The Effect of Consuming Grass Finished Lamb Meat on Omega-3 Blood Response Among Healthy Consumers
Phase: NA
Conditions: Healthy
Sponsor: Queen's University, Belfast

Human intervention study protocol version 19.08v2  
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Study protocol 
 
 
 
Official study title: The effect of consuming grass finished lamb meat on omega-3 blood 
response among healthy consumers 
 
 
NCT number: 19.08v2 
 
 
Date: 22/03/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Human intervention study protocol version 19.08v2  
Date: 22/03/2019  
 
Structured Protocol  
You are asked to ensure your protocol includes all of the headings listed.  If you feel the 
heading is not relevant to your protocol, then please specify the reason. 
 
Title of project: The effect of consuming grass finished lamb meat on omega-3 blood response 
among healthy consumers  
 
Aims and objectives 
Overall aim: To investigate the impact of consuming grass finished lamb meat on human health 
parameters when compared to concentrate finished lamb meat.   
Primary objective: To assess the effect of regular consumption of grass finished and 
concentrate finished lamb meat on the plasma and platelet omega-3 status of healthy 
individuals. 
Secondary objective: To assess the effect of consumption of grass finished and concentrate 
finished lamb meat on cardiovascular risk factors including blood pressure, body mass index 
(BMI), total cholesterol, HDL-cholesterol and triglycerides among healthy individuals.  
 
Background 
In recent years, consumers have become increasingly interested in the food they eat, given 
advances in nutrition science, the nutritional quality of food products and knowledge of non-
communicable disease aetiology. Currently, cardiovascular disease represents 31% of deaths 
worldwide (World Health Organisation, 2016) and incidence is predicted to rise given modern 
day lifestyle habits including smoking, physical inactivity and poor diet. Meat consumption can 
contribute a nutritionally significant as part of a balanced diet. Due to its high biological value, 
meat is an important source of nutrients for human health and wellbeing, e.g. iron, vitamin B12 
and zinc. However, there are concerns and controversy surrounding the fat content of meat, 
despite fat being a vital component directly influencing palatability, flavour and overall liking 
(Font-i-Furnols and Guerrero, 2014). Fat type and content varies greatly between species and is 
further influenced by age, sex, genetic makeup and nutrition of the animal (Khan et al, 2015; 
Scollan et al, 2017). Within the muscle, fat is distributed into neutral and polar fractions and 
deposited intermuscular, intramuscularly or subcutaneously, with type and amount of lipid 
varying within individual muscles (Listrat et al, 2016).   

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When compared to monogastric meat, ruminant meat contains high levels of saturated fatty 
acids (SFA) and trans-fatty acids (TFA) relative to monounsaturated fatty acids (MUFA) and 
polyunsaturated fatty acids (PUFA). However, all meat types are relatively low in the essential 
omega-3 (n-3) fatty acids for which naturally rich sources include fish and fish products. 
Nonetheless, by nature of its frequent consumption ruminant meat supplies not just dietary SFA 
but also makes considerable contributions to dietary MUFA and PUFA.  
Excessive intake of dietary SFA is known to increase risk of cardiovascular disease (CVD) and 
contribute to various other non-communicable disease incidence (Mensink, 2005). UK dietary 
reference values recommend, for adult males and females, total fat intake should not exceed 
35% of food energy, with 11% of this accounting for SFA (British Nutrition Foundation, 2018). 
Recommended intakes of total cis-PUFA are 6.5% food energy, of which there should be a 
minimum n-3 PUFA intake of 0.2% of food energy (British Nutrition Foundation, 2018). Similarly, 
The WHO (WHO, 2016) recommended that, for adults, total fat should not exceed 30% of total 
energy intake. More specifically, total SFA and TFA content should be no greater than 10% and 
1% of total energy intake, respectively. The WHO guidelines also recommend that total PUFA 
should reach 11% of total energy intake with 2% and 9% accounting for n-3 and n-6 PUFA, 
respectively. 
Despite these guidelines, global n-3 intake is sub-optimal, with western countries falling 
particularly short of optimal blood eicosapentaenoic (EPA; 20:5 n-3) and docosahexaenoic acid 
(DHA; 22:6 n-3; Stark et al. 2016). The European Food Safety Authority (EFSA; 2009) stated 
that for food products to be considered ‘a source of’ or ‘high in’ n-3 long chain PUFA (LC-PUFA) 
it must contain per 100 kcal 40 and 80 mg/ day of 20:5 n-3 and 22:6 n-3, respectively. In order 
to reach the recommended n-3 PUFA intake, UK guidelines suggest that two portions of fish per 
week should be consumed, one if which is oily. However, global fish consumption is lower than 
recommended, whilst poultry and red meat consumption is higher and represents a large 
proportion of fat intake.  
The relationship between n-3 PUFAs and human health is well established and generally 
accepted. Increased dietary levels of 20:5 n-3 and 22:6 n-3 have been documented to instigate 
physiological reaction cascades, thereby improving the inflammatory response, including 
alleviating arthritis and asthma symptoms (Yashodhara et al, 2009; Norling and Perretti, 2013; 
Li, 2015). Similarly, enhanced 20:5 n-3 and 22:6 n-3 has also demonstrated its ability to 
decrease CVD risk by reducing triglycerides in the bloodstream, lowering the risk of myocardial 

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infarction, stroke, atherosclerosis development and overall risk of death (Jacobson et al. 2012; 
Givens, 2015; Singh et al. 2016). Nevertheless, there is a level of controversy surrounding n-3 
PUFA exhibiting health promoting effects, with some studies finding little or no evidence of 
significant health benefits of consuming n-3 PUFA (Rangel-Huerta and Gil, 2018). 
Discrepancies could be explained partially by differences of bioavailability of n-3 PUFA between 
dietary sources found within foodstuffs and supplementary sources of n-3 PUFA (Harris et al, 
2017). 
Due to the generally accepted ability of n-3 to reduce non-communicable disease incidence, 
nutritional approaches have focused on increasing the amount of beneficial fats in meat and 
meat products with emphasis placed on increasing n-3 and specifically n-3 LC-PUFA. Improving 
the lipid composition of poultry and monogastric meat has been largely successful. However, 
the process for ruminant animals is more multifaceted due to the diversity of the rumen and 
resident microbial community. Consequently, large proportions of dietary PUFA are converted 
into SFA such as stearic acid (C18:0) or palmitic acid (C16:0) by lipolysis and biohydrogenation 
processes, which are controlled by complex microbial flora (Lourenco et al. 2010). Research 
has attempted to overcome the biochemical limitations of nutritionally enhancing ruminant meat 
by examining early life feeding, including microbial programming (De Barbieri et al. 2015) and 
finishing feed (Vestergaard et al. 2000). Emphasis has also been placed on the positive benefits 
of pasture vs concentrate feeding. 
It is widely accepted that pasture compared to concentrate based feeding is more efficient in 
enhancing beneficial n-3 PUFA in meat of ruminant animals (Nuernberg et al. 2005; Fisher et al. 
2010; Hajj et al. 2016; Scollan et al. 2017), with enrichment proving more efficient in lamb than 
beef (Bessa et al. 2015). More specifically, grass chloroplasts are rich in 18:3 n-3, which are a 
known precursor for longer chain n-3s such as 20:5 n-3, 22:5 n-3 and 22:6 n-3. In fresh grass, 
18:3 n-3 can represent up to 75% of the total lipid fraction (Blasko et al, 2010). This level can be 
influenced by season, species, geographical location, growth stage, nitrogen fertilization and 
senescence. In addition, pasture-based feeding provides increased concentrations of vitamin E, 
which contains valuable antioxidant properties, reducing the incidence of lipid oxidation and 
therefore minimising the opportunity for skewed flavours and odours to develop.  
The effect of animal-based foods enriched fatty acid profiles on human health has received 
relatively little attention and is largely unknown. In recent years, a single study explored the 
effect of consumption of either n-3 enriched pasture-based feeding or concentrate fed beef and 

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lamb on blood fatty acid profiles and circulatory parameters including blood pressure and 
cholesterol in an intervention trial among healthy individuals (McAfee et al. 2011). Results 
showed that the total PUFA and LC-PUFA plasma and platelet lipid composition of individual’s 
subject to the pasture fed meat treatment was significantly higher, when compared to subjects 
who consumed concentrate fed meat. These results highlight the ability of n-3 within enriched 
meat to be successfully taken up and utilised within the human body. However, this study is an 
isolated examination of the human health effects of n-3 enriched ruminant meat products and 
further and more in-depth research is required to fully establish a confirmed biological effect and 
contribute to the current limited literature. 
The research area of enriching n-3 PUFA concentration in ruminant meat is increasingly being 
developed and continues to expand. However, there is a significant lack of research 
demonstrating the effect of consuming grass finished lamb on n-3 blood status among free living 
healthy consumers. Therefore, identification of current knowledge gaps is an imperative to 
overcome the challenges in ensuring an optimal level of n-3 PUFA is present in muscle and in 
turn consumed by the public, ultimately contributing to human health by assisting with reducing 
cardiovascular and other non-communicable diseases incidences with little change to 
customary dietary habits. The present study proposal uniquely focuses on the consumption of 
lamb meat for a four-week period as a proof of principle technique to demonstrate preliminary 
biological effect of habitual lamb consumption on blood omega-3 status and cardiovascular risk 
factors. 
 
Plan of investigation  
 
Study design  
The study period will consist of a four-week, single blinded, randomised dietary intervention 
whereby participants will be required to eat three portions per week of lamb meat from one of 
two treatments for a four-week study period. The study will focus entirely on the consumption of 
lamb meat. This is because evidence in current literature suggests that lamb muscle has a 
higher capacity to store omega-3 in the muscle phospholipids when compared to other meats 
such as beef or pork (Bessa et al, 2015). Therefore, as a proof of principle study, volunteers will 
consume three portions of lamb per week for four weeks, substituting lamb for regular red meat 
consumption and therefore maintaining intakes of red meat within safe levels and not 
encouraging any adverse dietary pattern.   

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Lamb portions will include per week one portion of mince (raw weight 250g) and two portions of 
lamb chops (raw muscle weight 235g). Raw lamb portions will total 720g per week. Literature 
suggests cooking losses will represent approximately 32% (Matthew and Garrison, 1975), 
meaning around 490g of lamb per week will be consumed, keeping below recommendations by 
the World Cancer Research Fund (WCRF) to consume less than 500g of red meat/week and 
UK NHS recommendations to consume no more than 70g/day of red and processed meat. One 
group of participants will receive lamb meat from concentrate finished lambs and another group 
will receive lamb meat from grass finished lambs. The study will be single blinded meaning 
researchers will know what treatment participants are receiving however participants will not be 
told what dietary treatment they are receiving. 
 
Meat will be stored at AFBI Newforge, AFBI Stormont and North South Retail Belfast. The meat 
will be delivered to the participant’s residence frozen on a weekly basis. Days and times of 
delivery will be discussed and decided on an individual participant basis. Participants will also 
be expected to keep a three-day food diary which will be completed prior to the intervention start 
and then again during the mid-point of the intervention during week one and week three. This is 
where all food and drinks for the three days will be recorded with as much detail as possible 
regarding consumption (including left overs), time of day, quantity of food and cooking methods. 
In addition, participants will also be asked to keep a meat diary for the entire duration of the 
study as a record of whether all meat is consumed or whether some is not consumed. This will 
also include what cooking methods are used and whether participants consume the 
subcutaneous fat or not. Participants will receive recipe cards at the beginning of the four-week 
study period to provide cooking ideas; however, there is no obligation to follow the recipes. 
Participants may cook the lamb however they choose but will be reminded of the importance of 
following good food safety and hygiene practices e.g. defrosting meat safely and the importance 
of thoroughly cooking minced meat. 
 
Meat source 
The lambs which will provide the meat were reared for an 11-week finishing period at AFBI, 
Hillsborough, NI. The finishing period started 04th September 2018 and finished on 20th 
November 2018. The lambs were transported to Dunbia, Dungannon for slaughter on the 21st 
November 2019. The carcases were left to hang for 48 hours before the meat was processed 
into loins, shoulder and leg meat. Finer processing including separation of loins into chop 

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portions and mincing of meat was carried out at Loughery College, Cookstown on 26th 
November 2018 and 16th January 2018, respectively. The loins were band-sawed fresh creating 
lamp chops, individually packaged and then frozen. The shoulder and leg meat to create mince 
portions was frozen and then tempered to -1°C, minced, weighed into 250g portions, vacuum 
packaged and re-frozen. Alternative lambs have been raised and slaughtered to act as a back-
up option if any of the intervention study meat was to be unsuitable. A representative proportion 
of the meat from both the loins and the mince portions will undergo microbial analysis prior to 
being used in the intervention study.  
 
Sample size and justification  
Healthy volunteers (N=38) will be recruited and allocated to one of two treatments 
(n=19/treatment) balanced for age and gender using a computer-generated randomization tool, 
which will be completed with the assistance of Dr. Christopher Cardwell (statistician). Sample 
size was determined using power calculations based on changed in blood plasma and platelet 
PUFA levels from a similar study (McAfee et al, 2011). Based upon the SD of plasma PUFA of 2 
units (%), with 16 in each treatment there will be over 80% power to detect, as statistically 
significant at the 5% levels, a difference in mean PUFA of 2 units in the grass-fed lamb meat 
group and the concentrate fed lamb meat group. This will be determined using an independent 
samples t-test. This difference in plasma PUFA is similar (slightly less than) the difference seen 
in the previous study (McAfee et al, 2011), however it is anticipated a larger difference will be 
detected as only lamb meat is being used which is documented to be of increased capacity to 
store omega-3 PUFA (Bessa et al, 2015) and therefore it is expected that this will have a 
greater influence on blood plasma PUFA status. The power calculation for platelets will be more 
limited, however, based on the SD of platelet PUFA of 4 units (%), with 16 in each treatment we 
would have over an 80% power to detect, as statistically significant at the 5% level, a difference 
of 2 units in the lamb-fed meat group and concentrate fed meat group. In addition to the 32 
required participants established by statistical power calculations an additional six participants 
will be recruited to account for drop out and non-compliance. 
 
Volunteer recruitment 
Volunteers will be staff and students recruited from Queen’s University Belfast. Involvement in 
the study will be advertised via poster advertisement around university buildings initially in the 
David Keir Building (DKB), Sonic Arts Research Centre (SARC) and Northern Ireland 
Technology Centre (NITC). Following this, advertisement posters will be displayed in the 

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Medical Biology Centre (MBC). Volunteers will also be recruited via university mailing lists and 
through inclusion of the study advertisement on the staff round-up.  All potential volunteers will 
be given the information sheet and will be assessed for suitability and eligibility using a short 
screening questionnaire which will identify whether volunteers fit into the inclusion and exclusion 
criteria (see below). Subsequently, participants will be given 48 hours to decide whether or not 
to participate in the study. When participation is agreed written consent will be obtained.  
 
Inclusion and exclusion criteria 
Potential participants will be screened using an eligibility questionnaire (See attached 
questionnaire), which will identify suitable participants by applying specific inclusion and 
exclusion criteria outlined below. Male and female healthy volunteers aged between 18-64 
years old will be considered for participation. For eligibility of participation, volunteers must be 
non-smokers, must not be taking statins to lower LDL cholesterol levels and must not have high 
blood pressure (systolic >140/ diastolic >90mmHg). Moreover, participants with a BMI of <18.5 
kg/m² and greater than >35 kg/m² will be excluded. Participants who are taking prescribed 
medication and any form of dietary supplements will also not be eligible to participate The 
consumption of the lamb meat will act as a substitute for optimal habitual red meat consumption 
as recommended by the WCRF and UK NHS. Therefore, participants must be red meat 
consumers and willing to consume three portions of lamb per week. Participants who consume 
more than two portions of fish per month will also not be eligible to participate, as that may mask 
the ability of the lamb to affect n-3 PUFA status.  
 
Blood collection and handling 
Blood samples (25ml) will be taken at the beginning of week 0 and end of week 4 (end of dietary 
intervention) from both the grass-finished meat and concentrate-finished meat treatment groups. 
As total-cholesterol, HDL-cholesterol and triglyceride concentrations will be measured 
participants will be asked to fast for 12 hours prior to blood being taken. Samples will be taken, 
separated within 1 hour of collection and then stored at -80C until analysed at a later date. 
Plasma and serum will be harvested by spinning blood samples at 2500 g for 15 minutes, whilst 
platelets will be harvested by centrifuging blood samples at 150 g for 15 minutes. Samples will 
be analysed for the fatty acid composition of blood plasma and platelet, in addition to total 
cholesterol, HDL-cholesterol and triglycerides (by commercially available kit on a Randox 
Daytona autoanalyser). All blood will be taken by a trained phlebotomist and stored and 
analysed at the Centre for Public Health, Queen’s University Belfast.   

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Blood plasma, platelet and serum aliquots will be analysed for fatty acids using gas 
chromatography at the Centre for Public Health, Queen’s University Belfast. Lipids will be 
extracted using the Folch method (Folch et al. 1957) and methyl esters will be prepared using 
an adaption of the method outline by Morrison et al. (Morrison et al. 1964). Fatty acid 
composition will be analysed using gas chromatography (Hewlett Packard 6890 Gas 
Chromatograph) and flame ionizing detector. Peaks will be integrated using ‘Chemstation’ 
computing integrator. Fatty acid methyl esters will be identified by output retention times which 
will be compared to known and commercially available standards. Results will be presented 
initially as % of total fatty acid content and then will be quantified by looking at retention times, 
peak areas and the use of an internal standard.      
 
Blood Pressure and BMI  
Blood pressure and body mass index (BMI) will also be assessed at baseline (week 0) and post-
intervention (week 4) from both the grass finished meat and concentrate finished meat 
treatment groups. Blood pressure will be taken using an automated sphygmomanometer using 
established Centre for Public Health SOPs. Blood pressure from all participants will be taken 
from the same arm to ensure consistency. Systolic and diastolic pressure will be measured and 
recorded. BMI will be calculated by assessing the participant’s weight (kilograms) and height 
(metres squared). Assessment will take place at The Centre for Public Health, Queen’s 
University Belfast. Blood pressure and BMI data will be taken by Lynda Perkins (PhD 
researcher). 
 
Illness during the study 
During the study, if a participant feels unwell as a result of eating the lamb, participants should 
contact the researchers using the phone number or email address provided on the participant 
information sheet. There is minimal risk of discomfort as a result of consuming the lamb, 
however if discomfort does occur, participants can opt out of the study at any given stage.  
Participants should seek medical advice if it is felt this is appropriate. 
 
Incidental findings 
It will be made clear to participants that during data analysis the researchers are not looking for 
anything other than what is stated in the information sheet. Nonetheless, if any incidental 
findings are discovered (e.g. hypertension), the research team is obliged to disclose this 

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information with the participants. If an incidental finding occurs, the researchers will advise 
participants to consult a GP or a medical professional who will be able to guide them further.  
 
Planned statistical analysis of results 
The change in each outcome variable will be compared between intervention groups using an 
independent samples t-test. A significance level of 0.05 will be used to determine statistical 
differences. Any effect within treatments from baseline to end of intervention will be analysed 
using a paired t-test. A significance level of 0.05 will be used to determined statistical 
differences. All statistical analysis will be carried out with the assistance of Dr. Christopher 
Cardwell and using established statistical software such as SPSS. 
 
Project timetable 
Recruitment of volunteers is planned to commence in April 2019 using poster advertisement 
(See Table 1). Once participants have been recruited, they will commence the dietary 
interventions. Participants will be stagger started as and when they are recruited. Completion of 
volunteer study periods are planned to finish in July 2019, at which point laboratory, data and 
statistical analysis will occur. The study is due to be fully completed by November 2019. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 1: Proposed project timetable for recruitment, intervention study period, analysis and 
writing up 
 
 
Data protection  
The data collected will be treated securely and confidentially as necessary under the General 
Data Protection Regulation (updated May 2018) and stored as required by the University. All 
participant information will be kept within a locked filing cabinet and will be kept on password 
protected university computers and stored in secured access-controlled university buildings. All 
participants will be allocated a unique study number which will ensure anonymously during the 
study. Blood samples will be labelled using the participants unique study number only and will 
be stored within a locked freezer within secured, access only buildings. Blood samples will not 
be used for any further analysis and will be disposed of after completion of the study.  
 
 
 
 
 
Mar 
Apr 
May 
Jun 
Jul 
Aug 
Sep 
Oct 
Nov 
Dec 
Ethics 
committee 
 
 
 
 
 
 
 
 
 
 
Recruitment 
 
 
 
 
 
 
 
 
 
 
Commence 
study periods 
 
 
 
 
 
 
 
 
 
 
Complete study 
periods 
 
 
 
 
 
 
 
 
 
 
Laboratory, data 
and statistical 
analysis 
 
 
 
 
 
 
 
 
 
 
Writing up and 
publishing 
 
 
 
 
 
 
 
 
 
 
Study 
completion  
 
 
 
 
 
 
 
 
 
 

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