 30 28 18 40 42 August 38 29 25 57 47

Each month, feed samples (hay, chaff, lupins and pasture) were collected and analysed for fatty acid composition. The fatty acid profile of the feed ingredients used is shown in Table 4.2.2.

Milk production was measured weekly and samples of milk were collected and analysed for protein, fat and lactose. Subsamples of milk from each sheep were stored at -20°C before it was thawed and analysed for fatty acid composition.

Table 4.2.2. Fatty acid profiles of dietary ingredi ents (values i n g fatty acid/10 0g total fatty a cids).

Fatty acid Hay Lupins Chaff Pasture 8:0 0.56 nd 0.26 0.14 10:0 0.33 0.13 nd 0.13 12:0 1.48 0.20 0.63 0.35 13:0 0.31 0.20 0.39 0.51 14:0 2.34 0.39 2.46 0.73 14:1 0.49 0.14 0.26 0.28 15:0 0.95 0.14 0.49 0.39 16:0 31.89 11.81 28.95 17.11 16:1 0.89 0.14 0.63 0.47 17:0 0.81 0.15 0.47 0.46 18:0 5.71 6.03 2.41 2.12 18:1 t9 0.37 0.22 nd 0.14 18:1 c7/TVAδ nd nd nd 0.17 18:1 c9 10.88 32.03 9.76 2.52 18:2 29.84 42.62 35.65 15.72 18:3 10.25 5.23 16.61 58.17 20:0 2.91 0.79 1.16 0.85

The fatty acid composition of milk fat was determined by preparing methyl esters of fatty acids following base-catalysed transesterification (Christie, 1989). Fat in 5 mL of milk samples was extracted by adding 1 mL of ammonium hydroxide solution, 5 mL of ethanol and 12.5 mL of diethyl ether. After mixing, the top phase (lipid + solvent) was transferred into individually labelled test tubes, and the solvent was evapourated under nitrogen. Methylation was performed by adding 5 mL of freshly prepared sodium methoxide solution (Sodium methoxide/diethyl ether, 1:1 v/v) to the (dried) lipid sample. Methyl esters were extracted by adding 2.5 mL of diethyl ether to the solution and by removing an aliquot of the top phase into GC vials. Further details of our procedures are published elsewhere (Kitessa et al., 2003). For both milk and feed samples, individual fatty acids were determined by analysis of their fatty acid methyl esters (Christie, 1989) on a gas chromatograph (Perkin-Elmer AutoSystem, Sydney, Australia) fitted with a BPX70 capillary column (SGE Australia, Pty Ltd, Melbourne, Australia). Peak identification was performed using retention times of a mixture of standards of fatty acid methyl esters which included the two common isomers of CLA: 18:2 c9, t11 CLA and 18:2 t10, c12. Peak confirmation was done as in Kitessa et al. (2001a) by submitting selected samples for analysis by GC-MS (Gas Chromatography-Mass Spectrometry). We did not detect the CLA isomer 18:2 t10,c12 in any milk samples. All values reported here refer to the rumenic acid isomer (18:2 c9, t11 CLA). Δ9 - desaturase activity index was calculated as MUFA/(SFA+MUFA) (Corl et al., 2001). The fatty acids included were 14:0, 14:1 cis-9, 16:0, 16:1 cis-9, 18:0 and 18:1 cis-9.

A repeated measures analysis of variance was conducted on the variables milk yield, concentration of fat in milk, fat yield, CLA concentration and CLA yield. The variable days in lactation (DIL) was used to account for differences in lambing dates. The factors examined were time (DIL) and the breed of the animal together with the DIL by breed interaction. The repeated factor animal within breed was used.

4.3.3 Results

Over the eight-week study CLA concentration for the five breeds were 299±13.3, 312±21.2, 340±13.0, 342±17.2 and 382±23.5 mg per 100g milk fat for A, AM, EFA, EFM and M, respectively. The concentration of CLA in the milk fat declined significantly in the M, EFA and EFM breeds, but it declined very slowly in the milk of the A and AM ewes (P<0.05, Fig 4.3.1). In the latter, the decrease from beginning to end of lactation was not significant (P>0.05). The pattern of change in Δ9- desaturase activity index was similar among all breeds and it remained fairly constant throughout the duration of the study (Fig. 4.3.2).

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Fig. 4.3.1. Rate of decline of CLA concentration in the milk of five different breeds of sheep as predicted (least square means) by the statistical model throughout the experimental period. The rate of decline was significantly lower for the breeds with a fat tail (Awassi and Awassi x Merino).

9-desaturase activity index

Fig. 4.3.2. Change in Δ9-desaturate activity index calculated from milk from the whole sheep dairy flock comprising five different breeds.

The EFA ewes consistently produced more milk per day than the Merino ewes; the other breeds were intermediate (Fig. 4.3.3a). The overall average fat content of milk was 6.6 ± 0.11 g/100g. The Awassi ewes had consistently higher concentration of milk fat than the EFM on most sampling days; the other breeds were intermediate (Fig. 4.3.3.b). There was less divergence in milk fat concentration among breeds near the end of lactation (Fig. 4.3.3b).

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4.3.4 Discussion

In general, the concentration of CLA decreased as lactation progressed. However, its rate of decline with advancing lactation was different between breeds. The grouping of breeds into fat tails and thin tails in relation to the rate of decline in milk CLA concentration with advance in lactation has not been reported before. It may be due to the mobilisation of tail fat reserves in fat tail sheep. It is commonly accepted that stearic acid is a major constituent of fat reserves due to its high melting point (55°C for tristearine), as body fat is solid at body temperature. When body reserves are mobilised the mammary tissue Δ9- desaturase acts on the stearic acid to produce oleic acid. In fact, the presence of long chain unsaturated fatty acids in milk provides an indication of the nutritional status of lactating ewes, as these long chain unsaturated fatty acids are abundant in the milk of sheep that mobilise body reserves (Nudda et al., 2004). While the fatty acid composition of the fat in the tail is not known, Unsal et al. (1995) measured the melting point of the tail fat and found it to be around 36°C. This finding suggests that the fat deposits in the tail contain unsaturated fatty acids such as linoleic acid and TVA that may then be converted to milk CLA. However, it is difficult to reconcile this with the lack of difference between breeds in the computed Δ9- desaturase activity index. Whether or not such grouping exists, the observed breed difference in the rate of decline of CLA concentration in milk has important practical implications. Production of milk with consistent CLA levels that enable product specification (health claim) will require a dairy herd with minimal variation in their CLA output over the lactation period. Our results suggest that, at least in sheep, some breeds maintain the CLA concentration of their milk throughout their lactation better than others.

The results presented here indicate that the divergence in CLA concentration amongst breeds becomes minimal near the end of lactation. This raises two points of interest. First, the sampling point in a lactation period clearly affects the capacity to detect breed differences in the concentration of CLA in milk. This is perhaps why there are conflicting observations in the literature regarding the effect of breed on CLA content of milk. For instance, Lawless et al. (1999) reported significant effect of breed on CLA concentration of milk among Holstein 21

Friesian (Irish and Dutch), Montebeliardes and Normandes cows. On the contrary, Kelsey et al. (2003) who compared Holstein and Brown Swiss cows reported that the effect of breed was minor compared to individual variations within a breed. The former study had two sampling points while Kelsey et al’s (2003) data were based on a single point in the lactation curve.

Secondly, the selection of sheep breeds for CLA concentration of their milk provides us with a choice of using breeds with high CLA content (which declines relatively rapidly as lactation progresses) or breeds with modest CLA content that is fairly maintained through most of the lactation period. For instance, the Awassi sheep (fat-tailed) in this study did not have the highest CLA concentration in their milk, but they started low and maintained the CLA concentration as lactation progressed. Hence, an overall sheep breed strategy for high CLA milk needs to either capture the small window of opportunity near the beginning of lactation (Fig. 4.2.1), or select within breeds for animals with less steep decline in the CLA content of their milk. The idea of selecting animals within breeds is supported by Kelsey et al.’s (2003) observation that CLA content varied over threefold among individuals within a breed. Our data also support this claim.

The results in this study clearly bring into question the validity of previous conclusions on breed comparisons in CLA concentration of milk that were based on one or two sampling points in the lactation curve. However, although we introduced the DIL variable to account for differences in lambing dates, this may not be as good as having synchronised lambing dates. Furthermore, inferences from our results should take into account the fact that our results were based on data collected over two months near the end of lactation. Whole lactation data from these breeds may not agree with the trend suggested from our predictive lines. This said, the results provide a rationale for a whole lactation study of CLA concentration in milk from these breeds with synchronised lambing and larger numbers of ewes per breed. The levels of CLA in milk fat observed in this study were lower than those observed in most of the studies on dairy cows (Table 3). However, such across species and across experiment comparisons may not be valid due to the plethora of variables that would confound such comparisons.

Table 4.3.3. Reported conjugated linoleic acid levels in milk from different species of ruminants.

Reference Basal/supplement Animals Number Range of CLA, of breeds g/100g milk fat Lawless et al., 1999 Pasture Cows 4 1.47 - 1.86 Lock and Garnsworthy, 2003 TMR, Pasture Cows 1 0.8 - 1.9 Abu-Ghazaleh et al., 2001 TMR/(SBM or FM) Cows 1 0.39 – 0.72 Gulati et al., 2000 (Hay-grain)/CLA Goats 1 2.4 - 2.6 Offer et al., 1999 Silage-concentrate/oilsA Cows 1 0.16 – 1.55 Kelly et al., 1998 Silage-concentrate/ oilsB Cows 1 1.33 - 2.44 This study (Hay-grains), pasture Sheep 5 0.247- 0.989 A Linseed, Fish or Tuna oil. B Peanut, sunflower or linseed.

Our study clearly shows that sheep milk provides the ideal strategy for providing CLA- enriched milk that has high CLA concentration per serve because: 1) it has higher total fat content than cow’s or goat’s milk, and 2) the CLA content of its milk is equal, if not superior, to cow’s or goat’s milk (Banni and Martin, 1998).

4.4.1 Sunflower oil increases the level of Conjugated Linoleic Acid in the milk of different breeds of sheep

The recent interest in Conjugated Linoleic Acid (CLA) prompted this work, which aimed at increasing the concentration of CLA in the milk of our dairy sheep by feeding them sunflower oil supplements.

For humans, the principal dietary source of CLA comes from ruminant products (Chin et al., 1992). Production of CLA in ruminants is possible because of the microbial hydrogenation of the unsaturated fatty acids, linoleic and linolenic acid within the rumen (Harfoot and Hazlewood, 1997). Biohydrogenation of linoleic acid is a multi-stepped process that ultimately forms stearic acid (C18:0) (Hudson et al., 2000). Along the pathway two intermediates are formed that are of major importance to the production of CLA. The first is a conjugated diene of linoleic acid called cis- 9, trans- 11 CLA and the second is trans- 11 monoenoic acid or Trans Vaccenic Acid (TVA). These intermediate isomers enable CLA to be adsorbed and incorporated into the milk and tissue (Harfoot and Hazlewood, 1997). The major source of milk and tissue CLA is from endogenous (within tissue) synthesis that results from the activity of the enzyme Δ9 - desaturase (Kay et al., 2002). Endogenous synthesis of CLA by Δ9 - desaturase is estimated to supply between 64 and 100% of the CLA in milk fat (Kay et al., 2002; Griinari et al., 2000).

Among domestic ruminants, sheep’s milk has the highest concentration of fat and is the richest source of CLA (Banni and Martin, 1998). Sheep naturally produce about 27 mg of CLA per gram of milk fat whereas cows milk contains only about 7 mg/g of milk fat (Banni and Martin, 1998). However, the potential of dairy sheep to produce CLA is yet to be exploited. Kelly et al. (1998) suggested that it is important to increase the CLA content of milk fat rather than the absolute amount secreted in milk. Since the milk of sheep is high in fat, it would make an ideal “carrier” to deliver the recommended dose of CLA. Therefore there is a need to focus research on dairy sheep as potential suppliers of CLA. This project was undertaken by a 4th year student, Peter Hutton, to determine the potential for dairy sheep to produce CLA in their milk at concentrations that are high enough to provide health benefits to humans (Hutton, 2003). It was hypothesised that CLA could be increased in sheep milk by manipulating the biohydrogenation process in the rumen through the diet by feeding a sunflower oil supplement to dairy sheep. The increase in unsaturated fatty acids that have a lower melting point that those normally found in the milk fat could potentially affect the processing performance of the milk and may result in curds of lower consistency. Therefore we also investigated if the sunflower oil supplement would affect the clotting properties of the milk.

4.4.2 Material and methods

Six Merino and six Awassi ewes in mid-lactation were selected for the experiment. The selected sheep were moved to a feedlot area where they were provided with fresh water, shelter and an ad libitum supply of meadow hay for roughage. The sheep were introduced to a Qlamb (Macco Feeds) pelleted diet comprised of cereal hay and straw, cereal grains, lupins, canola meal, macro and trace minerals and vitamins. The pellets contained crude protein of 15.9% and 1.1% lipid of dry matter and had an energy value of approximately 9.9 MJ/ kg dry matter.

The sheep were randomly assigned to a treatment group in a Latin square experimental design. Each sheep was fed one of three treatments for 15 days followed by a rest period of six days before entering the next period. All sheep received three treatments over three periods of 15 days. The three treatments were comprised of Qlamb pellets with no sunflower oil, Qlamb 23

pellets with 2.5% sunflower oil of the total pellet weight and Qlamb pellets with 5% sunflower oil of the total pellet weight. Five percent sunflower was selected as the maximum dose because of the associated risk of milk fat depression at levels higher than this (Szumacher- Strabel et al., 2001).

Pellet refusals were weighed and subtracted from the weight of pellets offered to calculate daily intake. The relatively high levels of fat increased the risk of the breakdown of fermentation in the rumen. An acclimatization period for the introduction of sunflower oil to the diet was assigned to each treatment period to allow the microbial populations in the rumen time to adjust to the new diet. During acclimatisation the following protocol for the introduction of sunflower oil was used in each period:

• Day 1-3: 1/3 of the total oil required for the treatment • Day 4-8: 2/3 of the total oil required for the treatment • Day 9-15:The full requirement of oil for the treatment

The oil was poured over the pellets using a 60 mL plastic syringe and mixed well. The pellets were left to soak up the oil for approximately 20 hours prior to feeding. The composition of the sunflower oil is shown in Table 4.4.1.

Table 4.4.1 -. Composition of sunflower oil (from Budavari et al., 1996) Component per 100ml Energy 3400 KJ Protein 0 g Fat total 92 g - saturated 10.32g - trans 0.76g - polyunsaturated (linoleic acid) 61.0g - monounsaturated (oleic acid) 19.6g Carbohydrate 0 g - sugars 0 g Sodium 0 mg

Vitamin E was included in the sunflower oil diets at the rate of approximately 150g/ Kg of pellets as an antioxidant and to compensate for the extra lipid consumed (Kitessa et al., 2001a).

The sheep were milked twice daily (at 6:30 am and 3:30 pm) and milk was sampled for analysis on day 0, 8 and 15 of each treatment period. Milk samples were analysed for fat, protein and lactose and fatty acids were converted to methyl esters and extracted from the milk samples using the methoxide methodology modified from Bannon et al. (1982). Methyl esters were separated by gas chromatography using a Perkin Elmer autosystem equipped with an autosampler, split injector, flame ionization detector and a capillary column (BPX70-0.25 μM x 120 mm x 0.32 mm ID, SGE Australia PTY. Ltd.).

The renneting time, rate of firming and curd consistency of the milk samples were also measured with a lactodynamograph (Bencini, 2002).

Means from the Latin square design were tested for differences on Genstat version 6.1 using the linear mixed model analysis. This analysis used a Wald statistic to produce a chi-squared

probability. The factors used in the fixed model were ‘Period’ and ‘Breed by Treatment’. The random effects were ‘Sheep within Breed’. Fisher’s least significant difference tests were used to comp are treatment means.

4.4.2 Results

There was a significant increase in cis-9, trans-11CLA in the milk fat of the Merino and Awassi ewes that were fed 2.5 and 5% sunflower oil compared with the control (p<0.05, Table 4.4.2).

Table 4.4.2 - Mean concentration (mg/g of fat ± S.E) of cis-9, trans-11CLA in the milk fat of Merino and Awassi sheep that were fed three levels of sunflower oil over three periods. Milk was sampled for analysis on day 15 of each treatment period. Different letters indicate a significant difference (p<0.05) between means.

Sunflower oil 0 2.5 5 (% of pellet) Merino 10.6 (1.89) a 20.1 (5.76) b 15.5 (4.51) b Awassi 9.4 (2.77) a 19.7 (4.47) b 23.9 (7.25) b

There was no difference between the breeds in the concentrations of cis-9, trans-11CLA at each of the three treatment levels. Therefore there was no interaction between breed and treatment (P>0.05).

There was an increase in the concentration of TVA in the milk fat of the Merino sheep at the 5% treatment level when compared with the 0 and 2.5% levels of sunflower oil (p<0.05), but there was no difference in TVA concentration between the 0 and 2.5% treatments (p>0.05). The concentration of TVA increased in milk of the Awassi sheep at the 2.5 and 5% treatment levels compared with the 0% treatment (p<0.05) but the concentration of TVA was the same in the milk of the Awassi sheep at 2.5 and 5% treatments of sunflower oil (Table 4.4.3). There was no interaction in TVA concentration between breeds and treatments (P>0.05).

Table 4.4.3 - Mean concentration (mg/g of fat ± S.E) of TVA in the milk fat of Merino and Awassi sheep that were fed three levels of sunflower oil over three periods. Different letters indicate a significant difference (p<0.05) between means.

Sunflower oil 0 2.5 5 (% of pellet) Merino 42.8 (6.56) a 53.1 (15.10) a b 60.6 (11.20) b Awassi 40.4 (21.12) a 77.4 (16.15) b 75.3 (18.24) b

There was a linear relationship between the concentration of TVA and cis-9, trans-11 CLA in the milk fat of the Merino sheep (R2 = 0.83, Figure 4.4.1).

cis-9, trans-11 CLA mg/g of fat 35 y = 0.2809x - 2.0797 30 R2 = 0.8313 25

0 0 20406080100120 TVA mg/g of fat

Figure 4.4.1. Relationship between TVA and cis-9, trans-11 CLA (g/100 g of total fatty acids) in the milk fat of Merino sheep that were fed three levels of sunflower oil over three periods.

There was also a linear relationship between the concentration of TVA and cis-9, trans-11 CLA in the milk fat of the Awassi sheep (R2 = 0.94, Figure 4.4.2). The relationship was described by a slope of 0.31 between TVA and cis-9, trans-11 CLA.

cis-9, trans-11 CLA (mg/g of fat)40 y = 0.31x - 2.84 2 35 R = 0.94 30 25 20 15 10 5 0 0 20 40 60 80 100 120 140 TVA (mg/g of fat)

Figure 4.4.2. Relationship between TVA and cis-9, trans-11 CLA (g/100 g of total fatty acids) in the milk fat of Awassi sheep that were fed three levels of sunflower oil over three periods.

The renneting time of milk from the Awassi breed was shorter than from the Merino breed at the 5% level of sunflower oil (p<0.05). Renneting time decreased in the Awassi milk at the 5% level of treatment compared with the 0% treatment (p<0.05). Renneting time was not affected by the level of sunflower oil in the Merino sheep (p>0.05, Table 4.4.4).

Table 4.4.4. Average renneting time (minutes ± S.E) of milk from Merino and Awassi sheep that were fed three levels of sunflower oil over three periods. Different letters indicate a significant difference (p<0.05) between means.

Sunflower oil 0 2.5 5 (% of pellet) Merino 17:00 (1.77) a 14:06 (0.49) a 20:38 (3.97) a Awassi 19:21 (4.85) a b 20:10 (8.38) a, b 11:34 (2.99) b

The rate of firming decreased, but not significantly, in the milk from the Awassi breed at the 5% level of sunflower oil and there was no difference in the rate of firming between treatments in the milk from the merino sheep (p>0.05, Table 4.4.5).

Table 4.4.5. Average rate of firming (minutes ± S.E) of milk from Merino and Awassi sheep that were fed three levels of sunflower oil over three periods. Different letters indicate a significant difference (p<0.05) between means.

Sunflower oil 0 2.5 5 (% of pellet) Merino 1:23 (0.10) a 1:30 (0.90) a 1:38 (0.80) a Awassi 1:30 (0.01) a 1:25 (0.14) a 1:23 (0.99) a

The consistency of curd in the milk from the Merino or Awassi sheep was not affected by the level of treatment of sunflower oil (p>0.05, Table 4.4.6).

Table 4.4.6. Average curd consistency (mm ± S.E) of milk from Merino and Awassi sheep that were fed three levels of sunflower oil over three periods. Different letters indicate a significant difference (p<0.05) between means.

Sunflower oil 0 2.5 5 (% of pellet) Merino 64.70 (3.94) a 55.24 (0.82) a 53.53 (4.27) a Awassi 61.12 (3.64) a 53.98 (4.09) a 58.59 (7.26) a

Milk yield was not affected by the level of sunflower oil in the Merino or Awassi sheep (P>0.05). The Awassi ewes produced a higher milk yield on each sampling day and therefore over the total experimental period than the Merino ewes (p<0.05). The concentration of fat was higher in the milk from the Awassi breed compared with the Merino breed (p<0.05), but the concentration of milk fat within each breed did not change with the different levels of sunflower oil (P>0.05). There was no difference in the concentration of milk protein between the Awassi and Merino sheep over the total period of the experiment and the concentration of milk protein was not affected by the level of sunflower oil in either breed of sheep (P>0.1, Table 4.4.7).

Table 4.4.7. Mean yield (g/day ± S.E) and concentration of fat and protein (% ± S.E) in the milk from Merino and Awassi sheep. Values are averages for three levels of sunflower oil fed over three periods. Different letters indicate a significant difference (p<0.05) between means.

Breed Milk yield (g/day) Fat (%) Protein (%) Merino 363 (48) a 6.20 (0.14) a 6.18 (0.11) a Awassi 793 (56) b 7.09 (0.15) b 6.23 (0.06) a

4.4.3 Discussion

The addition of sunflower oil to the diet of lactating sheep increased the concentration of cis-9, trans-11 CLA and TVA in the milk fat of both Merino and Awassi sheep. Therefore, the hypothesis that linoleic acid in the diet increases CLA and TVA in the milk fat of sheep was supported. Biohydrogenation in the rumen is the most likely explanation for the increase in TVA and CLA. An isomerisation reaction by specific rumen bacteria converts linoleic acid to an intermediate form of CLA. Any CLA that escapes hydrogenation passes into the small intestine where it is adsorbed and circulated to the mammary tissue where it can be incorporated into the milk fat. However, most of the CLA is hydrogenated to TVA. TVA can then be hydrogenated to stearic acid by bacteria such as Fusocillus spp. or pass into the small intestine and circulate to the mammary tissue where it is desaturated by Δ9 - desaturase. High levels of linoleic acid have been shown to inhibit bacteria that the hydrogenate TVA. When these bacteria are inhibited TVA accumulates in the rumen and increased levels pass into the small and along the Δ9 - desaturase pathway.

Most of the CLA that was measured in the milk fat had very likely been produced along this second pathway of endogenous synthesis by Δ9 - desaturase pathway. However, the levels of cis-9, trans-11 CLA that were detected in the milk fat were not as high as expected. In addition, cis-9, trans-11 CLA did not increase between the 2.5 and 5% treatments of sunflower oil. Peake (2002) found that Merino and Awassi sheep that were fed pasture produced concentrations of cis-9, trans-11 CLA in the milk fat that were higher than the levels measured in this experiment. Linolenic acid is the major fatty acid in pasture and has been demonstrated to produce lower concentrations of cis-9, trans-11 CLA than diets that are high in linoleic acid (Kay et al., 2002). However, in this experiment production of cis-9, trans-11 CLA in the fat of both breeds of sheep was lower than the levels observed by Peake (2002) despite a higher intake of linoleic acid.

The stage of lactation and the length of the treatment period may have caused the lower than expected concentrations of cis-9, trans-11 CLA. The sheep used in this experiment were in late lactation and this is the most likely explanation for the relatively low concentrations of cis- 9, trans-11 CLA in their milk fat. In late lactation and the activity of Δ9 - desaturase can be low in the mammary tissue relative to adipose tissue (Song and Kennelly, 2002) and the lower production of CLA in the mammary tissue reduces the quantity that is incorporated into the milk fat (Griinari and Bauman, 1999).

The levels of stearic acid in the milk fat of sheep in this experiment remained constant, which supports the idea that the activity of Δ9 - desaturase is low in late lactation. If Δ9 -desaturase is active in the mammary tissue then stearic acid is desaturated to oleic acid (St John et al., 1991) and the concentration of stearic acid in the milk fat should decrease.

Another possible explanation for lower CLA and TVA concentrations than expected could be the length of the treatment period. The cis-9, trans-11 CLA in the milk fat of the sheep in this experiment may not have reached the maximum concentration because the treatment periods may have not been not long enough for the biohydrogenation and desaturation processes to be effective. Kelly et al. (1998) gradually acclimatised cows to diets of sunflower oil over 14 days and then fed this diet for another 14 days. The gradual increase over a longer period resulted in higher levels CLA in the milk fat than were observed in this experiment. This may explain why the 5% treatment of sunflower oil did not increase the production of cis-9, trans- 11 CLA above the level that was produced at the 2.5% treatment. The plateau in CLA production at the 5% level of treatment may have been due the acclimatisation period being too Anotheshort. r unexpected result was that the two breeds of sheep did not demonstrate differences in their ability to transform varying concentrations of linoleic acid into CLA. However, the 28

Merino sheep produced lower concentrations of CLA at the 5% treatment level than the 2.5% level. The Merino breed is known to have shorter lactations than the Awassi breed. Therefore the Merino sheep were in a later stage of lactation than the Awassi sheep. If Δ9 - desaturase activity is lower towards the end of lactation this may explain why the Merino sheep tapered off in the production of CLA more than the Awassi breed. This finding may be also related to the slower rate of decline in CLA concentration observed in the Awassi breed in the previous experiment (Sections 4.3.1 to 4.3.4).

The ratio of TVA:CLA in the milk fat was linear across all treatments which was expected and supports the conclusions of Knight et al. (2003) and Griinari and Bauman (1999) that TVA is an important precursor to desaturation in the mammary tissue by Δ9 - desaturase. The linear relationship in the TVA to CLA ratio across all treatment levels indicated that Δ9 - desaturase was active in converting TVA to CLA in the mammary tissue. The ratio was similar between breeds similarly to the report by St John et al. (1991) that the activity of Δ9 - desaturase is similar between different breeds of cattle.

Although there was no difference in the production of cis-9, trans-11 CLA between the breeds of sheep a wide range of concentrations was observed between sheep within treatment. Lawless et al. (1999) also found that there was large variation in the production of CLA between dairy cows within each breed and this indicates that factors other than breed influence the CLA content of milk.

The expectation that high concentrations of unsaturated fatty acids would worsen the processing properties of milk was inconclusive as the renneting time, rate of firming and the consistency of curd did not change in the milk samples from the Merino or Awassi sheep at any of the levels of sunflower oil tested. However, in light of the lower than expected concentrations of CLA and TVA in the milk fat the results for the processing properties were expected. The milk samples may not have contained concentrations of unsaturated fats that were high enough to affect the clotting properties.

Dairy sheep are naturally high producers of milk fat and also CLA within the milk fat. This experiment has demonstrated that the microbial environment of the rumen can be manipulated easily by dietary supplementation to increase the level of CLA in the milk fat. This opens the possibility of commercializing sheep milk and sheep dairy products with health enhanced properties.

4.5.1 Transfer of n-3 polyunsaturated fatty acids from tuna oil into sheep’s milk

There are various claims for the health benefits of consuming the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), also known as omega-3 fatty acids. The main claims include reduced risk of cardiovascular disease (Sheard, 1998), enhanced development of brain and visual acuity in infants (Hoffman et al., 1993), modulation of autoimmunity (Calder, 1998; Grimble, 1998), and modulation of inflammatory disorders (Kinsella, 1986, 1987; Simopoulos, 1991; Calder, 1998). The cardio-protective role of EPA and DHA is of a major significance, as cardiovascular disorder is one of the most important causes of death in the developed nations. Based on increasing epidemiological and clinical evidence about the protective role of omega-3 fatty acids against cardiovascular diseases, the 34th Annual Scientific Meeting of European Society of Clinical Investigation forwarded seven conclusions about omega-3 fatty acids and cardiovascular health. The first was that consumption of two fish meals per week is associated with reduced mortality from coronary

heart disease (Nordøy et al., 2001). However, it has been reported that daily consumption of omega-3 fatty acids in most developed nations is less than the recommended daily intake (650 mg; Simopoulos et al., 1991). Part of the problem is likely to be that omega-3 fatty acids are not abundant in food items that are traditionally consumed daily in developed nations, i.e. meat and milk from ruminants.

Various avenues have been pursued to increase the levels of omega-3 fatty acids in the western diet. For instance, many forms of vegetable oils and dairy spreads of high polyunsaturated fatty acid content, not necessarily of high omega-3 fatty acids content, have entered the food market in several countries. Previous studies have focused on production of n-3 PUFA- enriched milk (Kitessa et al., 2001a) and meat (Kitessa et al., 2001b) from ruminants through supplementation of protected tuna oil to lactating or growing ruminants. The oil is protected from degradation in the rumen by the rumen microorganisms by treating it with formaldehyde, which results in the encapsulation of the oil in a protein-aldehyde matrix (Scott et al., 1971). This approach aims to value-add to milk while retaining its other nutritional and health benefits. This approach also provides an option for natural incorporation of omega-3 fatty acids into dairy products as opposed to post-harvest chemical manipulations that are perceived to be less favoured by consumers.

Results from our earlier study (Kitessa et al., 2001a) provided apparent transfer of dietary omega-3 fatty acids into goat’s milk that were markedly different from that reported for dairy cows (Cant et al., 1997). This raised the question whether different ruminant species differ in their ability to transfer dietary EPA and DHA into milk. In this study we investigated the transfer of omega-3 fatty acids from protected tuna oil into sheep’s milk, as well as the effects of tuna oil supplementation on the yield and composition of milk in dairy sheep. The project was undertaken by 3rd year student David Peake, with the support of a CSIRO summer Scholarship. The work was subsequently published in Animal Feed Science and Technology (Kitessa et al., 2003).

4.5.2 Material and methods

Sixteen lactating sheep in their final month of lactation were selected from our dairy flock. These were divided into two groups of control and protected tuna oil-supplemented group. The two groups were balanced for breed and parity. The control group was kept in a paddock with the rest of the dairy herd, and the treatment group was kept in an adjacent paddock. Both paddocks had negligible pasture cover, as it was mid-summer. Both groups had ad libitum access to meadow hay in each paddock. Both groups were offered (in a communal trough) lupin grains after morning and afternoon milking (500 g/hd/day). At each milking, the control ewes were offered 50-100 g of a mixture of oaten chaff (0.70) and lupins (0.30) for contentment during milking. The treatment ewes were offered individually a protected tuna oil-supplemented chaff mixture during milking. This was made up of soybean-tuna oil (70/30, w/w). The soybean-tuna oil mixture was emulsified and treated with formaldehyde, which resulted in encapsulation of the oil in a protein-aldehyde matrix (Scott et al., 1971). The protection level in vitro (incubation in rumen fluid) was 82 %. The supplement had 304 kg/kg crude protein and 374 g/kg total fat. The supplement was coated with molasses by manual mixing (80/20, supplement/molasses, w/w). Table 4.5.1 summarises the total fat and fatty acid profile of the protected tuna oil supplement and other feed ingredients offered to the sheep. At the morning milking, each sheep was offered 150 g of molasses-coated supplement mixed in a bucket with 160 g of stem-cut oaten chaff (total DM = 310 g). After milking the residue from each sheep was stored separately and offered to the same sheep during the afternoon milking. The residue left after the afternoon milking by each sheep was weighed and recorded.

Table 4.5.1. Fatty acid composition of the protected tuna oil (PTO) supplement and feed components offered to dairy sheep during the experiment.

Variable PTO Lupins Hay Chaff Supplement Total fat (g/kg total DMa, w/w) 374 64 15 15 Fatty acids (g/kg total fat w/w): 14:0 20 24 20 28 16:0 175 118 313 294 16:1 28 ndb 27 8 18:0 52 56 55 22 18:1cis 177 316 154 87 18:2n-6 203 430 212 295 18:3n-3 45 52 100 206 20:5 n-3 31 nd nd nd 22:6 n-3 140 nd nd nd a DM, Dry matter; bnd, not detected

The sheep were milked twice a day and milk production was measured and milk samples were collected each day during the 10-day feeding period. In addition, production measurements and sample collections were done one day before feeding began, as well as 1, 4 and 6 days after the feeding of tuna oil supplement ceased. Table 4.5.2 summarises intake values for protected tuna oil supplement, EPA and DHA.

Table 4.5.2. Total daily supplement intake (70/30, soybean/tuna oil) and calculated intakes of EPAa and DHAb by sheep offered protected tuna oil (PTO) supplement mixed with chaff.

Protected tuna oil EPA DHA Days from start g s.e.m. g s.e.m. g s.e.m. 1 32 10 0.37 0.12 1.69 0.53 2 102 5.1 1.16 0.06 5.31 0.26 3 111 6.4 1.27 0.07 5.78 0.33 4 109 5.5 1.25 0.06 5.69 0.29 5 116 0.7 1.33 0.01 6.08 0.04 6 102 5.9 1.17 0.07 5.34 0.31 7 109 7.1 1.25 0.08 5.70 0.37 8 110 7.0 1.25 0.08 5.72 0.37 9 109 5.3 1.25 0.06 5.70 0.27 10 114 5.5 1.31 0.06 5.97 0.29 a) EPA, Eicosa pentaenoic acid; b) DHA, Docosahex aenoic acid

All milk samples were stored at 1-4oC and analysed for fat, protein and lactose. Total fat in feed samples was determined by Soxhlet extraction (AOAC, 1990, Method 963.15) using petroleum ether for lupin, hay and chaff, and chloroform-methanol (2:1, v/v) for the protected supplement. For both milk and feed samples, individual fatty acids were determined by analysis of their fatty acid methylesters (Christie, 1989) on a gas chromatograph (Perkin-Elmer AutoSystem, Sydney, Australia) fitted with a BPX70 capillary column (SGE Australia, Pty Ltd).

Differences in milk yield (g/d) and composition were evaluated from data gathered during pre- feeding, feeding and post-feeding periods. The fatty acid composition of fat in milk samples collected during each of these periods was also compared between treatment groups. The transfer of dietary omega-3 fatty acids into milk for the treatment group was determined on milk samples obtained on the last day of the feeding period (day 10). It was computed by 31

assuming dietary tuna oil supplement was the sole source of EPA and DHA in milkfat, although sporadically trace levels of EPA and DHA were found in milk from one or two animals in the control group. Differences between treatments for all variables were determined by using Student’s T-test (P<0.05).

4.5.3 Results

There were no significant differences between treatment groups in milk yield, or concentrations of fat, protein and lactose before or after supplementation period (Table 4.5.3). The tuna oil supplementation did not have a significant effect on milk yield or its gross composition (fat, protein and lactose) over the 10-day feeding period (Table 4.5.3).

Table 4.5.3. Yield and composition of milk from sheep fed control or control plus protected tuna oil (PTO) supplement (average of whole period, 10 days).

Treatme nt Variablea Control PTO s.e.m. Milk yield, g/d 722 716 32.3 Fat, g/kg 74 77 1.0 Protein, g/kg 54 56 0.8 Lactose, g/kg 52 52 2.1

The composition of the milk fat, however, was significantly affected by the tuna oil supplement. The tuna oil supplementation nearly doubled the total n-6 (P<0.001) and quadrupled the total n-3 (P<0.001) unsaturated and polyunsaturated fatty acids in the milk (Table 4.5.4). The ratio of total n-6 to n-3 unsaturated plus polyunsaturated fatty acids was significantly lower in milk from the treatment group. Tuna oil supplementation did not have a significant effect on fatty acids with <18 carbon atoms (Table 5). There was a significant reduction in the percentage of stearic acid (18:0, P<0.05) and oleic acid (18:1 cis, P<0.001) in the milk from the treatment group as compared to the control group. The group supplemented with tuna oil also had higher (P<0.001) concentrations of linoleic (18:2), linolenic (18:3), EPA and DHA than the control group (Table 4.5.4).

EPA and DHA appeared in milk in significant quantities 24 hours after feeding begun. There was a rapid linear increase in the percentage of both PUFA in milk fat up to day 7, after which it tended to plateau (Fig.4.5.1).

Table 4.5.4. Fatty acid profile of milk (g/kg milk fat) from sheep fed either control or control plus protected tuna oil (PTO) supplements. Data represent mean values for the last day of treatment (day10).

Treatment Fatty acid Control PTO S.E.M. 6:0 22 22 1.0 8:0 18 23 1.6 10:0 51 67 23 12:0 32 38 7.5 14:0 12 13 2.5 14:1 4.1 4.0 0.3 16:0 302 296 56.9 16:1 13 13 0.5 18:0 12 8.6 4.05* 18:1 cis 250 190 4.6*** 18:1 trans 4.0 4.6 0.1 18:2 n-6 29 56 0.8*** 18:3 n-3 8.0 13 0.4*** 20:4 n-6 3.8 4.9 0.3 20:5 n-3 0.4 4.7 0.2*** 22:6 n-3 0.4 19 0.4*** Total SFA 68 66 68.6 Total UFA 316 324 59.2 SFA:UFA 2.2 2.1 0.07 Total n-6 34 63 1.4*** Total n-3 8.7 36.7 1.1*** n-6: n-3 4.3 1.7 0.16***

* within row means were significantly different at P<0.05; ***within row means were significantly different at P<0.001. SFA, saturated fatty acids; UFA, unsaturated fatty acids.

Milk samples collected 6 days after the feeding of the protected tuna oil supplement stopped were still significantly enriched (EPA: 2.3±0.9 g/kg; DHA: 6.5±0.9 g/kg), both fatty acids were nearly as high as 50 % of the levels observed on the final day of feeding. In contrast, the levels of both linoleic and linolenic acids returned to pre-feeding period levels on the 4th day after feeding stopped (Fig. 4.5.1).

25 70

EPA DHA 60 18:02 20 18:03

15 40

30 10 18:2 and 18:3 in fat, g/kg

0 0 02345678910146 Days from start of feeding

Figure 4.5.1. Concentrations of selected fatty acids in milk from the tuna oil-supplemented group during and six days post the supplement-feeding period. Bars represent standard errors of the means.

4.5.4 Discussion

The use of the tuna oil supplement enabled the enrichment of milk with omega-3 fatty acids without the deleterious effects reported by other authors, such as reduction in protein content (Cant et al., 1997; Rotunno et al., 1998) and milk yield (Palmquist et al., 1977; Cant et al., 1997). It is unclear why the supplemental fat did not result in increased milk yield from the supplemented group. There may have been some substitution of hay consumption, as animals tend to eat less of low quality feed when given access to high quality feed (Grainger and Mathews, 1989).

Generally, about half of stearic acid (16:0) and all of the fatty acids with ≥18 carbon atoms in ruminant milk are of dietary origin. All those with ≤14 carbon atoms are synthesised de novo (Moore and Christie, 1979).

The reduced concentration of stearic acid (18:0) levels in the milk fat was probably due to differential effects of the supplements on hydrogenation of mono-, di- and polyunsaturated 18- carbon fatty acids. In all our previous works, tuna oil supplementation consistently reduced the level of 18:0 in ruminant products, with a consequent increase in 18:1, 18:2 and 18:3 (Kitessa et al., 2001a,b).

EPA and DHA were still present in significant concentrations in milk samples collected six days after supplementation stopped. Cant et al. (1997) who also observed a similar result suggested that this was due to storage of EPA and DHA in adipose tissue during the feeding period and release into milk during subsequent weeks by mobilising the adipose tissue. The question is why would EPA and DHA accrue in adipose tissue instead of further increasing the EPA and DHA levels during the feeding period? It is possible that there is an upper limit beyond which the proportion of EPA and DHA in milk fat will not rise. Due to a limitation in 34 the transfer from blood into the mammary gland (lipoprotein lipase activity) and/or incorporation into milk triacylglycerols (acylation of glycerol; 0.98 of milk lipids are in TG form) in the mammary gland.

The transfer of EPA and DHA were markedly higher than the 0.04 to 0.08 observed on dairy goats (Kitessa et al., 2001a). Cant et al. (1997) suggested that diet transfer calculations were confounded by the residual transfer of EPA and DHA from adipose tissue to milk. While this point cannot be discounted, its magnitude should be much lower for sheep in their final weeks of lactation. We propose that, once supplement consumption reaches a steady state, apparent transfer calculations based on a selected day during the steady state may provide comparable efficiency of transfer, as the residual EPA and DHA from the preceding day would most likely be of similar magnitude to that carried over to subsequent days from that absorbed on that day.

The difference in transfer of EPA and DHA between dairy sheep (this study) and dairy goats (Kitessa et al., 2001a) could partly be due to differences in the point of sampling in relation to the point where the transfer of EPA and DHA into adipose tissue and milk was at a steady state. In the dairy goat study, supplementation lasted only seven days, well short of the 10 days used in this study. Further research, using animals at the same stage of lactation, on the same basal ration, the same feeding periods, and the same sampling points, is needed to determine differences between species in the apparent transfer of EPA and DHA. The initial weeks of lactation where adipose tissue is mobilised to support milk yield may not be an ideal time to investigate this issue.

When computed from values on Day 10 of the feeding period, a cup (250 ml) of this omega-3 enriched milk will provide 437mg of EPA plus DHA, which is about 67 % of the recommended daily intake for healthy adults (Simopoulos et al., 1991). This makes sheep milk a much better option than goat’s or cow’s milk which only provide about a quarter of the recommended daily intake in one cup (Kitessa et al., 2001a). This was due to a combination of the presence of more fat in sheep milk (40 g/kg in goat’s milk versus 77 g/kg in sheep’s milk) as well as the greater level of EPA plus DHA (15 versus 24 g/kg total fat) observed in this study.

4.6.1 Product development

To address the second problem faced by the sheep milking industry we used the milk produced by the experimental ewes to develop a blue cheese.

4.6.2 Development of a blue (mould-ripened) sheep milk cheese

We developed a new mould-ripened blue cheese. Roquefort is one of the most famous sheep milk cheeses and is ripened in caves in the Roquefort area using the blue mould Penicillium roquefortii. Roquefort is protected by the DOC* trademark and although imported into Australia it has a very small market. This could be partially due to its high price of up to $90/kg, or to its strong taste.

* DOC means Denomination d’Origine Controlée, or “controlled denomination of origin”. Many European cheeses and wines are covered by this trademark, which involves that a particular product can only be made in a certain region or even area. The wine Brunello di Montalcino, for instance, has to be made on the hill of Montalcino. A few meters out and even if you crop the same grapevines and use the same technology your wine is worth $15/bottle instead of 50! The most famous case is that of Champagne. Roquefort, a blue cheese made with sheep milk is also DOC, and so are many Italian Pecorino varieties as well as Parmigiano (Parmesan) Reggiano cheese. 35

Roquefort is matured for 5 to 12 months, which gives the blue mould plenty of time to develop both inside (thanks to the piercing of the rounds aimed at allowing circulation of air, and hence development of the aerobic mould) and outside the cheese. This results in a piquant flavour because the mould has proteolitic and lipolitic activities that liberate strong tasting small chain fatty acids and small peptides (Resmini.

As long maturation cheeses such as those imported for the local ethnic communities have high storage costs and risk of spoilage during maturation, the industry has identified the need for short maturation products that can provide the cash flow for the enterprise.

The need to target the local market with a fresh cheese of relatively short maturation and long shelf life prompted this developmental work. Our initial aim was to produce a blue vein cheese by piercing the rounds similarly to what is done with the Roquefort, Stilton, Gorgonzola and other blue cheeses. However, the sheep milk curd proved to be too soft and piercing resulted in small channels that closed over very rapidly, preventing the formation of the desired blue veins. Initially we perceived this as a problem. We were giving away our failed cheeses and soon realised that people liked them. The final decision to not worry about how many blue veins developed inside the cheese came when potential American customers enthusiastically asked what was this wonderful cheese called and Svjetlana Mijatovic, the project’s technician, replied that it was called ”one-vein blue” because in the particular round that they were tasting there was only one solitary blue vein.

4.6.3 Materials and methods

To make this cheese the milk is first pasteurised at 72°C for 20 seconds, then cooled to 40- 42°C, filtered and transferred to the cheese vat at a temperature of 35-37°C. Mesophilic lactic starter cultures (CHR Hansen) are added (0.1g/l of milk) together with 0.1g of blue mould spore powder (Penicillium roquefortii sp). Both cultures are mixed thoroughly and left for 15- 30 minutes. Calf rennet (CHR Hansen) at the concentration of 0.8ml/l of milk is then added to coagulate the milk. After 35 minutes the curd is cut with curd knives into 12-15 mm cubes and left for another hour in order to expel part of the whey. The curd then is transferred into small hoops of 9 cm in diameter and 8 cm height. Each round is not filled to the top so that a small lid can be placed on top of the curd. Approximately 250-260g of curd are placed into each hoop. The cheeses are then incubated overnight at 24-26°C. The following day the cheeses are removed from the hoops and placed into a cold 20% brine solution, with a speck of mould powder added, for about 30 minutes. The cheeses are then removed from the brine and placed on a plastic rack to dry for 24 hours at room temperature (20°C). After this, the rounds are pierced with a stainless steel needle to allow the growth of the mould and the formation of the blue veins inside the cheese. The rounds are then put into plastic containers to maintain a humid environment at 15°C and stored for 10-15 days. The cheeses are fully covered with mould within 10 days and by this time they weigh approximately 200 g each. They are wrapped in clear plastic (cling-wrap) and stored in the fridge at 4°C. Under these conditions the cheeses can be consumed immediately but remain quite palatable for up to 4-6 weeks (depending on individual taste).

In total 10 batches of cheese were made. For each batch of cheese the milk was analysed for fat, protein, lactose, total solids concentrations. When production became consistent a sample of the cheese was analysed for protein with the Kjeldhal method, fat by acid hydrolysis and solvent extraction, moisture by oven drying at 100°C, ash by incineration in a furnace at 550°C. Sodium and Calcium were also determined by the atomic absorption method.

A small number of customers that turned up to purchase our dairy products* were asked to conduct a sensory evaluation of the cheese. Consumers were asked to taste the cheeses and evaluate the cheeses for aroma, colour, flavour and texture on a scale from 1 to 7 where 1 meant very bad and 7 meant excellent. They were also asked to give a mark, also of 1 to 7 for a comprehensive evaluation of the cheese if they would buy the cheese (yes or no) and to make any additional comments.

4.6.4 Results

Our initial aim was to induce the growth of blue mould inside the cheese round by piercing the rounds similarly to what is done with other blue cheeses. However, the sheep milk curd proved to be too soft and piercing resulted in small channels that closed over very rapidly, preventing the formation of the desired blue veins. With this method very few blue veins actually developed inside the cheeses.

The blue cheese has an average yield of 19. 5%, 24% fat and 50% moisture (Table 4.6.1). It has a high energy content (1268 kJ/100 g) and has proven to be very popular at The University of Western Australia, where the local community flocks every Friday to buy the cheese.

Table 4.6.1. Average composition (%) and nutritional information for the blue cheeses developed within the project.

Component Mean Standard error Kjeldhal Nitrogen (N) g/100g 3.41 0.015 Protein (N x 6.38) g/100g 21.7 0.10 Fat g/100g 24.3 - Water g/100g 50.5 - Ash g/100g 2.6 - Total Carbohydrates g/100g 0.9 - (by difference) Energy kJ/100g 1268 Sodium (Na) mg/100g 352 2.0 Calcium (Ca) mg/100g 644 12.5

Our survey reveled that 75% of the people interviewed would be prepared to buy the cheese. The cheese scored quite highly for all aspects of the evaluation, with a comprehensive evaluation mark of 5.55 (Table 4.6.2). Table 4.6.2. Average marks on a scale of 1 to 7 where 1=worse and 7=best awarded to batches of blue cheese by a small group of consumers (n=16). Values in brackets represent the standard errors. Aroma Colour Flavour Texture Comprehensive evaluation 5.13 5.47 5.47 5.47 5.55 (0.45) (0.26) (0.41) (0.27) (0.31)

* Throughout 2004 we commenced selling some of our dairy products at The University of Western Australia because our Industry Partners, Casa Dairy Products, changed hands and the new owners declined to participate in our project. They suddenly stopped purchasing our milk and the only alternative that we had was to sell the produce locally. Our cheese sales have proven to be popular and have generated considerable publicity for the sheep milking industry as well as for the project. 37

4.6.5 Discussion

This cheese is easy to make and it has a high yield from each litre of milk. It can be sold within two weeks from production. Such a short maturation time involves low risk of spoilage during maturation and low storage costs.

It is however, very mild and it is more similar to a brie or a camembert made using a blue mould rather than a traditional blue cheese. We see this as an advantage because we do not intend to compete or produce a recipe that may lead to competition with other blue cheeses already available on the market such as the Meredith Blue.

Currently we sell the cheese at a price of $6 per round ($ 30/kg). This is only done to cover partially project costs and industry would be likely to sell it at a higher price. A similar cheese made with cows’ milk in Tasmania sells for $37/kg and a blue cheese made with sheep milk sells for $55/kg (http://www.grandview.au.com).

The famous Meredith Blue cheese retails for $ 38-45/kg (depending on the retailer) suggesting that potential returns from each litre of milk processed may be very high.

5. General Discussion and implications

The results presented in this report have major implications for the Australian sheep milking industry.

The problem of the lack of a productive breed of dairy sheep was addressed by evaluating the dairy potential of the East Friesian and of the transgenic GH sheep. The East Friesian and East Friesian x Awassi sheep appear to be very promising genotypes as they produced large amounts of milk and had long and persistent lactations.

If until now the sheep dairy industry had been hindered by the lack of productive breeds of dairy sheep, this is no longer the case. It is hoped that the importers of the Awassi sheep will make this breed available to dairy farmers similarly to what has happened with the East Friesian sheep. Crosses of these two breeds are likely to be hardy and well suited to the Australian environment as well as highly productive.

Although the GH sheep seemed to be a promising genotype because they produced almost twice as much milk as the control animals, current legislation and consumers’ attitudes towards genetically modified organisms makes it unlikely to have commercial applications in the near future.

Our work has clearly shown that beneficial compounds such as the Conjugated Linoleic Acid (CLA) are already present in the milk of sheep and may be present in more constant concentrations throughout lactation in the fat tail breeds. The Mediterranean diet has been known for its health benefits for many decades now, and benefits have been so far ascribed to the use of olive oil, complex carbohydrates and red wine. Sheep dairy products are also a characteristic of the Mediterranean diet, and the fact that these products are high in CLA, that has been found to have anti-carcinogenic and anti-atherosclerosis properties in laboratory animals, may be partly responsible for the health benefits of the Mediterranean diet.

Our work on the CLA provides the sheep milking industry with a further reason to publicize its products to consumers. They are not just good to eat: they are also good for you.

Regardless of the health benefits of consuming sheep milk products, our work has demonstrated that it is also possible to adopt feeding strategies that increase the concentration of beneficial compounds in the milk. This opens the way for the production of designer sheep milk similar to designer milks suggested within the traditional cow dairy industry as methods to increase the marketability of cow’s milk.

The need to develop typically Australian sheep milk products was addressed by developing a blue cheese that is very promising in terms of consumer acceptance and has a high yield and short maturation time, a characteristic highly sought by dairy products manufacturers.

6. Recommendations

Our results clearly indicate that the East Friesian x Awassi sheep have exceptionally good productions of milk. These were not only greater than those of local sheep, but they were also greater than those of the specialised Awassi sheep. This could be the effect of the genetic improvement brought about by a superior ram, and selection for dairy production should still be an essential component of any sheep dairy enterprise. The choice of superior sires capable of improving the milk production of their progeny should be a major goal for any sheep milking enterprise.

Our work on the concentration of CLA in sheep milk has important implications. Firstly, we have identified the fact that fat tail genotypes have a slower rate of decline in milk CLA concentration throughout lactation. If the sheep milking industry wants to use the health claims associated with high CLA concentrations, having sheep in the flock that produce a consistent output of CLA throughout lactation may be important as CLA concentrations otherwise may fluctuate throughout the year making health claims difficult to maintain. Our work has also shown that milk CLA can be increased by feeding sunflower oil, and in the next project we will continue to investigate methods to increase CLA concentration in sheep milk and sheep milk dairy products. We have also shown that it is possible to produce milk that has increased concentrations of omega-3 fatty acids by supplementing the sheep with protected tuna oil supplements. This could be taken on by industry if they wished to develop a niche market for health conscious consumers.

The blue cheese developed within the project was well received by consumers. This mould- ripened cheese has a short maturation time, which is a desirable characteristic. It is hoped that dairy manufacturers throughout Australia will soon start to process sheep milk for the production of specialty dairy products such as this one, as this would provide the impetus for the development of more sheep dairy farms in our country. While traditional manufacturers refuse to purchase sheep milk, or demand to buy it at the same price of cow’s milk, a sheep milking industry is not likely to become established.

7. Communications Strategy

During the three years of this project, we have participated in field days and the Principal Investigator has given invited talks at industry and scientific meetings. This extension work will continue and we will endeavour to divulge our results as widely as possible. Liaison with industry has always been a strong characteristic of our projects, and it continued in this one.

This report has already resulted in the publication of honours theses, articles in scientific journals, and it is envisaged that other publications will stem from this project.

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