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Horses Used for Large-Scale Production of Immunoglobulins: An Inter-Species Approach Massage or Music Meant to be Relaxing Result in Lowering Salivary Cortisol Concentration in Race Horses A Vegan Diet for Dogs and Cats May Improve Health Outcomes and Longevity Pig Loss Analysis of Virulent ASFV Infections Under Different Test and Removal Success Scenarios Official Supporting Associations -

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CONTENTS 04 FOREWORD WATCH PAGES 06 Bovine & Ovine Colostrum The feeding and management of dairy calves directly impacts their future milk production, in addition to proper colostrum intake on calf health. Hesham Kamel, a Technical Expert, explains more about the adequate intake of colostrum in bovine and ovine.

MANAGING DIRECTOR Mark A. Barker EDITORIAL MANAGER Beatriz Romao beatriz@senglobalcoms.com

REGULATORY & MARKETPLACE 08 Pig Loss Analysis of Virulent ASFV Infections Under Different Test and Removal Success Scenarios African swine fever (ASF) is known as an infectious disease causing high mortality of pigs and is notifiable to the World Organization of Animal Health (OIE). The etiological agent in African swine fever virus (ASFV) has an incubation period varying from 4 to 19 days. Dr. Jiancong Yao at PIC analyses the virulent ASFV infections under different test and removal success scenarios.

RESEARCH AND CIRCULATION Virginia Toteva virginia@senglobalcoms.com DESIGNER Jana Sukenikova www.fanahshapeless.com BUSINESS DEVELOPMENT Jerome D’Souza info@senglobalcoms.com ADMINISTRATOR Jessica Dean-Hill jessica@senglobalcoms.com FRONT COVER © istockphoto PUBLISHED BY Senglobal Ltd. Unit 5.02, E1 Studios, 7 Whitechapel Road, E1 1DU, United Kingdom Tel: +44 (0) 2045417569 Email: info@senglobalcoms.com www.international-animalhealth.com International Animal Health Journal – ISSN 2752-7697 is published quarterly by Senglobal Ltd.

The opinions and views expressed by the authors in this Journal are not necessarily those of the Editor, Publisher or the Supporting Organisations which appear on the front cover. Please note that although care is taken in preparation of this publication, the Editor and the Publisher are not responsible for opinions, views and inaccuracies in the articles. Great care is taken with regards to artwork supplied, the Publisher cannot be held responsible for any loss or damage incurred. This publication is protected by copyright. Volume 8 Issue 4 Winter 2021 Senglobal Ltd. www.international-animalhealth.com

RESEARCH & DEVELOPMENT 12

Horses Used for Large-scale Production of Immunoglobulins: An Inter-species Approach The use of horse-derived plasma in large-scale production of immunoglobulins is currently adopted worldwide for treatment of various infectious diseases, such as, tetanus, rabies, botulism, and is the only treatment available for envenoming by several species of snakes, scorpions, and spiders. Ana Lucia Camphora and Manuela Pucca discuss procedures and techniques applied in the production of conventional antivenoms, breaking the customary silence on the millions of horses that have been essential in the historical developments of scientific institutions, such as the Pasteur Institute, in France, and the Butantan Institute, in Brazil.

16 A case-control study comparing the effects on calf health after use of a commercially available Mycoplasma bovis vaccine in dairy herds in Scotland Mycoplasma bovis is a common cause of bovine pneumonia in calves. Treatment and control of the disease is challenging. Treatment can be ineffective, and control is hampered by a lack of a licensed vaccine in Europe. Graeme Fowlie at Meadows Vets Centre, outlines the proof of concept for the vaccine’s commercial use in the UK. Further work on assessing the effect of this vaccine in UK cattle herds infected with M. bovis respiratory disease is warranted. 24 Massage or Music meant to be relaxing, result in lowering salivary cortisol concentration in race horses At the beginning of training routine, young racehorses are exposed to stressful stimuli. Janet Marlow at PetAcoustics aims to evaluate the influence of a relaxing massage which the horses received in the stable, and the influence of music piped into the stable, on the long- lasting stress level of the horses. International Animal Health Journal 1

CONTENTS 28 Muscle Abnormalities in Broilers Over the past decades, broilers have been growing at an increasing rate. This is not only due to changes in their genetic composition but also through improvement of housing, nutrition and management. The increase in the breast meat weight of broilers is particularly striking. As the slaughter weight of broilers increases, in combination with the growth curve required to achieve this slaughter weight, we are seeing several muscle abnormalities which influence the quality. Robert Jan Molenaar at Royal GD discusses the main abnormalities that often only become apparent during the slaughter process. FOOD & FEED 28 A Vegan Diet for Dogs and Cats May Improve Health Outcomes and Longevity In the past decade, men and women around the globe have transitioned to a vegan diet in record numbers, propelled largely by a growing body of research pointing to better health outcomes, and by catastrophic weather events and wildfires that fuel concerns about the contribution of animal-based food production to climate change. Jan Allegretti, author of The Complete Holistic Dog Book: Home Health Care for Our Canine Companions, explains how a vegan diet for dogs and cats may improve health outcomes and longevity. 32 Is Insect Farming Truly a Solution to the Animal Feed Problem? The European Union has started considering alternative feed, such as insect protein, to supply the

2 International Animal Health Journal

livestock industry. Insect-derived protein as animal feed is increasingly seen as a solution to diminish the use of imported soy linked to deforestation and to supplement the use of fishmeal from depleted oceans. Camilla Björkbom at Eurogroup for Animals analyses if insect farming is the solution to the animal feed problem. 33 Organic Trace Minerals– Enhancing Mineral Bioavailability Through Chelation The chemistry of complexation or chelation as it is commonly known has created a great deal of confusion in the animal feed industry. Terms such as metal amino acid complexes, metal amino acid chelates, metal polysaccharide complexes and metal proteinates abound, yet official definitions remain vague and unhelpful. Richard Murphy at Alltech’s European Bioscience Centre discusses important factors in mineral chelation. 40 The Value of Circularity in Sustainable Food Systems The circular economy, or circularity of a system, is an intentional effort to design out waste and pollution, keep products and materials in use and regenerate natural systems. Lara Moody at Institute for Feed Education and Research, and Anton van den Brink at European Feed Manufacturer’s Association address the role a circularity metric could have in assessing and valuing sustainability efforts for the animal food supply chain and consider measurement and assessment limitations that exist, which restrict the industry’s potential to accurately account for its environmental impact and role in a circular economy.

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FOREWORD There is little doubt, at least where I am writing this in the UK, that it is now winter. In the last few weeks we have had plummeting temperatures and we’ve been treated to the delights of Storm Arwen, the first of this year’s winter storms to hit the UK. This storm caused widespread destruction and mayhem and, sadly, three fatalities. Hopefully, as we head into December, we may be able to look forward to slightly calmer, if not warmer weather and then begin to look forward to the return of spring. Perhaps… Whatever, this is the winter edition of the Journal and, for those entering northern winter (or southern spring), it brings with it a series of articles designed to inform, provoke thought or provide insight for a wide variety of topics. I will focus on just one of these. These are the mycoplasmas and specifically Mycoplasma bovis. The mycoplasmas are fascinating organisms. They are the simplest and smallest free living prokaryotes and the outer boundary of their cells is the plasma membrane - they lack a cell wall. As a result of this, they can change their size and shape. They are heterotrophic, and while some can live as saprophytes, the majority are parasites and hence are pathogenic. They can infect plants and animals. These organisms are bacteria in the class mollicutes but, unlike other bacteria, they have no bacterial cell walls. However, like other bacteria, they possess 70S-type ribosomes. Both these characteristics are important for disease treatment.

Lacking bacterial cell walls, mycoplasmas are not susceptible to antimicrobial drugs that target bacterial cell wall synthesis as their mode of action. Hence, penicillin and related drugs are ineffective. However, because they have 70S ribosomes, a ribosome type common to bacteria, then antimicrobials which inhibit protein synthesis at the ribosome can be used to treat infections associated with mycoplasmas. These include the tetracyclines, macrolides and chloramphenicol. In veterinary medicine, only tulathromycin and florfenicol (in combination with flunixin meglumine) are indicated for disease associated with M. bovis. Thus, the therapeutic armoury is very limited and treatment of the established disease can be challenging. In this issue of the Journal, Graham Fowlie, a practising veterinarian from Aberdeenshire, describes his experiences with on-farm trials of a vaccine designed to protect cattle, specifically calves, against M. bovis. In the US, following cattle vaccination, there was a dramatic drop in the numbers of animals affected by arthritis and in the severity of the disease. The results of Mr Fowlie’s endeavours are therefore, eagerly anticipated and will be of interest to many readers of this publication. Kevin Woodword, Managing Director, KNW Animal Health Consulting

There are over 130 species of mycoplasma and at least 15 of these infect humans. For example, they are thought to be responsible for some sexually transmitted diseases, male sterility and infant mortality. M. pneumoniae is a cause of “walking” pneumonia, an atypical pneumonia not associated with its normal causative pathogens. A number of species are associated with the development of cancers in humans. For example, M. genitalium is associated with prostate cancer. These findings are not incidental because some mycoplasma species have been shown to cause malignant cell transformation in some cell cultures. There are a number of mycoplasma species that are pathogens of animals. These include M. capricolum which causes contagious pleuropneumonia in goats, M. gallisepticum a pathogen of chickens and other avian species and M. hyopneumoniae the causative organisms of porcine enzootic pneumonia in pigs. M. bovis is a pathogen of cattle. It is one of the causative organisms of bovine respiratory disease, bovine mastitis and arthritis in cattle. These conditions pose major animal welfare issues as well as being significant contributors to economic losses.

EDITORIAL ADVISORY BOARD Germán W. Graff - Principal, Graff Global Ltd Fereshteh Barei - Health Economist & Strategy Advisor, Founder of BioNowin Santé Avenue Association Carel du Marchie Sarvaas Executive Director Health For Animals Kimberly H. Chappell - Senior Research Scientist & Companion Animal Product Development Elanco Animal Health Dr. Sam Al-Murrani - Chief Executive Officer Babylon Bioconsulting & Managing Director at Bimini LLC Sven Buckingham - Buckingham QA Consultancy Ltd. Dan Peizer - Director Animal Health at Catalent Pharma Solutions Dawn Howard - Chief Executive of the National Office of Animal Health (NOAH) Jean Szkotnicki - President of the Canadian Animal Health Institute (CAHI) Dr. Kevin Woodward - Managing Director KNW Animal Health Consulting Norbert Mencke - VP Global Communications & Public Affairs Bayer Animal Health GmbH 4 International Animal Health Journal

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WATCH PAGES

Bovine & Ovine Colostrum

Introduction The feeding and management of dairy calves directly impacts their future milk production, in addition to, proper colostrum intake on calf health. So, the first and adequate intake of colostrum is critical.

Colostrum = Life

The dry period management is very important for the dam and the calf as well, so underfeeding during the last two months of gestation can increase mortality of the calf within the first two weeks of life and have an effect on the colostrum quality produced by the dam which can result in difference of colour, composition and function from normal milk produced after 72 hours from calving time (shown in Table 1).

Milking Number

Less than 5.0 mg/dl

colostrum deprived

Item

Milk

5.0–5.5 mg/dl

suspect deprived

Specific Gravity

1.056

1.040

1.035

1.032

5.5–7.5 mg/dl

colostrum satisfied

Solids, %

23.9

17.9

14.1

12.9

Protein, %

14.0

8.4

5.1

3.1

Casein, %

4.8

4.3

3.8

2.5

IgG, g/L

0.6

Fat, %

6.7

5.4

3.9

3.5

Lactose, %

2.7

3.9

4.4

5.0

Colostral Immunoglobulin 4Q/4H Calves are born without antibodies against diseases and need to absorb the immunoglobulin found in colostrum to build the local immunity. There are three types of immunoglobulins in colostrum of cattle: 70–80% IgG 10–15% IgM, and 10–15% IgA. Additionally, there are two isotypes of IgG: IgG1 and IgG2. These immunoglobulins work together to provide the calf with passive immunity until the calf's own active immunity develops. Colostral immunoglobulin (IgG) is absorbed most efficiently within the first 4 hours of life and gradually decrease within the first 24 hours of age equal zero. Detecting the Colostrum-Deprived Calf To determine if a calf has absorbed adequate levels of protective antibodies from colostrum a popular procedure for large dairies is measurement of serum total proteins by using Refractometer at the 3rd day of age. Serum fractions from calves containing less than 5.0 gm/100 ml indicate insufficient colostrum antibody absorption.

Table 2: Interpretation of serum total proteins.

Some suggestions to maximise the biological safety of colostrum: • Collect colostrum from cows known to be healthy. • John’s and staph positive cows’ cases should collect colostrum and discarded directly. • A surveillance program for infected cattle can reduce the risk of transmission of disease through colostrum. • Sanitise the udder prior to collecting colostrum – use the same methods that you would use for collecting milk. Dirty colostrum can be a significant source of disease to the calves during the first few hours. • Collect colostrum into a clean, sanitised and dry container used ONLY for the purpose of collecting colostrum. • Do not allow colostrum to sit at room temperature. If you are not feeding colostrum direct to the calves you should keep it frozen with the quality marked, date and batch number. • The frozen colostrum is valid to be used again for 6 months from freeze date.

Hesham Kamel Hesham Kamel is a well-respected Technical Expert in the Middle East region and beyond, currently focusing on Dairy Cattle Production Medicine. For the last few years he has worked as a Technical Manager Ruminants for MSD in the Middle East focusing on the Animal Pharmaceutical Industry, mainly the technical side of production medicine, and has been involved with the cutting edge science being improving animal health. Email: hesham.kamel_1@consultant.com

6 International Animal Health Journal

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International Animal Health Journal 7

REGULATORY & MARKETPLACE

Pig Loss Analysis of Virulent ASFV Infections Under Different Test and Removal Success Scenarios African swine fever (ASF) is known as an infectious disease-causing high mortality of pigs and is notifiable to the World Organization of Animal Health (OIE). The etiological agent is African swine fever virus (ASFV), with a main characteristic of high mortality, and it has an incubation period varying from 4 to 19 days. The main routes for disease transmission are direct contact between susceptible and sick animals or their fluids or excretions, and indirect contact through contaminated feed, pork, people, vehicles, or fomites.1 However, since the introduction of ASFV into China swine herds in 2018, the virus has seen diverse mutations.2 In the research published by Chinese scientists, mutations, deletions, insertions, or short-fragment replacement occurring in all 22 isolates isolated in seven provinces in China in 2020 compared with Pig/HLJ/2018 (HLJ/18), the earliest isolate in China. Some mutated strains have shown as highly lethal as HLJ/18. Some non-HAD strains were showing to have lower virulence causing non-lethal, sub-acute or chronic disease, and persistent infection. The emergence of lower virulent natural mutants and genetically engineered deletions brings greater difficulty to the early diagnosis of ASF and creates new challenges for ASFV control. While ASFV has been challenging the Chinese veterinarians and pig producers, the industry has tested and pioneered the strategy of test and removal to contain the disease. Though the success rates have varied, the test and removal strategy which highly hinged on early detection and people capabilities for biosecure and rapid removal, has helped many farms avoid being fully depopulated. In this study, by distilling the experience gained from Chinese veterinary practitioners, the author intends to decipher the loss of ASFV under different health management scenarios and readers can infer its economic impacts in farm situations, by comparing with benchmark production parameters. Test and Removal Strategy “Test and removal” is to detect the ASFV infected pigs and remove them biosecurely from the herds to avoid further

spreading diseases to other herds and pigs. This strategy uses the biology characteristic of ASFV which primarily transmits via direct contact of pig to pig, or via indirect contacts of contaminated fomites, thus transmits slowly. Early detection of the infected pigs needs three factors to be in place: 1) right samples from at risks pigs; 2) reliable diagnostic tools, such as fluorescent quantitative PCR and 3) a trustable veterinary diagnostic laboratory (VDL) and proficient staff who performs the testing. Very often, false positives can be generated by a VDL, and sometimes, a truly infected animal is missed. The strategy has been widely utilised by pig veterinarians in China, but the publications are rare. Dr. Jiancong Yao have authored two papers on this subject and two success cases were all involved full depopulation of one barn in the setting of the big production compound.3,4 Typical Baseline Breeder Herd Production Parameters Without Economically Important Diseases Without major economically important diseases infections, in a typical well performing breeder farm with 1,000 production sows, we expect to give birth to 24,000 live pigs, and average mortality in a year in this herd is expected to be 15.62%. The cross-sectional average mortality rate (include one turn in each stage) is expected to be 6.07%. Three ASFV Infection and Health Management Scenarios Gleaning the experiences of different pig veterinarians, three case scenarios are categorized (Table 2). Scenario 1: Optimistic with successful test and removal

Scenario 2: Expected parameters with test and removal

Scenario 3: Pessimistic with depopulation of the herd

Sow loss %

15%

Sow loss %

25%

Sow loss %

100%

Boar loss %

15%

Boar loss %

25%

Boar loss %

100%

Preweaning loss %

25%

Preweaning loss %

40%

Preweaning loss %

100%

Nursery loss %

12%

Nursery loss %

30%

Nursery loss %

100%

G-F loss %

10%

G-F loss %

30%

G-F loss %

100%

Table 2 – Loss Simulations of Different Level of Success with Test and Removal

Baseline mortality (without major disease situations)

Parameters

Herds

Example/Herd size, 1000 Mortality No. sows

Sow mortality %

8.00%

Sow

1000

Boar mortality %

1.00%

Boar

0.5

Preweaning mortality %

12.50%

Preweaning

2000

250

3000

Nursery mortality %

3.50%

1500

52.5

368

G-F mortality

2.50%

G-F

3000

300

Current inventory

7550

458

15.62%

M%: accumulated death/annual output

6.07%

M%: cross sectional deaths/current stocks

Turns

Death No.

Total Live Pigs Born

80 1

3748

24000

Table 1 – Baseline Production Parameters and Mortality Rates in Different Herds 8 International Animal Health Journal

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REGULATORY & MARKETPLACE • • •

Scenario 1: Optimistic, successful test and removal Scenario 2: Expected parameters Scenario 3: Pessimistic, whole herds need to be depopulated Each scenario comes with different sets of loss parameters.

In ASFV infection cases, we would expect a certain number of pigs to be removed in a definitive period, normally 21 days, or 42 days, assuming one incubation period for ASF to be 21 days of maximum. So, we use the current stock of pigs in the farm as the denominator in calculating loss percentages (Table 3). Loss projections: • • •

Scenario 1: Optimistic, 15.07% Scenario 2: Expected parameters, 31.95% Scenario 3: Pessimistic, 100% ASFV Scenario 1

ASFV Scenario 2

ASFV Scenario 3

Current Mortality With stock (w/o Turns turns)

Mortality with (w/o Turns turns)

Mortality With (w/o Turns turns)

Sows (heads)

1000

150

250

1000

Boars

7.5

12.5

Pre-Weaning

2000

500

6000

800

9600

2000

24000

Nursery

1500

180

1260

450

3150

1500

G-F

3000

300

1200

900

3600

3000

Subtotal

7550

1137.5

8617.5 2412.5

Death % (accumulated/ current)

15.07%

16612.5 7550

31.95%

100.00%

–

Table 3 – Loss Projection of the Number and Percentages of Pigs in Three Scenarios

The surveyed pig veterinary practitioners agree that the likelihood of ASFV infection in a typical farm with sound biosecurity is 30%. The probability for each of the ASFV test and removal scenarios is expected to be: Scenario 1, 10%, Scenario 2, 60% and Scenario 3, 30% (Table 4). Baseline 70%

ASFV infections Probability

30% Scenario 1 Scenario 2 Scenario 3

Probability

10%

60%

30%

15.07%

31.95%

100.00%

50.68%

Loss% With ASFV

4.25%

0.45%

5.75%

9.00%

19.45%

Loss% All scenarios

15.20%

Loss% ASFV with P

Table 4 – Probabilities of Different Scenarios and Loss Percentage Projections (Note: P=probability)

Results If a farm is confirmed with ASFV classic high virulent strains, we expect 50.68% loss rate, when adopting test and removal strategy and assuming different probabilities (optimistic 10%, expected normal 60% and pessimistic 30%) for 3 disease containment scenarios. When 30% likelihood of ASFV infection is assumed, in the era of ASFV presence in China, a farm would expect 19.45% loss compared with 6.07% baseline loss without major disease impacts. ASFV itself, with 30% chance of infection, contributing 15.2% in the overall expected loss rate. www.international-animalhealth.com

Discussions This simulation model assuming different test and removal strategy success levels used in this paper, empirically, can be applied to field classic virulent ASFV infection cases, because of reasons such as the slowness of transmission of ASFV via direct contact of infected pigs or via contaminated fomites, and the onsets of observable clinical signs such as reduced feed intake, loss of appetite, lethargy, skin reddening, vomiting, fever, propensity to lie down, in some cases bloody faeces etc. The success hinges on early detection of infected pigs without omissions, and ensure that the infected pigs, at risks ones and the manure being removed in a biosecure way, usually the pigs being euthanised and wrapped in plastic film in the infected pen to avoid further spread of liquid, or pigs evacuated via a sealed corridor (microenvironment sealed with plastic film to make leakage impossible), and contact surfaces being thoroughly cleaned and decontaminated to avoid residual viruses on the surfaces and avoid introduction International Animal Health Journal 9

REGULATORY & MARKETPLACE

of pigs until there is evidence that no viable virus or DNA of ASFV is still present in the barn. Scientists have found that animals infected with the strains of reduced virulence may cause latent infections, or subclinical signs or showing no clinical signs,5 coupling with the facts that the pigs may intermittently shed the virus via oral-nasal routes, making it a challenge to identify the infected pigs. Thus, can make eliminating the lower virulent viruses via test and removal difficult. The sustainable success of a farm depends on its ability to prevent entry of viruses into the farm by enforcing strict biosecurity measures and supporting people to comply with it and do right things all the time. Doing the right things one day, or 7 days is not enough, and the viruses do not take a break on the Sundays. In our interviews with pig practitioners who had worked on ASFV containment with test and removal approach, their investigations frequently pointed to carrying of virus into farms by workers, because of the improper contact with transport trucks, couriered goods which had exposed to ASFV contaminants, or was contaminated by contacting with pork harbouring the virus. As a result, no matter the infection pressure is high or low, a farm needs to have a mindset to enforce good biosecurity practices consistently and compliances should be monitored all the time, and being reported, and gaps being closed in a responsive manner to avoid ASFV entry. Acknowledgement Thanks for Dr. Zhi Lai who provided insights on the characteristics of ASFV strains of different virulence, and Dr. Butch Baker and Dr. Dan Tucker for reviewing this paper. 10 International Animal Health Journal

REFERENCE 1. 2.

3. 4.

Jeffrey Zimmerman et al., Diseases of Swine. African Swine Fever Virus, p 443 Sun, E., Zhang et al., Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020. Sci China Life Sci 64, 752–765. https://doi. org/ 10.1007/s11427-021-1904-4 Jiancong Yao et al., A Case Study: Use Test and Removal Strategy to Contain an ASFV Outbreak in a Farm. International Animal Health Journal 40, volume 7 issue 1 Jiancong Yao et al., Successful Depopulation and Reopening of an Isolation Facility within a Large Production Compound without Allowing ASFV Spread to the Main Breeding Herd. International Animal Health Journal 44, volume 8 issue 1 Wales and Davies, Disinfection to control African swine fever virus: a UK perspective. Journal of Medical Microbiology 2021; 70:001410

Dr. Jiancong Yao Dr. Jiancong Yao currently works with PIC as its Health Assurance Director for Asia region. He had studied virology and holds a Master Degree in Preventive Veterinary Medicine from China Agricultural University; he also holds Master degrees in Agricultural Economics and in General Management, each from University of Purdue and the University of Indiana. Dr. Yao have authored numerous articles on swine health management subjects.

Email: miles.yao@genusplc.com

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International Animal Health Journal 11

RESEARCH AND DEVELOPMENT

Horses Used for Large-Scale Production of Immunoglobulins: An Inter-Species Approach Abstract This paper seeks a better understanding of the use of horses as serum producers for the pharmaceutical industry over the last 120 years. We discuss procedures and techniques applied in the production of conventional antivenoms, breaking the customary silence on the millions of horses that have been essential in the historical developments of scientific institutions, such as the Pasteur Institute, in France, and the Butantan Institute, in Brazil. By examining selected literature on the modern and contemporary history of antivenom therapy, we highlight prospects for the future, and in particular of the next-generation of antivenoms that eliminate the use of equines. Introduction The use of horse-derived plasma in large-scale production of immunoglobulins is currently adopted worldwide for treatment of various infectious diseases, as tetanus, rabies, botulism, and as the only treatment available for envenoming by several species of snakes, scorpions, and spiders.1 Beginning in 1894, in Europe and across the world, a revolutionary treatment to cure diphtheria, a widespread disease with high mortality rates that mainly affected children, wrote heterologous serotherapy into the history of modern medicine, through the contribution of renown scientists as von Behring, Kitasato, and Roux.2,3 In the hyperimmunisation process, horses are inoculated with several doses of a specific antigen (e.g. toxin) that induces an active immunological response in them by creating a level of antibodies high enough to neutralise the antigen lethal effects. When the horse serum presents high antibody levels (i.e. satisfied antibody titles), the animal is bled to extract the antibodies from the plasma.4 Seeking for new angles in the history of serotherapy, from an integrative viewpoint of the humanities and the health sciences, we review the routine of the early equines used as living source of antibodies, in France of the late 19th century, and in Brazil of the 20th century. To the extent that today’s hyper-immune equine plasma production system remains so similar to the procedures adopted 120 years ago, early records may be used to disclose aspects of the past use of equines by the pharmaceutical industry and to prospect further considerations on current large-scale production system. Concerning its contemporary constraints, we also shed light on the limits and risks associated to the induced-adverse effects of heterologous immunoglobulins, prospecting trends and expectations regarding novel envenoming therapies. It is not the use of animals in therapeutics that distinguishes modern medicine from the healing methods of antiquity or traditional practices. In fact, animals have long been associated with some sort of power in benefit of human health. Over the early decades of the 20th century, the Brazilian pharmaceutical industry offered a wide range of biological medicines. In 1920, the pharmaceutical repertory of the Vital Brazil Institute included 29 different types of horse therapeutic sera, and 31 opotherapics, which were pills composed of 12 International Animal Health Journal

desiccated spleen, liver, ovary, mammary gland, red blood cells, and thyroid gland.5 In late 19th century, even in Europe, the medical industry worked closely with slaughterhouses to obtain such raw material.2 As a significant contribution to the history of animals in modern medicine,6 the challenges and trends resulting from intense and critical use of equines as living producers of antitoxins confront us with ethical concerns, as well as with the risks and limits of pharmaceutical products derived from horses. It is a history that has been largely ignored by those who, in other ways, have so fervently sought a better understanding of the multiple entanglements involving horses and human societies. “At the cost of giving up all their blood…”2 Jonathan Simon2,7 brings elucidative benchmark on the participation of equines on the Pasteur Institute, in Paris of the late 19th century. The early experimental use of horse-based antitoxin to treat ill children was carried out in early 1894, and consisted in the immunisation of horses with recovered bacteria from diphtheria infected children. These horses were given the names of children who had passed through the wards of the Hôpital des Enfants Malades. Stables became laboratories used for performing the novel routine of largescale serum production. As experimental subject, each individual horse, as a manufacturing unit, was submitted to long-term and large-scale verification of several events aimed at techniques for maximising production of both the quantity and quality of the heterologous sera. In 1894, the national newspaper Le Figaro announced that: “if you go to the Pasteur Institute, at the foot of the garden, you can see around ten young cab horses between six and nine years old. They are comfortably installed in their stalls, marvellously well cared for and groomed, admirably fed, and can't possibly miss their exhausting billets in Batignolles or Montrouge, to which fate had initially destined them” (7:4). Considering the brutal reality of the horses explored in the streets of European cities, this scenario became partially true, thus comprising suitable publicity that was motivating generous donations in a national fund-raising campaign for the new treatment. In 1894, a retired Thoroughbred, Salifou, who had previously enjoyed success at the racetracks, was donated to the Institute's diphtheria service. It was the first time that a Thoroughbred horse was admitted to the Institute stables. The symbolism of such initiative expressed a healthy philanthropic competition among the French bourgeois. Yet the fate of these horses was better captured by the literal words of the physiologist Émile Roux, who in the face of the urgency that led to increasing demand for serum and the fact of its slow production process, exclaimed, “our poor animals, even at the cost of giving up all their blood, would not be capable of furnishing one hundredth of the required quantity” (2:69-70). In early 1895, the Pasteur Institute had about 136 horses, and a production of over 7,000 litters of blood. The true fortune of these animals would be better expressed later by the French writer, Marguerite Yourcenar (8:144): "the handsome steeds of the Garde Républicaine when they are old and broken, sent away to die, sometimes over a period lasting as long as two years, in a stall of the Institute Pasteur, where their sole diversion is to be bled (…), until Volume 8 Issue 4

RESEARCH AND DEVELOPMENT finally, void of blood, they crumble, equine tatters which are the victims to our progress in immunology. The men of the Garde themselves exclaim that they would much rather see them sent straight to the butcher shop.” At that time, the new and vast field of antivenom therapy has also been developed by French scientists, as Calmette, Phisalix, and Bertrand, to face envenoming snakebites, a common threat to human survival in the tropical colonial territories. Concomitantly, studies developed by the Brazilian doctor, Vital Brazil, rectified Calmette’s principle of polyvalent serum, by demonstrating that an antivenom would only be effective against the same venom (or at least against the same snake genus) that had caused the envenoming.4 Bleeding as an efficient matter It is widely known that the first Brazilian public serotherapy institutions, such as the Butantan Institute, founded in 1901, were inspired in the technical expertise of the Pasteur Institute.9 Over the first decades of the 20th century, Vital Brazil was the head of the two major Brazilian serum manufacturers, the Butantan Institute, and the Vital Brazil Institute. In the Brazilian scientific literature, we found descriptions of experimental procedures which pushed the limits of horse’s physiology. In this sense, the early death (usually from liver disintegration, in consequence of amyloidosis), hyper-immunisation process performed by Vital Brazil for subcutaneous inoculation of pure scorpion venom into horses, is emblematic. Over a three-month period, twenty-four injections totalling an amount of venom equivalent to 1,512 scorpion bites was inoculated into one horse, an animal considered to be extremely sensitive to even small amounts of it. The bleeding was performed 11 days after. The horse’s reactions throughout the immunisation process were described in detail: “With each injection, [the horse] demonstrated intense reaction to pain, showing widespread trembling, breathlessness, nasal and tear hypersecretion, a rise in body temperature, intense sweating. Such symptoms lasted no more than 12 hours” (10:49).

Vital Brazil11 observed that horses do not always react in the same way to the induction of antitoxin production. So, rest may prove beneficial, while in others, after a few years of immunisation, the animal becomes a poor producer of antibodies, reacting little to the toxins. This matter was further addressed in a study that emphasised the hyperimmunisation as a concern of economic efficiency.12 Physiological constraints, as weight loss, unresponsiveness to increased feeding, and rest after immunisation, sudden death (usually from liver disintegration, in consequence of amyloidosis), cardio-hepatic-renal disorders caused by vast subcutaneous edemas, and fractures due to the accidental falls taken by weakened animals, indicated that the horse would be apt to be submitted to ‘total bleeding’. So, when the risk of animal’s death would represent consequent loss of a significant amount of blood for the serum production supply, total bleeding was performed to guarantee whole blood extraction. The method consisted of inoculating the animal with a saline solution that kept circulatory mechanics going, delaying haemorrhagic shock to allow the animal to survive until all the (diluted) blood could be withdrawn, in a procedure that could take as long as two days. This study also presents one of the rare records on deaths of horses used as serum producers by the Butantan Institute, during the years 1947 and 1948, (Figure 1). From the 1970s, some refinements in the process of hyperimmunization guaranteed an increasing of horse’s survival. Nonetheless, the continuous process of injection of toxins imposes critical and ruthless consequences on the horse physiology and welfare which have been conveniently obscured by the industry of heterologous immunoglobulins. Currently, the lack of transparency of the Brazilian serum producer institutions can be verified from the absence of data available on the amount and origin of these animals, mortality rate, and destination for the horses unresponsive to the hyperimmunization, between other aspects related to their management. Is noteworthy that considerations on the welfare and ethical compliance

Figure 1. Number of horses that died at the Butantan Institute, over the years 1947 and 1948 (12:282). The red columns present the number of horses that underwent total bleeding, and the brown columns present the number of horses who died without total blood extraction, in 1947 (A) and 1948 (B). The study did not explain the increasing of total bleeding, in 1948. www.international-animalhealth.com

International Animal Health Journal 13

RESEARCH AND DEVELOPMENT

Figure 2. Disadvantages of horse-derived serumtherapy. (A) Patients may develop early adverse reactions (within 24 h) resulting from exposure to animal antibodies, due to IgE mediated anaphylactic reactions. (B) Horse-derived antibodies combined with elicited human anti-horse antibodies (IgGs and IgMs) may result in high production of immune complexes, which are often deposited in target organs promoting serum sickness (1-2 weeks after administration of antivenom therapy). (C) Horse-derived serum can be contaminated during the production process with endotoxins, which can induce anaphylaxis since endotoxin can directly activate mast cells. (D) Around 70% or more of the horse-derived antibodies are not directed towards the antigen.

concerning humane use of horses in the production in the production of antivenoms was only included in the ‘2016’s Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins’ of the World Health Organization.1 The horse-derived serum and induced-adverse effects In the early 1900s, adverse effects of products derived from horses, such as sera, were described,13 including anaphylaxis, serum sickness, and pyrogenic reactions, (Figure 2). Anaphylactic reactions are known as early adverse reactions (up to 24 h) and can be either IgE-mediated or non-IgEmediated. In the second case, a study demonstrated that 48% of snakebite victims that received horse-derived antivenoms developed anaphylaxis. The main problem of anaphylaxis is the severe anaphylaxis that can be lifethreatening.14 Serum sickness is a late reaction (1–2 weeks after serum administration) caused by immune complexes (ICs) deposition in target tissues (e.g. blood vessels, glomeruli, and joints), mediating inﬂammation.15 Products derived from horse serum and administered as serotherapy are historically the most common cause of serum sickness. The incidence of serum sickness can be expected to be greater than 50% in cases receiving large number of vials of serotherapy.22 Although serum sickness is not life-threatening, it can have multi-organ involvement including acute renal failure.15 14 International Animal Health Journal

Pyrogenic reactions are neither rare. Characterized by contamination of the serum preparation by endotoxin or lipopolysaccharide (LPS), this response also contributes to the development of anaphylaxis since LPS can directly activate mast cells.16 Pyrogenic reactions were described in 27% of snakebite victims after receiving horse-derived serum.14 There are also uncertainty concerning to the heterologous serum efficacy. Most of the antibodies in horse-derived antivenoms are not directed towards venom components but are work against antigens that horses have encountered over the course of their lives (microorganisms, parasites, etc). Thus, most antivenoms contain about 70% of antibodies not directed against venom components, that is, they are not neutralizing antibodies.4 Underlying this significant problem is the question as to whether the remaining 30% of horsederived antibodies targeting the key antigens are 100% neutralising molecules? For instance, the remaining 30% of specific antibodies cannot neutralise important toxins responsible for envenomings.17 Unfortunately, Brazilian scientific study has tapped very little into these accounts of the induced-adverse effects of heterologous antibodies. Within Brazilian epidemiology and snakebite records, the occurrence of adverse effects is a matter that has been entirely ignored within most publications.18 A study demonstrated that the anticrotalid antivenom produced by Butantan Institute, since Volume 8 Issue 4

RESEARCH AND DEVELOPMENT 1901, keeping the same protocol. It also concluded the absolute ineffectiveness of the anti-crotalid antivenoms from Butantan Institute and Vital Brazil Institute antivenoms to immunorecognize crotamine.19 In the aftermath of the publication, the Butantan Institute improved its horse immunisation protocol, including only crotamine positive rattlesnake venoms. Yet, no study has yet been conducted to evaluate its change. Finally, a structural aspect of heterologous antibodies pertained to the fact that horses are not a unit of production, but a biological system with a significantly diverse genetic and immune system which cannot provide a guarantee for uniform product quality at the end of the manufacturing process. Such constraint outlines continuous challenges faced by manufacturers to ensure the effectiveness of serum quality. The continuous increase in regulatory standards for biological medicines products has imposed real hindrances to ensure the supply of national (and global) demand for animal-derived immunoglobulins.1 The next-generation serum therapy: overcoming resistances in a current reality Very limited efforts have been done to change the ancient use of horses as an antibody machine, especially in the ﬁeld of antivenoms. Nevertheless, there were many progresses in antibody discovery technologies, antibody engineering approaches, and antibody manufacturing. The development of monoclonal antibodies (mAbs) was first described in 1975, with the discovery of the hybridoma technique. Although the hybridoma technique was described in 1975, only 10 years later, in 1985, the first monoclonal antibodies were released for therapeutic use in humans. In the last years, the pharmaceutical market with monoclonal antibodies had a remarkable growth.3 With 136 mAbs already marketed in 2021, there is an expectation of more than 200 in 2022. Indeed, mAbs (whether murine, chimeric, humanised or human) in

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the pharmaceutical market is a reality, being used as the most advanced therapies for the treatment of a diversity of diseases (e.g. cancer, allergy, autoimmune diseases, etc). The current global therapeutic monoclonal antibody market was valued at approximately US of $150 billion in 2019 and is expected to generate revenue $300 billion by the end 2025.20 Even so, with many investments and mAbs available, many horse-derived sera are still used in therapy. Although we do not have any recombinant antivenom available, composed by a mixture of monoclonal antibodies, researchers from all over the world have been working in the field. There is a vast literature exploring basic research and pre-clinical tests with mAbs targeting venomous animals’ toxins. So, further steps are expected to renew focus on snakebite as a neglected tropical disease from research efforts within novel envenoming therapies. The way forward to overcome structural limits of the conventional serotherapy can be uncovered into the contemporary developments of safer, healthy, and ethical therapy. The biopolitics of equine blood has led to the establishment of historical agreements between scientists, governments, industries, and societies. The Brazilian largescale production of heterologous antibodies has been characterised by recurrent institutional and economic crisis, shortage, increasing the vulnerability of population in remote regions.21 Yet horses themselves have been part of the constellation of circumstances, expectations, and experiments which define both the scope and limits of the serotherapy over time. We present here a critical approach on the past and present production system of heterologous antivenoms, by examining structural harms specifically related to the centrality of the equine physiology in the serotherapy industry. The intense worldwide demand for hyper-immunized plasma has written a crude trajectory of horse exploitation which are still not entirely dimensioned.

International Animal Health Journal 15

RESEARCH AND DEVELOPMENT Rashes. In:Zaoutis, L.B. & Chiang, V.W.(ed) Comprehensive Pediatric Hospital Medicine, Mosby:1005-1011. 14. Stone, S.F. et al(2013) Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation, Cytokine Production and Mast Cell Degranulation. Plos PLoSNegl.Trop.Dis 7(7):e2326. https://doi.org/10.1371/ journal.pntd.0002326 15. LoVecchio,F. et al (2003) Serum Sickness Following Administration of Antivenin (Crotalidae) Polyvalent in 181 Cases of Presumed Rattlesnake Envenomation. Wilderness & Environmental Medicine,Vol14,Issue 4:220-221. https://doi. org/10.1580/1080-6032(2003)14[220:SSFAOA]2.0.CO;2. 16. Mo X,Y.C. et al.(2012) Lipopolysaccharide enhances FcepsilonRI-mediated mast cell degranulation by increasing Ca2+ entry through store-operated Ca2+ channels: implications for lipopolysaccharide exacerbating allergic asthma. Exp Physiol 97:1315–1327. 17. Boldrini-França, J. et al (2010) Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management.Journal of Proteomics,Volume 73, Issue 9:1758-1776.https://doi.org/10.1016/j.jprot.2010.06.001. 18. Bochner, R. The international view of envenoming in Brazil: myths and realities. J Venom Anim Toxins Incl Trop Dis 19, 29 (2013). https://doi.org/10.1186/1678-9199-19-29 19. Herrera,L.G. et al (2013) Pathogenic mechanisms underlying adverse reactions induced by intravenous administration of snake antivenoms. Toxicon. Dec15;76:63-76. doi: 10.1016/j. toxicon.2013.09.010. 20. Lu, R.M. et al.(2020) Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27,1.https://doi. org/10.1186/s12929-019-0592-z 21. Fan, H.W. and Monteiro, W.M.(2018) History and perspectives on how to ensure antivenom accessibility in the most remote areas in Brazil. Toxicon 151(2018):15–23. 22. Dart RC, McNally J. Efficacy, safety, and use of snake antivenoms in the United States. Ann Emerg Med 2001;37:181–8.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

World Health Organization (2016) WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. WHO Press: Switzerland. Simon, J. (2007) The origin of the production of diphteria antitoxin in France, between philanthropy and commerce. Dynamis, 27: 63-82. Pucca, M.B. et al (2019) History of Envenoming Therapy and Current Perspectives. Front. Immunol.| https://doi. org/10.3389/fimmu.2019.01598 Laustsen, A.H. et al (2018) Pros and cons of different therapeutic antibody formats for recombinant antivenom development. Toxicon 146: 151-175. Castro, A.J.W. (2011) Documentos contam a história do Instituto Vital Brazil. Ed. Rio Books: Rio de Janeiro. Woods, A. et al (2018) Animals and the shaping of Modern Medicine: one health and its histories. Palgrave-MacMillan: University of Manchester, UK. Simon, J. (2008) Monitoring the stable at the Pasteur Institute. Science in Context, vol 21, Issue, 2: 181-200. Yourcenar, M.(1992) That might sculptor, time. NY, Boston Public Library. Dias, C.E.S.B. (2010) As relações Brasil-França na criação do Instituto Butatan. In: Cadernos de História da Ciência – Instituto Butantan – vol. V (1) jan-jul 2010: 108-127. Brazil, V. (1918) Soro Anti-escorpiônico. In: Memórias do Instituto Butantan, tomo 1, fasc 1:pg 47-52. Pereira Neto, A. F. (Org) 2002. Vital Brazil: obra científica completa. Niterói, Instituto Vital Brazil. Vaz,E. & Araújo,P.(1949) Da sangria de animais de imunização. Memórias do Instituto Butantan,21:275-298. Bernard,L.A. et al (2007) Chapter 159 - Drug-Associated

16 International Animal Health Journal

Ana Lucia Camphora Ana Lucia Camphora has degree in Psychology, Master’s in Psychosociology of Communities and Social Ecology, and PhD in Social Sciences. As independent scholar, investigates historical interlinkages between animals and human societies. Her current investigations are oriented to modern and contemporary equine cultures. The author thanks the Center for Contemporary Equine Studies for the support received for funding this research. Email: alcamphora@gmail.com

Manuela Pucca Manuela Pucca holds a bachelor in Biomedicine, MS.c and Ph.D. in Immunology at the University of São Paulo, Brazil. She is a full professor at the Federal University of Roraima, and her research is focused on Immunotoxinology, on human recombinant antivenoms and bioprospection of venom-derived peptides. She leads clinical projects aiming to understand the severity of snakebite envenomings in Roraima. Web: www.snakebiteroraima.com

Volume 8 Issue 4

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International Animal Health Journal 17

RESEARCH AND DEVELOPMENT

A Case-Control Study: Comparing the Effects on Calf Health After Use of A Commercially Available Mycoplasma bovis Vaccine in Dairy Herds in Scotland

Background Mycoplasma bovis is a common cause of bovine pneumonia in calves. Treatment and control of the disease is challenging. Treatment can be ineffective and control is hampered by a lack of a licensed vaccine in Europe. Results This retrospective observational case-control study looked at evaluating, mortality and antimicrobial usage for 932 calves born into 4 herds over 2 time periods – before and after using a 3-strain M. bovis vaccine (Myco-B One Dose, American Animal Health, Grand Prairie, Texas, USA) programme in commercial herds in the UK which demonstrated previous prevalence of M. bovis. The vaccinated groups demonstrated a significant reduction (P< 0.02) in post-weaning mortality in calves aged 70200 days old. No significant changes in pre-weaning or total mortality were observed. A significant reduction in antimicrobial usage post-vaccination (P< 0.05) was observed when compared to 2 control farms with qualifying data. Conclusions Due to study design weakness and data gathering insufficiencies in a commercial setting, the significant reductions in post-weaning mortality and antimicrobial usage can only be broadly suggestive of an effect of the vaccine in this preliminary case study. The study does outline the proof of concept for the vaccine’s commercial use in the UK. Further work on assessing the effect of this vaccine in UK cattle herds infected with M. bovis respiratory disease is warranted. Keywords Mycoplasma bovis, vaccine, dairy calves, mortality, antibiotic reduction Introduction Mycoplasma bovis (M. bovis) is a significant bacterial pathogen of cattle with rising significance in the UK (APHA 2020, Burr 2018). It has recently been cited as the most commonly identified pathogen in bovine respiratory disease diagnoses (APHA 2020, SRUC 2019). Morbidity and mortality rates from pneumonia outbreaks in calves have been reported up to 74% and 30% respectively (Maunsell 2009, Mahmood and others 2017). M. bovis infection can cause acute disease and create chronic carrier animals. Clinical signs are varied: pneumonia, septic arthritis, otitis media, mastitis and less commonly meningitis, caesar wound seromas, keratoconjunctivitis and subfertility (Calcutt and others 2018, Maunsell and others 2011, Nicholas 2011, Nicholas and others 2006). Dudek and others (2013) found M. bovis infection facilitated systemic distribution and the immune system became ineffective in clearing M. bovis from the host. M. bovis spread in an endemically infected herd can be via aerosol, colostrum or milk (Dudek 2020). Calves can be infected from a young age: in one study nasal swabs from calves and vaginal swabs from their dams found 40% were positive for M. bovis at 4 18 International Animal Health Journal

days of age, suggesting possible pre or peri-partum infection (Stipkovits and others 2001). Treatment of M. bovis is unreliable, treatment failures are high and increasing antimicrobial resistance patterns have been documented over the last 30 years (GautierBouchardon and others 2014). M. bovis has no cell wall rendering penicillins and cephalosporins ineffective and M. bovis also has natural resistance to other antibiotics including trimethoprim-sulphonamides (Lysnyansky and Ayling 2016). Confirmation of the ability of M. bovis to produce a biofilm helps explain the lack of response to antibiotics and the hosts’ immune system (McAuliffe and other 2006). Bovine mycoplasmosis is a challenging disease to firstly identify and then control. Bacterial culture can be unreliable, time-consuming and be affected by faster growing polymicrobial infections resulting in poor bacterial recovery rates (Calcutt and others 2018). Control methods of infection include extended antibiotic courses metaphylactically (Williams 2010), segregating and culling infected animals (Nicholas and others 2016) and pasteurising colostrum (Gille and others 2020). Herd biosecurity to prevent disease incursion for M. bovis negative herds has been recommended – either a closed herd policy (Calcutt and others 2018) or screening added replacements (Nicholas and others 2016). Autogenous vaccines have been used to prevent M. bovis infection, some successfully in finishing cattle (Nicholas and others 2019), whilst others have demonstrated higher rates of morbidity than the control groups when used in young calves (Maunsell and others 2009). In the UK autogenous vaccines may only be manufactured with recovered isolates epidemiologically linked to the site where the vaccine is to be used (Ridgeway Biologicals 2020). Isolation of M. bovis can take several weeks in some cases (Nicholas 2011). Autogenous vaccine manufacture takes 8-10 weeks followed by an on-farm safety test at the herd of origin for every batch manufactured (Ridgeway Biological 2020). Good results can be achieved when vaccines are used correctly and other BRD pathogens are either well controlled or not present (Nicholas and others 2019). The author is unaware of any published data on the efficacy of US manufactured commercial M. bovis vaccines, none of which are licensed for use in very young calves (Nicholas and others 2006), (Maunsell and others 2009). Although they have been licensed for some time in the US, they have not been available in the UK, until now. In April 2019 the author acquired authorisation from the Veterinary Medicines Directorate to allow importation of Myco-B One-Dose (American Animal Health, Texas, USA), a 3 strain M. bovis bacterin vaccine into the UK. The first use of the vaccine in the UK was in this study. Therefore the objectives of this study were to evaluate this commercial vaccine under commercial UK field conditions in terms of: safety, ease of incorporation into pre-existing vaccine protocols on commercial UK dairy farms (i.e. ‘proofof-concept’), reducing mortality and reducing antimicrobial use in calves up to 200 days old. Volume 8 Issue 4

RESEARCH AND DEVELOPMENT Materials and Methods 2.1 Herd Selection and Vaccine Acquisition The selection criteria for enrolment included: a previous herd history of calf pneumonia in dairy calves confirmed or clinically suspected of M. bovis and a willingness to participate in the observational study. Only herds with uniformly positive serological findings from 5 home bred calves over 5 months of age (tested positive by M. bovis serology, SRUC, Aberdeen, UK) or confirmed M. bovis infection in calves via bacterial culture or PCR were selected. Maternal antibody is likely to make a significant contribution to the titres in animals aged less than 4 months and may inhibit seroconversion (Geraghty, personal communication). Three dairy herds from North East Scotland were enrolled in the study following positive calf serology screening results: DH, TY, and TB. A fourth dairy farm, CU, was enrolled 2 months later after M. bovis was confirmed in a clinical outbreak by PCR (M. bovis PCR SRUC, Aberdeen, UK) in pre-weaned calves. Most pre-vaccination calves (time period 1) in this study were born in the ‘winter’ (October/November to April). Most post-vaccination calves (time period 2) were born in the ‘summer’ (June to Oct). To offset this confounding timepoint difference control farms datasets were included in the study. Four farms of comparable management practices and size were selected. Two tested positive for M. bovis during the course of the study (PCR and serology – PG and MK respectively), two were not tested for M. bovis presence (CS and CG). Pre-vaccination absolute mortality risk data (time period 1) analysis showed no significant difference (P = 0.3703) to reduce any herd selection bias effect. All 8 farms were located within the same region to account for differing geography and seasonal weather variations. 2.2 Study Populations Vaccinating Farms All 8 farms made no material changes to their respective health plans during the 2 time periods. Farm

Milking Herd size

Cow Management

Cow Dry Off vax

Calf Rearing

RSV/ PI3 vax @ 10d

M. haem + H. somni vax @ 4wks

170

Summer grazed

Yes

Buckets

Yes

280

Housed

Yes

Machine

Yes

140

Summer Grazed

Yes

Buckets

Yes

240

Housed

Yes*

Machine

Yes

MK*

260

Housed

Yes

Machine

Yes

CG*

180

Housed

Machine

CS*

240

Housed

Machine

PG*

170

Summer Grazed

Yes

Machine

Yes

Table 1: Farm Management Summary (* - control farms)

Housed – all-year round housed milking/dry cows, summer grazing from May till September, otherwise housed. Dry cow vaccination – Rotavec Corona vaccination (MSD Animal Health UK Ltd, Milton Keynes, UK) given at time off dry off, 8 weeks pre-calving. * CU gave dry cow vaccinations at 4 weeks pre-calving. Calf-Rearing – buckets – paired calves reared on twice daily milk feeding via buckets with teats before weaning into larger groups. Machine – individually reared for up to 10 days before added to a larger rearing group fed by milk machine with an www.international-animalhealth.com

identification collar for each calf. RSV/PI3 vaccination – Rispoval Intranasal (Zoetis UK, Leatherhead, UK) used routinely from 10 days of age in all calves. M. haem + H. somni – Hipra Bovis Somni Lkt (Hipra UK Ltd, Nottingham, UK) at one month old. 2.3 Study Design and Reactive Changes All cows at drying off (or 4 weeks pre-calving – farm CU only) received vaccination with 2mls of Myco-B One-dose injected subcutaneously in the neck area, alongside the herds' routine Rotavec Corona vaccination (MSD Animal Health UK Ltd, Milton Keynes, UK). Calves born to these dams were fed dam's colostrum as per each farms' usual management system. These calves then received a Myco-B booster vaccination at approximately 60 days of age as per datasheet recommendation, alongside the calf vaccine plan described in table 1. Calves at farm DH began showing mild clinical signs suspicious of M. bovis at 6–7 weeks of age (unilateral ear droop, nasal discharge, reduced milk intakes and inconsistent responses to treatment) in the first batch of calves born to Myco-B vaccinated dams. In response to this the age at first booster for Myco-B at all vaccinating farms was immediately reduced to 4 weeks. No other changes were made in routine herd management on either vaccinating or control farms. 2.4 Study Data Collection Mortality data was collected for pre-vaccination calves born on all farms between October to April (time period 1) and for post-vaccination calves born (time period 2) between June to October. Calves were observed until 200 days old. Farm CU was enrolled in July. Farm CU’s post-vaccination data was collected from July to October. Farm records supplied for individual calf treatment records and mortality due to suspected pneumonia could not be used due to substantial inconsistencies. Therefore, all mortality records for calves born in each cohort observation period and followed up to 200 days old for each calf, were taken from official farm movement records supplied by Scot EID, Huntly, UK. The control farms were recorded in the same way. Antimicrobial sales recorded by the author's practice to both vaccinating and control farms were analysed. The four vaccinating farms and two of the control farms had specific calf pneumonia treatments that were not used on other classes of stock and were sold at regular intervals onto farms making them suitable for recording and analysis. Volumes of these direct sales of antimicrobials during each of the two time periods were recorded and converted to a population correction unit/100kg body weight, representing the approximate average liveweight of calves throughout the observation periods (PCU= mg of antimicrobial/100kg bodyweight). Two farms, CG and CS had unusual medicine bulk-purchasing activities rendering this method of data analysis unusable and were judged as non-qualifying. On at least one occasion during the study, on all vaccinating farms only, up to 6 calves aged 2–7 days old were blood sampled for total protein levels. This was to assess the level of passive immunity, in line with their usual monitoring practises, outlined in their existing health plans. All farms had at least 1 calf measured at below the recommended level of 5.6g/dl TP (MacFarlane and others 2015). 2.5 Statistical Analysis Mortality data was assessed on relative risk of mortality International Animal Health Journal 19

RESEARCH AND DEVELOPMENT before and after vaccine use (time period 1 – pre-vaccination, time period 2 – post-vaccination). A Kruskal-Wallis Rank Sum Analysis was performed on pre-weaning, post-weaning and overall relative mortality risks.

Figure 1 shows individual weaned calf mortality as % of calves born during each recording period pre and post vaccination. All vaccinating farms had reduced post-weaning mortality; all controls had similar or increased mortality.

Antibiotic usage was analysed as an absolute usage change with a Student’s Independent T-Test two-sample assuming unequal variances.

Table 1. Kruskal Rank Sum Test Analysis of mortality risk across all farms during both time periods (TP). TP1 describes time period 1, before any vaccine used. TP2 describes time period 2, when vaccinating farms began using the vaccine. When the relative risk of mortality is equal to 1 then no difference in risk has been observed pre and post vaccination. If the relative risk is below 1 then a reduction in mortality has been observed.

A Post-Hoc Dichotomous Endpoint, Two Independent Sample Study Power Calculation yielded a power of 0.937 for calf mortality during time period 2, with p<0.05. Results 1. Safety No adverse reactions were recorded when using Myco-B One-Shot on any of the vaccinating farms in either cows or calves. 2. Incorporating Myco-B One-Shot into existing Vaccine Plans During the time each farm remained enrolled in the study all farms successfully completed the vaccine protocols routinely and timeously in both cows and calves. Farmers continued to use the vaccine after the observation period of the study had been concluded, possibly confirming Myco-B’s ease of use. Calf Mortality

Figure 2: Box and Whiskers of Post-weaning Mortality significant reduction on post-weaning mortality on vaccinating farms p <0.02

Figure 1: 4 vaccinating farms on the left, control farms on the right

Post-weaning mortality risk showed a significant reduction after vaccination. There were no other significant changes in risk of mortality after vaccination.

Farm

Mortality Stage

Vacc

Calves TP1

TP1 Mort

Risk TP1

Calves TP2

TP2 Mort

Risk TP2

Relative Risk

Prewean

106

0.009

0.024

2.67

Prewean

192

0.021

176

0.045

Prewean

113

0.035

0.09

2.57

Prewean

123

0.049

Prewean

122

0.033

101

0.02

0.61

Prewean

0.035

0.031

0.89

Prewean

0.021

0.145

6.9

Prewean

0.063

Postwean

106

0.057

Postwean

192

0.042

176

0.01

0.25

Postwean

113

0.088

Postwean

123

0.057

Postwean

122

0.049

101

0.04

0.82

Postwean

0.105

0.154

1.47

Postwean

0.094

0.091

0.97

Postwean

0.063

0.131

2.08

Table 2: mortality data before and after vaccination on all farms Relative risk = <1 = reduction in risk of mortality in second time period >1 = increase in risk of mortality in second time period, TP = Time Period 20 International Animal Health Journal

Volume 8 Issue 4

RESEARCH AND DEVELOPMENT Antimicrobial Usage Calf Pneumonia Antibiotic Use*

Figure 3: Calf pneumonia antibiotic usage as population correction unit/100kg liveweight (approximated average weight throughout observation periods) Significant reduction in post-vaccination antibiotic use on the vaccinating farms (p<0.05) compared to non-vaccinating farms with qualifying data.

Reduction in antibiotics after vaccination compared to 2 control farms using an Independent T-Test two-sample assuming unequal variances saw a significant reduction (P <0.05). All vaccinating farms showed a reduction in the calf pneumonia antibiotic use PCU/100kg of calf pneumonia specific antimicrobials purchased by farm per live calf born during observation period.

and others 2020). Seasonal calving herds have seen a higher bulk tank PCR M. bovis presence 5-8 weeks after starting calving suggesting the post-calving period is a higher risk time for M. bovis shedding (Parker and others 2017) so potentially important to target in a vaccination programme. Further work with this vaccine on assessing the transfer of colostral M. bovis antibodies to neonatal calves and reduction in M. bovis shedding in colostrum is necessary. However, Maunsell and others (2009) showed no correlation with higher levels of M. bovis specific post-colostral antibodies and reduction in clinical signs associated with M. bovis infection. Pre- and post- calf vaccination M. bovis serum antibody levels would have been of interest especially given the ‘off-label’ use of the vaccine at 4 weeks of age complicated by the potential interference of maternally derived antibodies. The study design and data gathering were sub-optimal because all calf mortalities recorded on official movement records had to be utilised, therefore non-respiratory causes of death were included. This was due to a lack of reliable cause-of-mortality data from all the farms involved. However, one study showed the most common cause of Holstein heifer mortality between 101 and 160 days was respiratory disease (Zhang and others 2019). This would suggest the reduction seen in this current trial in this comparable age group (time period 2, post-vaccination) was due to a reduction in respiratory disease. Better data gathering for farmer-recorded cause of mortality alongside on-farm post-mortems and pathogen identification would have been helpful to confirm this. The significant reduction in antimicrobial use needs to be analysed in context. The author is unaware of any standardised PCU for rearing/weaned calves so the arbitrary weight of 100kg throughout the 2 times periods was used. The AMU measurements were antimicrobials prescribed by the author’s practice to each farm during the 2 observed time periods, classed as calf-pneumonia specific as laid out in each farms’ existing health plan. The precise time of use and which group received the medicines were not made available for the study. A period of overlap existed from May till October when either cohort could have received pneumonia treatments, potentially reducing the drop in AMU seen post vaccination.

Figure 4: Antibiotic usage on vaccinating and non-vaccinating control farms

The four vaccinating farms had a combined antibiotic usage reduction of 70.2% against a 33.9% rise in antibiotic usage on the combined non-vaccinating control farms with qualifying data. Overall Results The commercial 3-strain M. bovis bacterin vaccine assessed in this study caused no adverse reactions, was easily incorporated into different pre-existing vaccination protocols and was effective in reducing post-weaning mortality as well as antimicrobial usage on the vaccinating farms. Discussion The vaccination programme used in this study was ‘off-label’ from the supplied datasheet (calves mostly vaccinated at 30 days of age, instead of recommended 60 days). Dams were vaccinated during the dry period to potentially prevent M. bovis infection of the calf before birth (Stipkovits and others 2001) and reduce the shedding of M. bovis and potentially increase M. bovis specific antibodies in the colostrum (Gille www.international-animalhealth.com

Previous M. bovis vaccine studies have recorded individual calf records (Maunsell and others 2009, Nicholas and others 2019). However, these studies observed 373 and 141 calves respectively. This study observed 1581 in total, including 398 vaccinated calves. More accurate recording of calf medications and morbidity would have been beneficial in this study. Previous vaccine studies have been based on single strain bacterin preparations (Maunsell and others 2009, Nicholas and others 2019). Using a multiple strain vaccine, as in this trial, has previously been suggested as a way of improving M. bovis vaccine efficacy (Calcutt and others 2018). It has been suggested that vaccine development should be based on conserved recombinant proteins, not bacterinbased vaccines (Perez-Casal and others 2017); trials using recombinant protein vaccines have been unsuccessful to date (Prysliak and others 2018). This would suggest that currently bacterin-based vaccines, such as Myco-B One-Shot, provide the most promise for effective vaccination against M. bovis. The immediate availability of this commercial vaccine may prove useful in giving practising vets another control option International Animal Health Journal 21

RESEARCH AND DEVELOPMENT for M. bovis on commercial UK dairy farms when combatting rising calf mortality rates (Mee 2013) and increasing Dairy AMU in the UK (VMD 2019).

15.

Conclusion Due to study design weakness and data gathering insufficiencies in a commercial setting, the significant reductions in post-weaning mortality and antimicrobial usage can only be broadly suggestive of an effect of the vaccine in this preliminary case study. The study does outline the proof of concept and safety for the vaccine’s commercial use in the UK. Further work on assessing the effect of this vaccine in UK cattle herds infected with M. bovis respiratory disease is warranted.

17.

Acknowledgements Michael Kerby BVSc CertAVP DBR MRCVS of Synergy Farm Health, Evershot, Dorset, UK

16.

18.

19. 20.

REFERENCES 1.

6. 7. 8. 9.

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Animal & Plant Health Agency GB cattle quarterly report; Disease surveillance and emerging threats Volume 23:Q1 January-March 2019 https://assets.publishing.service.gov.uk/ government/uploads/system/uploads/attachment_data/ file/806066/pub-survrep-c0119.pdf Accessed June 03 2020 Burr P, (2018) Bovine respiratory disease what can we learn from current diagnostic tests? Proceedings of the British Cattle Veterinary Association, Leicestershire, UK 18-20 October 2018 26(2):156 Calcutt MJ, Lysnyansky I, Sachse K, Fox LK, Nicholas RAJ, Ayling RD 2018. Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements. Transbound Emerg Dis. 2018;65 Suppl 1:91-109. Dudek K, Bednarek D, Ayling RD, Szacawa E 2013. Immunomodulatory effect of Mycoplasma bovis in experimentally infected calves Bull Vet Inst Pulawy 57 2013, 499-506 Dudek K, Bednarek D, Lisiecka U, et al 2020. Analysis of the Leukocyte Response in Calves Suffered from Mycoplasma bovis Pneumonia. Pathogens. 2020;9(5):E407. Published 2020 May 24. Gautier-Bouchardon AV, et al. 2014. Overall decrease in Susceptibility of Mycoplasma bovis to Antimicrobials over the Past 30 years in France. PLos ONE; 9(2) e87672 Gille L, Evrard J, Callens J, et al 2020. The presence of Mycoplasma bovis in colostrum. Vet Res. 2020;51(1):54. Published 2020 Apr 16. Lysnyansky I, Ayling RD 2016. Mycoplasma bovis: Mechanisms of Resistance and Trends in Antimicrobial Susceptibility. Front Microbiol. 2016; 7:595. Published 2016 Apr 27. Mahmood F, Khan A, Hussain R, et al 2017. Patho-bacteriological investigation of an outbreak of Mycoplasma bovis infection in calves - Emerging stealth assault. Microb Pathog. 2017; 107:404-408. Mee JF 2013 Why Do So Many Calves Die on Modern Dairy Farms and What Can We Do about Calf Welfare in the Future? Animals (Basel). 2013;3(4):1036-1057. Maunsell FP, Donovan GA, Risco C, Brown MB 2009. Field evaluation of a Mycoplasma bovis bacterin in young dairy calves. Vaccine 2009; 27(21):2781-2788 Maunsell FP, Woolums AR, Francoz D, Rosenbusch RF, Step DL, Wilson DJ, Janzen ED 2011. Mycoplasma bovis Infections in Cattle. Journal of Veterinary Internal Medicine25(4): 772-83. McAuliffe, L., Ellis., R. J., Miles, K., Ayling, R. D. & Nicholas, R. A. J. 2006 Biofilm formation by mycoplasma species and its role in environment persistence and survival. Microbiology 152, 913-922 Nicholas RAJ, Ayling RD, Woodger N, Wessells ME, Houlihan MG 2006. Mycoplasmas in adult cattle: bugs worth bothering about? Irish Veterinary Journal Vol 59 (5) 301-304.

22 International Animal Health Journal

21. 22. 23. 24.

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Nicholas RA 2011. Bovine mycoplasmosis: silent and deadly. Vet Rec. 2011;168(17):459-462. Nicholas RA, Fox LK, Lysnyansky I. Mycoplasma mastitis in cattle: To cull or not to cull 2016.Vet J. 2016;216:142-147. Nicholas, Robin & Gr, Loria & Catania, Salvatore & Piccinini, Renata. (2019). Effects of an inactivated vaccine for bovine mycoplasmosis on calves naturally affected with Mycoplasma bovis. Animal Husbandry, Dairy and Veterinary Science. 3. 10.15761/AHDVS.1000161. Parker AM, House JK, Hazelton MS, Bosward KL, Morton JM, Sheehy PA 2017. Bulk tank milk antibody ELISA as a biosecurity tool for detecting dairy herds with past exposure to Mycoplasma bovis. J Dairy Sci. 2017;100(10):8296-8309. Perez-Casal J, Prysliak T, Maina T, Suleman M, Jimbo S 2017. Status of the development of a vaccine against Mycoplasma bovis. Vaccine. 2017;35(22):2902-2907. Prysliak T, Maina T, Perez-Casal J 2018. Th-17 cell mediated immune responses to Mycoplasma bovis proteins formulated with Montanide ISA61 VG and curdlan are not sufficient for protection against an experimental challenge with Mycoplasma bovis. Vet Immunol Immunopathol. 2018;197:7-14. Ridgeway Biologicals - Information for Veterinary Surgeons 2020, https://www.ridgewaybiologicals.co.uk/informationfor-veterinary-surgeons/ Accessed June 03 2020 SRUC: Pneumonia Review April 2019 https://www.sruc.ac.uk/ info/120144/farm_animal_diagnostics/2071/pneumonia_ review accessed June 03 2020 Stipkovits L, Ripley PH, Varga J, Palfi V 2001. Use of valnemulin in the control of Mycoplasma bovis infection under field conditions. Vet Rec. 2001;148(13):399-402. UK Veterinary Antibiotic Resistance and Sales Surveillance Report 2018, published Oct 2019. https://assets.publishing. service.gov.uk/government/uploads/system/uploads/ attachment_data/file/842679/PCDOCS-_1705150-v1-UKVARSS_2018__2019__Highlights_Report_FINAL_v2.pdf Accessed June 09 2020. Williams, M. 2010 The seven deadly sins of Mycoplasma bovis. Veterinary Practice 41, 1-2 Zhang H, Wang Y, Chang Y, et al 2019.Mortality-Culling Rates of Dairy Calves and Replacement Heifers and Its Risk Factors in Holstein Cattle. Animals (Basel). 2019;9(10):730. Published 2019 Sep 26.

Graeme Fowlie Graeme Fowlie is a director of Meadows Vets Centre, a practice based in Aberdeenshire. Graeme lives in rural Aberdeenshire and in between work and looking after his young family has time for the odd game of cricket with the MCC (Methlick Cricket Club) at Lairds. Meadows is a very mixed practice with strong links to the local family farms of the area (which supply many of the current staff, Graeme included). Graeme has always had a strong interest in pneumonia prevention since helping 'dose beasts as a young loon' on the family farm before graduating from Glasgow Vet School. The mycoplasma vaccine project finally got off the ground when Graeme had an unexpected 'break' from clinical work due to a broken leg one winter. Utilising the practice's strong trading links with Kernfarm and with the assistance of the extremely helpful staff at the VMD the successful importation of a US-manufactured multi-strain mycoplasma bovis vaccine to the UK was finally achieved in April this year. Email: thevets@meadowsvets.co.uk

Volume 8 Issue 4

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International Animal Health Journal 23

RESEARCH AND DEVELOPMENT

Massage or Music Meant to Be Relaxing, Result in Lowering Salivary Cortisol Concentration in Race Horses

Summary At the beginning of the training routine, young racehorses are exposed to stressful stimuli. The aim of the study was to evaluate the influence of a relaxing massage which the horses received in the stable, and the influence of music piped into the stable, on the long- lasting stress level of the horses. 120 Purebred Arabian horses were studied. They were examined during the first racing season, which lasted for two years. At the beginning of the study, the horses were 28–31 months old. The horses were brought to Słužewiec Horse Race Track (Warsaw, Poland) from their familiar studs and were randomly assigned to music (n=48), massage (n=48), or control (n=24) groups. All horses were regularly trained and competed in official races. Once a month, saliva samples were collected from each horse to determine the cortisol concentration. Both music and massage resulted in significantly lowering salivary cortisol concentration compared to the control treatment. We see them move to music through Dressage routines. As riders, we partner with our rhythmic friends eliciting their tempos in two and three beats, walking, trotting and galloping, defining the equine species as one of nature’s musicians. The equine hearing range is higher than human hearing. Our human frequency hearing range is 20Hz to 20,000Hz. The frequency hearing range of a horse is 55Hz to 33,500Hz. In fact, horses and humans share the most closely related hearing ranges than any other mammals on the planet. A whisper in their ear from your voice or a personalised whistle from the barn to come in from the field is a significant part of the deep bond with our horses and strong connection through sound. This understanding has been an integral part of the research of Pet Acoustics Inc. with the innovation of a special speaker system that modifies sound disbursement for animal hearing and specifically designed science-based music for dogs, cats and horses by Janet Marlow, internationally recognised composer and Sound Behaviourist. In 2015, a two-year study was initiated by Witold Kędzierski1, Iwona Janczarek, Anna Stachurska and Izabela Wilk of the Department of Biochemistry, University of Life Sciences in Lublin, Lublin, Poland Department of Horse Breeding and Use, University of Life Sciences in Lublin, Lublin, Poland. The study used the Pet Acoustics speaker and Janet Marlow’s equine specific music in the following study. Introduction Young Purebred Arabian horses in Poland are routinely submitted to race training. At the age of 2.5 years, they are moved from familiar studs to an unfamiliar race training centre. Much of the stimuli accumulated at the beginning of the training routine is associated with transport, change of residence, physical effort and participation in races, and can elicit chronic stress in horses (Alexander and Irvine 1998). Moreover, the commercialisation of racetracks alters the living conditions towards circumstances inconsistent with the biology of horses (MacTaggart et al. 2010). Keeping a racehorse in a box for most of the day, results in restricted freedom of movement (Henderson 2007). An unfamiliar 24 International Animal Health Journal

environment, isolation, and short feed intake can also deepen the stress level (Waters et al. 2002). It is known that longlasting stress has a negative influence on the organism. The factors generating stress in trained horses, however, can only be partially eliminated. Therefore, negative factors should be controlled and their effects should be mitigated (Evans 2003). Various relaxing methods may be used, e.g. free movement in the paddocks, massage and music. Massage promotes general body relaxation and increases the sense of an animal's well-being (Scott and Swenson 2009). In horses, the heart rate measured during and immediately after a massage was reduced, and improved behavioural responses were noted (McBride et al. 2004). Horses are generally sensitive to music. The most visible sign of the influence of music on horses is the horse’s ability to synchronise their movement to musical rhythm (Bregman et al. 2012). According to Carter and Greening (2012), the effect of the music on a horse’s behaviour depends on the music genre. Stachurska et al. (2015) showed that relaxation music positively affected the emotional state in racehorses. However, little is known how these kinds of relaxation methods reduce long-lasting stress in racehorses (Scott and Swenson 2009). The most common approach used to evaluate the stress level in horses is measuring cortisol release (Peeters et al. 2010, Schmidt et al. 2010a, b). The cortisol is a natural glucocorticoid hormone synthesised by the adrenal cortex, which in turn, is stimulated by sympathetic nervous system activity. The main function of the hormone is to increase blood glucose level during effort and stressful conditions. In horses, the blood plasma cortisol concentration reflects not only the response to exertion (Desmecht et al. 1996, Nagata et al. 1999) but also the mental stress level (Cayado et al. 2006, Fazio et al. 2008). In recent years, in cortisol determination, more attention has been devoted to saliva sampling. This form of sampling is less stressful for the horse than blood sampling (Peeters et al. 2010, 2011). The level of the saliva cortisol correlates well with its level in the blood (van der Kolk 2001, Peeters et al. 2011, Bohak et al. 2013). The salivary cortisol concentration was successfully used as an indicator of the stress level in horses in response to stabling (Harewood 2005), road transport (Schmidt et al. 2010a, b) and exercise (Kędzierski et al. 2013, 2014a). A significant increase in the salivary cortisol concentration was stated in naïve horses during the initial training. The increase was particularly significant in response to mounting by a rider (Schmidt et al. 2010a, Kęd- zierski et al. 2014b). Thus, measuring the cortisol concentration in saliva samples was assumed to be a useful marker of mental stress in horses. The aim of the study was to evaluate the influence of a relaxation massage, and music piped into the stable, on the horse’s long-lasting stress level gauged with the salivary cortisol concentration. Materials and methods This article presents data collected within a larger research project designed to determine the influence of chosen relaxation techniques on the emotional state of young racehorses. The study tested the hypothesis that relaxation techniques such as massage and music used over a long period of time, decrease the salivary cortisol concentration in racehorses. Volume 8 Issue 4

RESEARCH AND DEVELOPMENT Horses In the study, 120 Purebred Arabian horses were examined over two years: 57 horses in the first year and 63 in the second year. All the horses participated in the study during their first racing season. Each horse was studied for six months, which covered the full race season for three-year old Purebred Arabian horses. At the beginning of the study, the horses were 28–31 months old. The horses were brought to Słužewiec Horse Race Track (Warsaw, Poland) from their familiar studs about three months earlier to acclimatise to the new conditions. After a month of acclimatisation, the horses began the initial training. On the first few days, they were bridled and saddled inside their boxes. Next, they were walked and trotted in an automated horse walker for 30 minutes a day. After that, the horses were mounted. The caretaker held the reins and another caretaker assisted the rider to first lay over the back of the horse and then to move to a sitting position in the saddle. This initial training lasted nine to twelve days, depending on the horse's behaviour. Finally, the riders walked and trotted the horses in a paddock for 30 minutes a day for about six weeks. The horses were randomly divided into three groups: control group (n=24), music group which listened to music meant to be relaxing (n=48), and massage group (n=48) which regularly received a relaxing massage for about 30 minutes, three days a week. The only criterion of dividing the horses into the groups was that the groups were to include a similar number of mares and stallions. During the whole testing period, the horses were housed in four stables under comparable social and environmental conditions. Each horse was kept in a box stall measuring 3.5 m × 4 m. Straw bedding allowed the horse to comfortably lie down. To reduce the influence of nonspecific factors on the animal’s emotional reaction, all the horses were fed the same diet according to dietary guidelines and cared for by the same caretakers in a manner which was typical for racehorses. This means that all

primary race training was conducted on the sand track. The speed and duration were individually adapted to the level of each horse’s performance. An exception was made for the measurement days. On the measurement days, each horse had to cover a distance of 1800m at a speed of 6.4m/s. After the exercise, the horses were put on an automatic horse walker for 45 minutes. At the end of the third month of the study, the horses started to compete in official races at least once a month. A day before and two days after the race, the horses were only given 60 minutes of exercise in the automatic walker. On those days, they were not examined. All persons included in the training and maintaining of the studied horses didn’t know the purpose of the study. At the end of both race seasons, the horses’ performance was estimated with four parameters based on official race records: (1) number of races, in which a horse won; (2) prize per race, i.e. sum of prizes won to the number of races a horse took part; (3) success coefficient, i.e. sum of prizes won by a horse to sum of prizes won by all horses at the same age in respective race season; (4) general handicap, i.e. theoretical weight (in kg) a horse should carry in a race to equal the horse’s chance to win the race, with the chances of other horses at the same age.

horses had equal exposure to all the caretakers. To minimise the influence of the year and stable factors, the experiment was arranged in a manner shown in Table 1. For the first year of the study, the massage was introduced in stables 1 and 2, and the music in stables 3 and 4. The schedule was revised in the second year of the study. The control group also continued in respective stables in the consecutive years. Before the study, all of the horses were clinically sound according to a veterinarian. All the horses showed normal behaviour according to the trainer. Not all of the horses remained in training for the whole racing season because, in some cases, the owners decided to end their horse’s career before the end of the season. Thus, the number in the data for each month was lower than the number of horses included in a group, and amounted to 62.5%–89.6%.

My Pet Speaker®, Pet Acoustic Inc, Connecticut, USA) also designed to be within the range of horse-hearing. The speaker system design limits sound frequencies into 200 Hz ~

Listening to Music For approximately five hours a day, the music group listened to music piped into their stalls. The music was played in the stable from 1 to 6 o’clock p.m. The music used was specifically composed and recorded by Janet Marlow, a specialist in the scientific use of music for animals. The rhythms and sound frequency of each composition were specifically composed to be within the sensitivity of the equine hearing range (Saslow 2002, Wells 2009). The relaxation music contained 10 tracks of equine-specific music, composed by Janet Marlow, Sound Behaviourist and Founder of Pet Acoustics. The music was heard in the stable through a special sound speaker device:

Training and Racing The experiment was performed according to the regular race-training schedule, and for two race seasons. The training sessions were performed for about one hour a day, six days a week. The riders saddled the horses then walked the horses for approximately 10 minutes as a warm-up exercise. The www.international-animalhealth.com

International Animal Health Journal 25

RESEARCH AND DEVELOPMENT 12 KHz and features a soft bass designed for equine listening comfort. The speaker also disbursed the music in 360 degrees resonating from the four open areas of the top of the speaker. The equine music by Janet Marlow was played every day in the “Repeat All” mode. The volume was set in the mid-range; at about 65–70 decibels. The equine hearing comfort levels are between 60–80 decibels in general. The results of the massage group were significantly lower than the results for the music group. To simplify the graphs, in both Figures, the values of SD are presented as only upper or lower bars, nevertheless, each value of SD above the mean was equal with those below the mean. The race performance parameters are presented in Table 2. Horses in the massage group achieved better results than those in the control group with regard to the number of races won, prize per race and success coefficient. Music group had significantly higher prize per race than the control group. All the studied performance parameters did not significantly differ between music and massage groups.

Discussion The results of the present study clearly show that both stress-coping methods (music meant to be relaxing in the stable and relaxing massage), brought positive effects. Introducing those methods significantly decreased the release of cortisol. The amount of cortisol was estimated on the basis of the salivary cortisol concentration. The use of this kind of stress-level estimation is commonly accepted in current research (Schmidt et al. 2010 a and b, Peeters et al. 2010). The most interesting results concerned the determination of the cortisol level in saliva samples, taken at rest. The lack of differences in the first measurement taken at rest, showed that, as was expected, the three groups showed similar cortisol levels at the beginning of the study. The similarity of the groups was in accordance with the trainer’s opinion on the normal behaviour of all of the horses. Both of the

26 International Animal Health Journal

relaxation methods significantly decreased the cortisol release as compared to the control group, during the whole racing season, excluding the fifth month of the study. It is worth noticing that the massage was performed after the training sessions, so the horses were massaged at noon, and they listened to music in the afternoon. The horses were investigated at rest, in the early morning, hence the influence of the relaxation methods was analysed many hours after the relaxation treatments. Our results indicate the long-lasting effect of the relaxation methods on the salivary cortisol level in the racehorses. The positive effect the massage had on the salivary cortisol level determined just after a treatment, was described earlier (Scott and Swenson 2009). In another study, massage reduced the stress level which had been evaluated on the basis of heart rate and behaviour of the horses also during a treatment (McBride et al. 2004). In the fifth month of the study, the horses which listened to music had a significantly higher salivary cortisol concentration than those horses which received a massage. An analysis of heart rate variability in horses which listened to relaxation music, also showed that the effectiveness of this method decreased after some months of the treatment (Stachurska et al. 2015). Perhaps animals get accustomed to the music as time passes. Moreover, the horses in general get accustomed to the training. A tendency to decrease in resting cortisol values was seen in the control group during the study. Thus, probably, the salivary cortisol concentration dropped in the music group in the sixth measurement in comparison to the fifth measurement, because of a general tendency to adapt to the environment. However, the adaptation effects of long-lasting music treatment in humans and animals have been studied less. In fact, many reports describe beneficial effects of short-time use of relaxing music (Kıyıcı et al. 2013, Bowman et al. 2015, Linnemann et al. 2015, 2016). The analysis of those salivary cortisol samples taken after the daily training sessions, and taken in the fifth and sixth month of the study, showed the clear positive effect of the massage treatment over the music treatment. Exercise performed by racehorses during training increases the salivary cortisol concentration (Kędzierski et al. 2013, 2014a), however, endurance exercise has a stronger effect (Desmecht et al. 1996). Some studies suggest that salivary cortisol level determined after exercise, can indicate the relative intensity of exercise in racehorses (Kędzierski et al. 2013). Generally, the intensity of exercise increases with the duration of training. Therefore, the values of salivary cortisol obtained after the end of training sessions tended to increase, especially in control and music groups. In the following months of the study, we compared horses which were subjected to similar amounts of intensive exercise. Thus, the differences in the salivary cortisol concentration found between massage group and other groups were the evident effect of the massage treatment. Listening to music only appears to influence mental relaxation in the horses, whereas massage has an effect on both the mental and muscle relaxation (Haussler 2009). It is not surprising that the estimated cortisol release after physical activity showed that

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RESEARCH AND DEVELOPMENT

massage brought more beneficial effects than the music. From the economic and management point of view, though, providing music is much easier than providing a relaxing massage. Conclusion In conclusion, both the relaxation massage and the music treatment significantly decreased the cortisol release in Purebred Arabian horses trained for racing. The massage treatment gave better results than listening to music which was meant to be relaxing. Playing music, though, being easier to provide, may be widely introduced to improve the welfare and performance of racehorses. Funding statement The Polish National Centre for Research and Development (grant number 180061) sponsored this study. Citation Kędzierski W., Janczarek I., Stachurska A., Wilk I. (2017) Massage or music meant to be relaxing, result in lowering salivary cortisol concentration in race horses. Pferdeheilkunde 33, 146-151; DOI 10.21836/PEM20170206 Correspondence Witold Kędzierski PhD, Department of Biochemistry, University of Life Sciences in Lublin, Akademicka 12, 20-033 Lublin, Poland; e-mail: witold.kedzierski@up.lublin.pl www.international-animalhealth.com

Janet Marlow Janet Marlow, M.A., Sound Behaviourist is internationally known as a researcher, composer and author. In 1997, Janet Marlow innovated a science-based method of altering the frequency and decibel levels in music, “species-specific music”. This series of music for animals has been clinically proven to balance behaviours in dogs, cats, horses and birds. Her peer reviewed music studies have been published in the Journal of Equine Veterinary Science and the International Animal Health Journal. Ms. Marlow has been a consultant for Boehringer Ingelheim, (Equine) Ceva Animal Health (Canine) and Purina Friskies (Feline). In 2009, she founded Pet Acoustics Inc., an award-winning global brand with multiple products that have helped thousands of animals worldwide to diminish stress in their living environment for better animal welfare. Janet Marlow was named Woman of Influence in the Pet Industry by Pet Age Magazine. She has authored several books on animal hearing, most recently, What Dogs Hear: Understanding Canine Hearing and Behaviour. Email: janetmarlow@petacoustics.com

International Animal Health Journal 27

RESEARCH AND DEVELOPMENT

Muscle Abnormalities in Broilers

Over the past decades, broilers have been growing at an increasing rate. This is not only due to changes in their genetic composition but also through improvement of housing, nutrition and management. The increase in the breast meat weight of broilers is particularly striking. As the slaughter weight of broilers increases, in combination with the growth curve required to achieve this slaughter weight, we are seeing a number of muscle abnormalities which influence the quality. We shall discuss the main ones here. None of these muscle abnormalities are clearly visible in the living chicken, and therefore often only become apparent during the slaughter process. Deep pectoral myopathy Deep pectoral myopathy (DPM) is also known as green muscle disease. This is not a form of contagious disease, but rather a wasting of the deep pectoral muscle due to blood supply issues. How does it occur? The deep pectoral muscle is located between the breastbone and the breast meat (the superficial pectoral muscle) and is used when chickens move their wings. Broilers are extremely muscular and this muscle therefore has little space for expansion. If the broiler flaps its wings energetically, there is extra blood supply to this muscle. This is the body’s way of ensuring that the muscle has sufficient oxygen supply and sufficient disposal of waste products. All that extra blood results in the muscle becoming temporarily expanded. When the muscle contracts, it is smaller than when in a relaxed state. If the muscle then swells excessively in this state due to the extra blood, the situation can arise whereby the muscle no longer has enough space to relax normally. The pressure will then increase to such an extent that the blood vessels around the muscle are compressed, stopping the blood from flowing out of the muscle. No new blood can be pumped in, resulting in myopathy of the muscle tissue due to a lack of oxygen and the accumulation of waste products. This may colour the muscle tissue green, hence the name green muscle disease.

Green muscle disease (Source: NVWA)

White striping and Wooden breast White striping (WS) is characterised by white stripes running parallel to the muscle fibres of the breast meat (the large pectoral muscle/pectoralis major). These stripes can vary in thickness and give the meat a marbled appearance. Wooden breast (WB) is characterised by a pale and extremely sturdy breast, which can sometimes be as hard as wood (hence the name wooden breast). WB and WS are two possible manifestations of the same underlying problem and are therefore discussed together here. How does it occur? While the exact cause is unknown, the abnormality seems to be the result of a localised oxygen deficiency. Unlike deep pectoral myopathy however, there is no complete blockage of the oxygen supply in WS and WB. It seems more likely that there is a long-term slight shortage of oxygen and ineffective discharge of waste products. This is probably due to the growth of the system of blood vessels being incapable of keeping pace with the rapid growth of the muscle tissue. This rapid growth of muscle tissue primarily takes place in the frontal (thick) side of the broiler breast. If there is a slightly insufficient blood supply over a longer period of time, muscle tissue will waste away. It is replaced by connective tissue. This connective tissue is much harder than muscle fibre and has poorer blood circulation. Consequently, as more and more muscle fibre is replaced, the muscle becomes increasingly hard and pale. A period of rapid growth is a risk factor, and WS and WB are both found in standard farmed broilers and in slower growing broilers. The abnormality is not stress-related, nor is there any contagious disease involved. Spaghetti meat In the case of spaghetti meat (SM), the outer layers of the breast fillet fall apart into wet, loose strips when mechanically processed. This disorder gets its name from the fact that these strips somewhat resemble spaghetti.

Early phase of green muscle disease (Source: GD) 28 International Animal Health Journal

How does it occur? SM is the result of a poor connection between the bundles of muscle fibre as a result of underdeveloped connective tissue within the muscle. This is not a contagious disease. SM Volume 8 Issue 4

RESEARCH AND DEVELOPMENT contracting muscle fibres (fast twitch muscle fibres). Slow twitch fibres have a much higher volume of blood vessels, due to these muscle fibres having an oxygen-rich energy uptake, while the fast twitch muscle fibres require less oxygen for their energy uptake. Although the blood supply is therefore generally more effective, the muscle is much less capable of coping with an oxygen deficiency. Animals suffering from dorsal cranial myopathy probably have an issue with the blood flow to this muscle, resulting in a decreased supply of oxygen. In some cases, this muscle is possibly sometimes pinched by the strongly developed muscles of the broilers. The disorder is particularly seen in extremely muscular broilers. Once again, this is not a contagious disease.

White striping (Source: GD)

Identification of muscle disorders in animal health monitoring Broilers suffering from the disorders described here will appear healthy in the house and will not demonstrate any abnormal behaviour. They will not show any signs of pain. There is therefore no reason to submit these animals to GD for pathological examination. These disorders often only become visible at the abattoir. Abattoir staff can submit carcasses to GD, and the staff at NVWA (the Netherlands Food and Consumer Product Safety Authority) also submit carcasses, due to a new or striking pathology, for example, or a drastic change in prevalence. Animal health monitoring therefore has no insight into the prevalence of the disorders, though we do keep a close eye on any sudden new pathologies or drastic changes.

Wooden breast (Source: GD)

is not apparent during the life of the broilers and is a typical abattoir discovery; it is regarded to be a quality problem for the meat rather than a health problem in the broiler.

The skin of these carcasses has been opened: dorsal cranial myopathy can be seen in the underlying muscles of the left-hand and middle carcass; the right-hand carcass shows no abnormalities (Source: GD)

Spaghetti meat (Source: GD)

Dorsal cranial myopathy In dorsal cranial myopathy, there is continuous wasting of muscle tissue in the back muscle (anterior latissimus dorsi; ALD). This is a well-known abnormality in broilers, first detected in 2002. Up to 6 percent of the animals may have the abnormality in an affected flock (percentage based on 2009 research). This high prevalence is seen in combination with wooden breast and with heavier slaughter-weight broilers. How does it occur? There is one main difference between this disorder and the muscle disorders described above, namely the type of muscle fibre. The ALD comprises slowly contracting muscle fibres (the so-called slow twitch muscle fibres), unlike the pectoral muscle (in WB and WS) which contains quickly www.international-animalhealth.com

Robert Jan Molenaar Robert Jan Molenaar, DVM, FCRPath gained his veterinary qualification at the University of Ghent in 2008. In 2017 he became a fellow at the Royal College of Pathologists in London, after passing the veterinary pathology exams with a focus on avian pathology. His job as a senior poultry veterinarian and poultry pathologist at Royal GD has included research for Salmonella, Influenza and IBV and responsibility for the mink disease monitoring in the Netherlands.

International Animal Health Journal 29

FOOD & FEED

A Vegan Diet for Dogs and Cats May Improve Health Outcomes and Longevity In the past decade, men and women around the globe have transitioned to a vegan diet in record numbers, propelled largely by a growing body of research pointing to better health outcomes, and by catastrophic weather events and wildfires that fuel concerns about the contribution of animal-based food production to climate change. Many are driven by ethical considerations, given revelations about treatment of animals in slaughterhouses and feedlots, alongside increased awareness of the sentience and cognition of species of all kinds, including those used for food. The result is an emerging cultural shift – one that is also gaining prominence in the conversation regarding dietary choices for companion animals. As a result, animal health professionals are called upon to join this conversation with open eyes, an open mind, and the flexibility to acknowledge and respond effectively to clients and consumers as they explore plant-based options for their dogs and cats. Consumers who reduce their consumption of animal products for health reasons are motivated by reports of increased longevity, reduced risk of cancer, better weight management, increased energy, and more. As they begin to feel better, lose weight, and see improvements in their own health metrics, they often wonder if their dogs’ or cats’ health might benefit from a similar dietary change. Even among those who eat meat themselves, the steady stream of news about the health benefits of a vegan diet weigh heavily after grieving one more beloved dog or cat who succumbed too early to cancer, or who seemed to age too soon or deteriorate too quickly. For a caregiver who chooses a vegan diet due to environmental concerns, it’s a short step to recognise that a companion’s meat-based diet also contributes to depletion of water supplies and rainforests, and to the production of methane and other greenhouse gasses. The higher proportion of animal products in the typical meat-based diet for a dog or cat means the impact is greater as well. A 70-pound canine eating a raw meat diet consumes roughly twice as many calories from animal sources as a meat-eating human, with twice the environmental impact.1 The effects of a feline diet are even more pronounced. Many who chose a vegan diet out of concern for the animals used as food struggle daily when they feel compelled to include meat in their companions’ meals. Many times, I’ve heard the strain of conflicting priorities as a caregiver chafes against the perceived need to feed meat, in direct opposition to a desire to extend compassion to all species. For that individual, the revulsion that comes with handling animal flesh at mealtimes is not only unpleasant but can itself become unhealthy. Certainly, there is good reason for caregivers to explore options for feeding a vegan diet to their animal friends – and to seek ways to do it safely and sensibly. It’s incumbent upon us, then, to examine the options and understand the issues so we can provide sound recommendations and resources in support of that effort. 30 International Animal Health Journal

CNAT

Is It Natural? A discussion about whether a vegan diet is safe for a dog or cat often begins with concerns that it’s not natural, based on an assumed need for what has been called the “ancestral diet.” It’s true that canines and felines descended from predators who relied on the flesh of other animals for sustenance, so – it might seem – a vegan diet is clearly not natural, and therefore cannot possibly be safe. But in fact, the dietary needs of our household companions are decidedly different from those of their ancestors, due to changes wrought by thousands of years of domestication. Clearly a Dachshund is not a wolf, and a Tabby is not a tiger. Consider our dogs, whose genetic profile diverged from wolves as they evolved alongside humans. A wolf does rely on other animals as his primary food source. But he also routinely ingests grasses, acorns, berries and other fruits, and the stomach contents of his herbivorous prey. Dogs are even better suited for eating plants, including the grains and other higher-carbohydrate foods they have for millennia shared with humans. A comprehensive study of the genomes of wolves and dogs found changes in ten genes related to the digestion of carbohydrates. Because of these differences, dogs are far better able to convert starch into glucose, and more efficient at transporting glucose from the intestines into the bloodstream.2 Plainly, dogs are quite capable of digesting the grains,3 vegetables, and legumes that constitute a vegan diet. The wild ancestors of cats are, of course, true carnivores. However, our domestic cats have also evolved a greater Volume 8 Issue 4

FOOD & FEED ability to metabolise carbohydrates. Caregivers are familiar with their cats’ propensity to eat grass (and the occasional favourite houseplant), and their intriguing affinity for corn, melon, and other plants. Indeed, commercial cat foods are typically 20% to 40% carbohydrates, with ingredients like grains, potatoes, and legumes necessary for processing.4 Still, cats lack the ability to produce some essential nutrients, such as taurine and arachidonic acid, that are not widely available in plant foods. Nevertheless, if we assess a cat’s needs in terms of nutrients rather than ingredients, we can construct a nutritionally sound diet using plant-based ingredients augmented with appropriate supplementation. There’s good reason to consider doing so, as we’ll see. Domestication has also changed the animals we feed to dogs and cats, therefore the meats we use in today’s “ancestral” or “species appropriate” diets are substantially different from the foods our companions’ ancestors ate. A wolf or tiger might prey on a water buffalo, antelope, quail, beaver, or wild hare, all decidedly different from the cows, chickens, and sheep we feed our companions. Furthermore, today’s feed animals are often dosed heavily with antibiotics, eat grains that are genetically modified and treated with chemical pesticides and fertilisers, and face the stresses inherent in modern feed and slaughter operations – all of which are present in the meat consumed by companion animals, and none of which should be considered part of a “natural” diet. Can a Dog or Cat Thrive Without Meat? When we evaluate the diet based on nutrients rather than ingredients, we find all necessary macro- and micronutrients readily available using plant foods and, for cats, adding appropriate supplementation. (See guidelines below.) What may be less obvious is that a plant-based diet may in fact be healthier for our companions. It is heartrending to hear

windflower

long-time veterinarians describe the dramatic increase in cancer among their patients. These dedicated professionals who have been treating dogs and cats for decades suffer along with caregivers as they see more and more animals, at younger ages, succumb to the ravages of the dreaded disease. What could be causing the proliferation of cases? We live in a toxic environment; industrial and agricultural chemicals are pervasive. They’re in our air, our soil, our rivers, our groundwater, in the rain as it falls. Inevitably, they’re also in our food supply. Even organically grown crops are tainted—it’s impossible to avoid contamination when these substances are ubiquitous. Even more troubling, due to bioaccumulation these toxic substances become more concentrated as we move higher up the food chain, so toxicity in the flesh of an animal is exponentially greater than in the plants he consumes.5 That’s not the worst of it. Consider the animal who eats the flesh of the animal carrying such high levels of contamination. Remember, a canine typically consumes a greater proportion of meat than an omnivorous human; a feline consumes even more. Both ingest toxic chemicals at disturbing levels every single day. It’s a frightening reality and may be a critical factor in the tragic increase in cancers those seasoned veterinarians describe. When I give lectures on this topic, it’s not unusual to see heads nodding as I look around the room, as many recognise at last an explanation for what they’ve been struggling to address in their practices.

Carlyn Montes De Oca www.international-animalhealth.com

It’s well known that in humans, a vegan diet can reduce the risk of cancer, as well as diabetes, liver and kidney disease, gastrointestinal disturbances, arthritis, and obesity – all conditions that affect our dogs and cats. Whether this is due International Animal Health Journal 31

FOOD & FEED

Patrik Kraus/Shutterstock.com

to lower levels of toxicity, higher levels of antioxidants and phytonutrients, improved gut microbiome, or a direct result of eliminating animal proteins, it’s impossible to say. Sadly, there is little data available to tell us whether eliminating animal products from our companions’ diets would provide similar benefits. It’s reasonable to expect it would. It’s certainly consistent with changes I see when clients transition their animals to a vegan diet. Here are a few examples: •

•

Obesity. Grace was decidedly overweight when rescued from a shelter in New Mexico, U.S., but easily lost 16 pounds within the first year on her vegan diet. The reduced fat and calories and ample portions of fruits and vegetables allowed her to trim down without ever lacking in nutrition or feeling deprived. Gastrointestinal disturbance. I often see gastrointestinal disturbances resolve quickly with no treatment other than a change to a vegan diet, usually without an extended transition period. Atticus’ case was particularly dramatic. This sweet and mellow Cane Corso had ongoing upper and lower GI disturbances throughout his first 6½ years. He typically moved his bowels four times a day, and most stools were liquid. He had frequent bouts of anorexia and vomiting, on two occasions requiring emergency veterinary intervention. No illness, obstruction, or other causative factor could be identified. It was only when he was switched to a vegan diet that his GI function normalised. Food sensitivity. Patches was a long-haired calico cat who had suffered from food sensitivities most of her life.

32 International Animal Health Journal

Soon after eating, she’d dash around her apartment, stopping only to frantically lick her coat or scratch. Initially, changing to a different commercial food solved the problem, but within a few months symptoms reappeared. By age nine Patches was reactive to every meat-based commercial or home-prepared food her caregiver offered. When she was switched to a vegan diet, the itching stopped and did not recur. •

Reduced mobility. At age 11½, Ocean had difficulty getting up, and walking was painful. Radiographs showed advanced arthritis in his hips and spine. Within two months on a vegan diet, Ocean’s mobility and energy level improved, and he and his dad were able to enjoy their walks along the coast at Figueira da Foz.

•

Seizure disorder. Mira was having multiple seizures each day, while suffering severe side effects of potassium bromide and phenobarbital. She was put on a vegan diet and treated with homeopathy, and within weeks was free of seizures; she remained so after discontinuing pharmaceuticals.

•

Age-related degenerative changes. At 14, Dakota, a Chow cross, exhibited S-I joint pain, muscular soreness in the lumbar region, reduced hind-leg strength and motor control, inability to ascend stairs without panting, low energy, and a rough coat; lab results indicated early signs of kidney disease. After two weeks on a vegan diet, Dakota’s mobility had improved; she had less lumbar pain and a softer coat. After three weeks she was stronger, more playful, with less pain, and climbing stairs more easily and with no panting. After six months, her kidney function had normalised. Her caregiver reported that a Volume 8 Issue 4

FOOD & FEED friend they hadn’t seen in some time saw Dakota playing and asked if this was a new puppy – and was amazed to learn that the “puppy” was a revitalised Dakota. A Vegan Diet in Practice and Production As we’ve seen, a vegan diet can play an important role in treatment and provide support for preventive care and longevity. But how best to guide veterinary clients in implementing it? Like humans, dogs and cats thrive on a diet based on fresh, whole foods, with plenty of variety, drawing on an everchanging selection of ingredients. The Fresh and Flexible™ meal plan offers a simple guide for caregivers that applies these principles to optimise nutrition and is well suited to a plant-based diet.6 (For more on this approach, see “A New Approach to Optimizing Nutrition for Dogs and Cats” by this author in the Summer 2021 edition of this publication.7) For most vegan dogs on the Fresh and Flexible meal plan, supplementation is not necessary if the plan is followed carefully using a variety of ingredients and the recommended nutrition boosters. However, since some breeds are unable to manufacture taurine, it’s advisable to add it, and essential when the dog’s breed is not certain. Since a plant-based diet tends to be lower in phosphorus, consider reducing or eliminating calcium supplementation typically included in meat-based diets, to avoid in improper calcium-phosphorus ratio. The supplement Vegedog™, made by Compassion Circle (www.CompassionCircle.com), is designed to accompany a vegan diet, and offers an added safeguard. The Fresh and Flexible plan is also well suited to cats. However, since cats on a vegan diet do require supplementation, Vegecat™, also available from Compassion Circle, is highly recommended to ensure all needs are met. For those who rely on commercial foods, there is only a handful of vegan dog foods currently on the market, and fewer cat foods – with virtually none formulated for puppies, kittens, or seniors. Many are made with highly processed ingredients; as with any commercial diet, products made from whole foods are optimal. The limited number of options complicates efforts to feed a varied diet by rotating among several different brands. This, of course, reveals an excellent opportunity for commercial food developers to fill an unmet, growing need. From 2019 through 2020, grocery sales of plant-based foods grew 43%, to $7 billion in 2020, while total grocery sales grew by just 17%.8 Since companion-animal food trends mirror those of their caregivers, a line of vegan foods made from whole foods and formulated for different life stages would meet a ready, burgeoning, underserved market. A Simple Contribution with Far-Reaching Benefits With caregivers’ growing interest in vegan diets for their companions, we have an opportunity to expand our options for addressing health issues at the most fundamental level of care. The meals our dogs and cats eat every day play a crucial role in their health, vitality, and longevity. If we can eliminate a potential cause of devastating illness and shortened lifespan with a simple change in diet, our clients and customers – and their companions – will benefit, as will we as we see them thrive. If at the same time we can make a contribution to a healthier planet and the well-being of all species, we will all take a welcome and worthwhile step toward a brighter future. www.international-animalhealth.com

AMBERLIGHTPHOTO/Shutterstock.com

REFERENCES 1.

3. 4.

5. 6. 7. 8.

Pitcairn, Susan Hubble. How Your Dog Can Help Save Our Planet, in The Complete Holistic Dog Book: Home Health Care for Our Canine Companions, Jan Allegretti. Tenacity Press: Upper Lake, CA, 2018. Axelsson, E., Ratnakumar, A., Arendt, ML. et al. The genomic signature of dog domestication reveals adaptation to a starch-rich diet. Nature 495, 360–364 (2013). https://doi. org/10.1038/nature11837. Allegretti, Jan. Grains Are Good Food: Helping Caregivers Move Beyond the Grain-Free Diet Trend. International Animal Health Journal, Vol. 8, issue 3, 26-28. Verbrugghe A, Hesta M. Cats and Carbohydrates: The Carnivore Fantasy?. Vet Sci. 2017;4(4):55. Published 2017 Nov 15. doi:10.3390/vetsci4040055, https://www.ncbi.nlm. nih.gov/pmc/articles/PMC5753635/#B72-vetsci-04-00055 accessed October 22, 2021. Pitcairn, Richard, et. al. Are Vegan Pet Diets Significantly Less Polluted? Caring for All Animals: Exploring Vegan Diets for Dogs & Cats. Sedona, Arizona, US, January 13, 2017. Allegretti, Jan. The Complete Holistic Dog Book: Home Health Care for Our Canine Companions, Tenacity Press: Upper Lake, CA, 2018. Allegretti, Jan. A New Approach to Optimizing Nutrition for Dogs and Cats. International Animal Health Journal, Vol. 8, issue 2, 30-32. U.S. retail market data for the plant-based industry. Good Food Institute. https://gfi.org/marketresearch/ accessed October 15, 2021.

Jan Allegretti Jan Allegretti, D.Vet.Hom., is the author of The Complete Holistic Dog Book: Home Health Care for Our Canine Companions. She has more than thirty years’ experience as a teacher and consultant in holistic health care for nonhuman animals, offering workshops and consultations to caregivers, rescue organizations, veterinarians, and other animal health professionals. Jan can be reached through her website at www.JanAllegretti.com. Email: jaa@pacific.net

International Animal Health Journal 33

FOOD & FEED

Is Insect Farming Truly a Solution to The Animal Feed Problem? The European Union has started considering alternative feed, such as insect protein, to supply the livestock industry. Insect-derived protein as animal feed is increasingly seen as a solution to diminish the use of imported soy linked to deforestation and to supplement the use of fishmeal from depleted oceans. In the EU’s commitment to tackle the climate crisis, the promises of insect farming have been embraced by policy makers but it does not seem to be the sustainable solution the EU is looking for. The Farm to Fork strategy, a key initiative under the European Green Deal, was presented by the European Commission (EC) in 2020. It aims to accelerate the transition to a fair, healthy and environmentally-friendly food system with a neutral or positive environmental impact that helps to combat climate change and reverse biodiversity loss while ensuring safe, nutritious and sustainable food for all. As part of the EU’s commitment to sustainable food systems, the Farm to Fork strategy will reduce the European Union’s dependency on imported feed such as soy, which is linked to deforestation. Instead, EU produced feed materials, including both plant protein and alternatives such as insects, should be promoted.1 In addition to reducing soy dependence, the use of insect protein as feed is seen as having the potential to contribute to a circular economy under the condition that insects can be fed on organic waste. In this editorial we explore the effects that insect farming for feed can have on farming systems and animal welfare, including on insect welfare. Processed animal protein (PAP), including from insects, has been prohibited for use in animal feed in the EU since 2001. The EU feed ban followed the outbreaks of bovine spongiform encephalopathy (BSE) which led to the killing of over one million cattle in the UK in the early 1990s. Insect protein has been allowed as feed in aquaculture since 2017, as well as for fur animals and in pet food,2 but the feed ban has restricted the insect industry’s possibilities to produce insect protein for the animal feed market and thereby their possibilities to scale up. The EC now notes that the risks of BSE are reduced. In combination with the Farm to Fork strategy’s aim to reduce the dependency on soy and make better use of protein and other feed materials produced in Europe, the EC has revised the feed ban. In August the use of PAP of porcine origin in poultry feed, and of poultry origin in pig feed was authorised. In this context, the EC also authorised insect PAP in pig and poultry feed3 under the same conditions as required for aquaculture feed. The decision was announced as “another small step in our journey towards more sustainable feed chain”4 and was communicated as a way to achieve “another milestone in the Farm to Fork strategy’s ambition towards the use of quality and sustainable feed”.5 The recent decision to allow insect derived protein in pig and poultry feed conflicts with the goals of the Farm to Fork strategy. On the one hand, insect protein is promoted by 34 International Animal Health Journal

the EC as an alternative feed material with the potential to reduce Europe’s dependence on soy and reduce the negative environmental consequences of livestock farming. On the other hand, the Farm to Fork strategy recognises the urgent need for a shift towards healthy, sustainable diets with more plant-based food. Moreover, it emphasises the role of improved animal welfare: less and better meat. This attention given to improved animal welfare in the Farm to Fork strategy is also linked to One Health and the target of halving the sales of antimicrobials by 2030 to curb antimicrobial resistance. The density of animals in intensive, indoor, large-scale production sites facilitates the spread of pathogens and can lead to an increased risk of disease due to stress which makes the animals more susceptible to illness.6 That the insect industry predicts major growth of insect farming after the authorisation of insect protein for pig and poultry feed.7,8 The insect industry is likely to expand after the authorisation is reflected in the European Commission Agricultural Outlook 2020–2030 which assessed the impact that insect farming would have, when fully commercialised and if all existing restrictions were lifted. It forecasts that the increased supply of insect meal and lower prices could support conventional intensive animal production.9 A rapid expansion of insect farming for animal feed seems, therefore, to go against the Farm to Fork strategy’s objectives of improved animal welfare and real food systems transformation. Instead, insect farming for feed seems to favour intensive, indoor, large-scale production and, as a consequence, make the reduction target for antimicrobial sales more difficult to achieve. By supplying the intensive pig and poultry farming sector with insect protein, the insect industry runs the risk of sustaining these systems rather than transforming them into the higher welfare systems envisioned in the strategy and expected by EU citizens.10 It has been argued that insect protein in pig and poultry feed contributes to improved animal welfare as insects are part of the natural diets of both poultry and pigs. However, while good nutrition is one component, a focus only on nutrition for animals in intensive systems is a too narrow view of what improved animal welfare means. From the perspective of the Five Domains model,11 a positive affective state of an animal is also influenced by a good environment, good health, and opportunities to express appropriate behaviour, such as rooting and scratching. It accounts for not only the physical needs to be met but also ethological and psychological ones, and promotes the role of positive mental experiences. One example of the latter is foraging for food: searching for insects through scratching is a positive experience for poultry and therefore contributes to their welfare,12 in line with the Five Domains model. This is not, however, what is achieved by promoting the use of insect PAP for animals confined indoors in intensive systems. The welfare of insects also needs to be considered. Since they are bred to produce PAP they are defined as farmed animals in EU legislation.13 The European Food Safety Authority (ESFA) considers that the “general animal (vertebrate) health Volume 8 Issue 4

FOOD & FEED and welfare rules should also apply for insects”.14 However, Council Directive 98/58/EC concerning the protection of animals kept for farming purposes excludes any invertebrate animal.15 A major difficulty in assessing insect welfare is the current lack of scientific knowledge about the welfare needs of insects, as well as about their experiences of suffering and pain.16,17 There are an estimated 5.5 million insect species in the world of which only about one million species are currently known.18 The species allowed for animal feed in the EU are: • • • • • • •

Black soldier fly (Hermetia illucens) Common housefly (Musca domestica) Yellow mealworm (Tenebrio molitor) Lesser mealworm (Alphitobius diaperinus) House cricket (Acheta domesticus) Banded cricket (Gryllodes sigillatus) Field cricket (Gryllus assimilis)19

Of these seven species, black soldier fly, mealworm and house cricket are the species most commonly reared in Europe.20 The welfare requirements of each species would need to be assessed in order for the housing systems to meet each species’ environmental and species-specific needs.21 A recent review concludes that many insect species currently raised for both feed and food are assumed or confirmed to be capable of a range of cognitive abilities. The study finds that there is reason to believe that crickets can feel important emotions such as stress, and emphasise that the lack of knowledge about the impact of the various slaughter methods on different species of insects should be reason for special attention. Boiling is, for example, known to be painful to decapod crustaceans, and there is a strong possibility that this slaughter method is painful also to other invertebrates.22 While insects are often slaughtered by heating or boiling, mincing or freezing, the insect industry itself recognises that “the current lack of scientific evidence around invertebrate welfare makes it very difficult to develop science-based welfare rules for insect production”.23 It is also unknown if there are differences in sentience between the larvae and the grown insect. In the absence of scientific evidence, scientific literature recommends that we should apply the precautionary principle and treat insects as if they were sentient.24,25 We should not repeat past mistakes of developing large-scale farming without knowledge about the possible welfare consequences. With fish it was only recently scientifically established that they feel pain.26 By then, intensive aquaculture practices and slaughter methods with animal welfare problems had developed widely. These systems cause considerable suffering, pain and stress. Moreover, once developed and established they are difficult to reverse. The lack of scientific knowledge about sentience, pain and welfare needs of each insect species should be solved before insect farming is allowed to scale-up.27 Not only are there the consequences on animal welfare, large scale insect farming may also bring unforeseeable consequences for local farming and ecosystems. With climate change and the increase of extreme weather events, there is a greater risk of accidental mass-releases from insect farms leading to the introduction of invasive alien species when farms are damaged by extreme weather.28,29 Moreover, a warmer climate can be more conducive to the establishment of invasive alien species. The economic consequences could be significant, considering that invasive species are the cause of a 14% reduction of global food production.30 An increased risk of insect-borne pathogens should also be considered as it can pose an additional threat to the already www.international-animalhealth.com

struggling wild-living insects such as bees, that are essential for pollination and other ecosystem services.31 The impact on local ecosystems can thereby, beyond the economic impact, compromise both biodiversity and food security, at least locally. Many factors have changed the interactions between people, animals and the environment in fundamental ways. This has made the One Health approach even more important in recent years:32 One Health captures the idea that human health and animal health are interdependent and bound to the health of the ecosystem in which they exist.33 A concept closely related to One Health is One Welfare, which reflects the benefits of high animal welfare standards also on humans and considers the relation between animal welfare, human well-being and sustainability.34 These concepts can be used together as One Health-One Welfare.35 The Farm to Fork strategy encompasses many of these elements as it connects improved animal welfare to sustainability. Applying One Health-One Welfare as a perspective can initiate a more critical debate on the introduction of industrial insect farming, a debate that has been largely absent throughout the discussions on the Farm to Fork strategy and the subsequent lifting of the feed ban for insect protein. It is high time that this emergent system of industrial farming is assessed from a more critical standpoint, considering the serious lack of knowledge on several crucial aspects of management and ecology. Insect farming for feed seems to be a false solution to the EU’s sustainability challenge. Without a critical debate, the risk is that it will sustain intensive pig and poultry farming rather than lead to truly sustainable food systems where humans consume more plant-based protein as part of healthy, sustainable diets and where pigs can root and poultry are able to scratch the soil. REFERENCES 1.

2. 3.

4. 5. 6.

7. 8.

European Commission, 2020, A Farm to Fork Strategy for a fair, healthy and environmentally-friendly food system. https:// eur-lex.europa.eu/resource.html?uri=cellar:ea0f9f73-9ab211ea-9d2d-01aa75ed71a1.0001.02/DOC_1&format=PDF Commission Regulation (EU) 2017/893 as regards the provisions on processed animal protein, https://eur-lex. europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32017R0893 Commission Regulation (EU) 2021/1372 as regards the prohibition to feed non-ruminant farmed animals, other than fur animals, with protein derived from animals, Official Journal of the European Union, Volume 64, L295, 18 August 2021, https://eur-lex.europa.eu/legal-content/EN/TXT/ PDF/?uri=OJ:L:2021:295:FULL&from=EN

“Feed ban: Commission authorises use of certain animal proteins,” European Commission, 17 August 2021, https:// ec.europa.eu/newsroom/sante/items/718842/en “Feed ban: Commission authorises use of certain animal proteins,” European Commission, 17 August 2021, https:// ec.europa.eu/newsroom/sante/items/718842/en EMA and EFSA, 2017, “Joint Scientific Opinion on measures to reduce the need to use antimicrobial agents in animal husbandry in the European Union, and the resulting impacts on food safety (RONAFA),” EFSA Journal, 15(1): 4666, https://doi.org/10.2903/j.efsa.2017.4666 “Edible insects on the European market,” IPIFF, June 2020, https://ipiff.org/wp-content/uploads/2020/06/10-062020-IPIFF-edible-insects-market-factsheet.pdf “The insect sector milestones towards sustainable food supply chains,” IPIFF, October 2020, https://ipiff.org/wpcontent/uploads/2020/05/IPIFF-RegulatoryBrochure-

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10.

11.

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15. 16.

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update07-2020-1.pdf European Commission, 2020, EU agricultural outlook for markets, income and environment, 20202030, Directorate General for agriculture and rural development, https://ec.europa.eu/info/sites/default/ files/food-farming-fisheries/farming/documents/ agricultural-outlook-2020-report_en.pdf “Special Eurobarometer 442: Attitudes of Europeans towards Animal Welfare”, 2015, Directorate General for communication, https://data.europa.eu/data/datasets/ s2096_84_4_442_eng?locale=en Mellor, D.J. et al., 2020, “The 2020 Five Domains model: including human–animal Interactions in assessments of animal welfare,” Animals, 10(10):1870, https://doi. org/10.3390/ani10101870. Star, L. et al., 2020, “Gradual provision of live black soldier fly (Hermetia illucens) larvae to older laying hens: effect on production performance, egg quality, feather condition and behavior,” Animals, 10(2), https:// doi.org/10.3390/ani10020216 Regulation (EC) No 1069/2009 laying down health rules as regards animal by-products and derived products not intended for human consumption, https://eur-lex.europa.eu/legal-content/EN/TXT/?uri= CELEX%3A02009R1069-20191214 EFSA scientific committee, 2015, “Scientific opinion on a risk profile related to production and consumption of insects as food and feed,” EFSA Journal, 13(10):4257, https://doi.org/10.2903/j.efsa.2015.4257 Council Directive 98/58/EC concerning the protection of animals kept for farming purposes, https://eur-lex.europa. eu/legal-content/EN/TXT/?uri=CELEX%3A31998L0058 Pali-Schöll, I. et al., 2019, “Edible insects – defining knowledge gaps in biological and ethical considerations of entomophagy,” Critical Reviews in Food Science and Nutrition, 59(17), https://doi.org/10.1080/10408398.2018. 1468731 van Huis, A., 2019, Welfare of farmed insects, Journal of Insects as Food and Feed, 5(3), https://doi.org/10.3920/ JIFF2019.x004 Stork, N.E., 2018, How many species of insects and other terrestrial arthropods are there on Earth? Annual Review of Entomology, 63: 31, https://doi.org/10.1146/annurevento-020117-043348 Commission Regulation (EU)142/2011 laying down health rules as regards animal by-products and derived products not intended for human consumption, Annex X, Chapter II, https://eur-lex.europa.eu/legal-content/EN/ TXT/?qid=1590506734572&uri=CELEX:02011R0142-20191214 “The insect sector milestones towards sustainable food supply chains,” IPIFF, October 2020, https://ipiff.org/wpcontent/uploads/2020/05/IPIFF-RegulatoryBrochureupdate07-2020-1.pdf Gamborg, C., Röcklinsberg, H. and Gjerris, M., 2018, “Sustainable proteins? Values related to insects in food systems.” In A. Halloran et al. (Eds.) Edible Insects in Sustainable Food Systems, Springer... Lambert, H., Elwin, A. and D’Cruze, N., 2021, “Wouldn’t hurt a fly? A review of insect cognition and sentience in relation to their use as food and feed,” Applied animal behaviour science, 243 (105432), https://doi.org/10.1016/j. applanim.2021.105432 “Ensuring high standards of animal welfare in insect production,” IPIFF, 2019, https://ipiff.org/wp-content/ uploads/2019/02/Animal-Welfare-in-Insect-Production. pdf Pali-Schöll, I. et al., 2019, “Edible insects – defining knowledge gaps in biological and ethical considerations of entomophagy,” Critical Reviews in Food Science and Nutrition, 59(17), https://doi.org/10.1080/10408398.2018

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.1468731 25. van Huis, A., 2019, Welfare of farmed insects, Journal of Insects as Food and Feed, 5(3), https://doi.org/10.3920/ JIFF2019.x004 26. “Fish welfare”, Eurogroup for Animals, 2016, accessed 30 September 2021, https://www.eurogroupforanimals.org/ files/eurogroupforanimals/2020-02/E4A-Briefing-FishFinal-Oct-2016.pdf 27. Pali-Schöll, I. et al., 2019, “Edible insects – defining knowledge gaps in biological and ethical considerations of entomophagy,” Critical Reviews in Food Science and Nutrition, 59(17), https://doi.org/10.1080/10408398.2018. 1468731 28. Rzymski, P. et al., 2021.” COVID-19 pandemic is a call to search for alternative protein sources as food and feed: a review of possibilities,” Nutrients, 13 (150), https://doi. org/10.3390/nu13010150 29. Berggren, Å. Jansson, A. and Low, M., 2019, ”Approaching ecological sustainability in the emerging insects-asfood industry,” Trends in ecology and evolution, 34(2), https://doi.org/10.1016/j.tree.2018.11.005 30. Berggren, Å. Jansson, A. and Low, M., 2019, ”Approaching ecological sustainability in the emerging insects-asfood industry,” Trends in ecology and evolution, 34(2), https://doi.org/10.1016/j.tree.2018.11.005 31. Rzymski, P. et al., 2021.” COVID-19 pandemic is a call to search for alternative protein sources as food and feed: a review of possibilities,” Nutrients, 13 (150), https://doi. org/10.3390/nu13010150 32. “One health basics”, Centers for Disease Control and Prevention (CDC), November 2018, https://www.cdc.gov/ onehealth/basics/index.html 33. “One health”, World Organisation for Animal Health (OIE), accessed 30 September 2021, https://www.oie.int/en/ what-we-do/global-initiatives/one-health/ 34. “About One Welfare,” accessed 30 September 2021, https://www.onewelfareworld.org/about.html 35. Garcia, R., 2017, “One welfare: a framework to support the implementation of OIE animal welfare standards,” OIE Bulletin, 1, http://dx.doi.org/10.20506/bull.2017.1.2589

Camilla Björkbom Camilla Björkbom is Political Adviser at Eurogroup for Animals, a European umbrella organisation for animal protection NGOs.

Volume 8 Issue 4

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Organic Trace Minerals – Enhancing Mineral Bioavailability Through Chelation The chemistry of complexation or chelation as it is commonly known has created a great deal of confusion in the animal feed industry. Terms such as metal amino acid complexes, metal amino acid chelates, metal polysaccharide complexes and metal proteinates abound, yet official definitions remain vague and unhelpful. As an example, definitions of the most common organic trace minerals used in agricultural practice as laid down by the Association of American Feed Control Officials (AAFCO, 1998) are illustrated in Table 1. In deference to the AAFCO definitions, table 1 also gives an overview of the EU classification of organic mineral products from which one can appreciate the stark differences between the official terminologies used for regulatory control and the obvious confusion that can occur when comparing products.

AAFCO Metal Proteinate (57.23)

Typically speaking, chelates are prepared by reacting inorganic mineral salts with, for example, enzymatically prepared mixtures of amino acids and small peptides, under controlled conditions. Such amino acid and peptide ligands bind the metal at more than one point ensuring that the metal atom becomes part of a biologically stable ring structure. Many different assertions are made as to the relative merits and suitability’s of amino acids versus peptides in forming mineral chelates, with an even greater number of arguments existing in relation to the so-called bioavailability of such products. The role of bond strength on OTM stability Most amino acids and peptides bind metal ions though either nitrogen, oxygen or sulphur atoms. Individual amino acids exhibit a range of stabilities when complexed with mineral and these can be assessed in a variety of EFSA

The product resulting from the chelation of a soluble salt with amino acids and/or partially hydrolysed protein. It must be declared as an ingredient as the specific metal proteinate e.g. copper proteinate, zinc proteinate etc.

Metal chelate of protein hydrolysates

A powder with a minimum content of x % metal where x = 10% copper, iron, manganese and zinc. Minimum of 50 % copper, iron, manganese and 85% zinc chelated. Chemical formula: M(x)1-3 . nH2O, M =metal, x = anion of protein hydrolysates containing any amino acid from soya protein hydrolysate.

Metal Polysaccharide Complex (57.29)

The product resulting from complexing of a soluble salt with a polysaccharide solution declared as an ingredient as the specific metal complex e.g. copper polysaccharide complex, zinc polysaccharide complex etc.

Metal Amino Acid Chelate (57.142)

The product resulting from the reaction of a metal ion from a soluble metal salt with amino acids with a mole ratio of one mole of metal to one to three (preferably two) moles of amino acids to form coordinate covalent bonds. The average weight of the hydrolysed amino acids must be approximately 150 and the resulting molecular weight of the chelate must not exceed 800. The minimum metal content must be declared. When used as a commercial feed ingredient, it must be declared as a specific metal amino acid chelate e.g. copper amino acid chelate, zinc amino acid chelate etc.

Metal Amino Acid Complex (57:150)

The product resulting from complexing a soluble metal salt with an amino acid (s).Mineral metal content must be declared. When used as a commercial feed ingredient, it must be declared as a specific metal amino acid complex e.g. copper amino acid complex, zinc amino acid complex etc.

Metal (specific amino acid) complex (57.151)

The product resulting from complexing a soluble metal salt with a specific amino acid. Minimum metal content must be declared. When used as a commercial feed ingredient, it must be declared as a specific metal, specific amino acid complex e.g. copper lysine, zinc methionine etc.

Metal chelate of amino acids hydrate

Metal amino acid complex where the metal and the amino acids derived from soya protein are chelated via coordinate covalent bonds, as a powder with a minimum content of 10 % copper and zinc, 9 % iron and 8% manganese. Chemical formula: M(x)1-3 . nH2O, M = metal, x = anion of any amino acid from soya protein hydrolysate. Maximum of 10 % of the molecules exceeding 1500 Da.

Metal chelate of glycine hydrate (liquid) Metal chelate of glycine hydrate (solid)

A liquid with a minimum content of 6 % copper or 7 % zinc. Chemical formula: M(x)1-3 . nH2O, M = Cu or Zn, x = anion of glycine A powder with a minimum content of 15 % copper, iron, zinc and manganese and a maximum of 13 % moisture for copper and 10 % moisture for iron, zinc and manganese. Chemical formula: M(x)1-3 . nH2O, M = metal, x = anion of glycine

Table 1 Official terminology for organic trace minerals (AAFCO and EFSA) 38 International Animal Health Journal

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FOOD & FEED databases. It is reasonable to expect that peptides which have a greater number of donor atoms and hence the potential to form a number of chelate rings when binding to a metal ion would have higher stabilities than simple amino acids such as glycine. This is however dependent on the peptide being able to actually form more than one chelate ring. As in the case of amino acids, peptides also exhibit a range of stabilities.

Bonding group

Relative Stability

Propionic acid (74Da)

1x10–6

Methionine Hydroxy Analogue (150Da)

2.63x10–6

Met (m.wt. 149Da)

0.5

Consider the data in the table below which compares a range of ligands when complexed with copper under the same physiological conditions.

Gly (m.wt. 75Da)

His-Ser (m.wt. 260Da)

2.5

What this indicates is that the size of the bonding group is not the most critical factor influencing bond strength and ultimately stability of a chelate. Claims of superiority based on size clearly have little merit. However, simply increasing the number of amino acids in a ligand may not increase the stability of the metal complex and thus may not necessarily increase the relative proportion of bound mineral. Ultimately, not only does the type of amino acid influence the stability of a given chelate but the position of amino acids in a peptide can also significantly influence how the ligand and mineral interact.

His-Met (m.wt. 304Da)

2.5

Gly-Cys (m.wt. 196Da)

Gly-Lys (m.wt. 221 Da)

2818

Tyr-Trp (m.wt. 385Da)

3235

Ala-Lys (m.wt. 238Da)

9549

Tyr-Lys (m.wt. 327Da)

186208

EDTA

5.6 x 1010

From a production standpoint, it is important to note that the extent and type of hydrolysis of a protein source to form short chain peptides can very significantly influence the sequence of amino acids present in these peptides. The production of an ‘optimal’ protein hydrolysate for mineral chelation can be affected through careful selection of the hydrolysis conditions. This ensures that the final mix of hydrolysed peptides will have the necessary properties to ensure constancy and mineral binding stability under conditions of changing pH. A recent study (Byrne et al., 2021) using potentiometricbased techniques analysed a range of commercial OTMs using a Cu ion-selective electrode to determine their in vitro stabilities over a pH range reflective of physiological conditions (Figure 1). In this work, samples were reconstituted and suspended prior to titration of the supernatants, with subsequent measurement of the percentage bound copper over a pH range of 3 to 8. This confirmed that notable differences exist in the pH-dependant stability of commercial OTMs, with the amount of bound copper varying considerably between samples. Furthermore, the data indicates that some OTMs have low or no capacity for stable mineral bonding at acidic pH, with obvious impacts on the bioefficacy of the products. These differences can be attributed to not only the type of bonding group used but also to the production process used to generate same.

Table 2 Relative stabilities of organically bound copper complexes. Adapted from: Critically selected stability constants of metal complexes, NIST Database 46.

Ultimately, the stability of an OTM is of paramount importance to its bioavailability. During transit through the GI tract and as the pH decreases or acidifies, all OTMs are subjected to physiological forces which can result in the bound mineral complex dissociating and releasing free mineral ions. Effectively this means that complexes or chelates with low stabilities will potentially have reduced bioavailability and reduce the effectiveness of the product to that of the corresponding inorganic salt. Maximising the pH dependant stability of OTMs will increase mineral bioavailability and uptake in the intestine. In essence, the higher the stability of an OTM, the greater its bioavailability is likely to be. Influence of OTM stability on relative bioavailability In addition to differing by virtue of the bonding group used in the chelation process, OTM’s also differ greatly in terms of how well they are absorbed and utilised by an animal. Significant time and effort have been utilised by researchers in understanding the relative bioavailability’s of organic minerals, usually by comparison with inorganic mineral sulphates using a range of assessment parameters. There are numerous definitions of bioavailability, but in terms of trace minerals, this may be considered as the relative proportion of an ingested mineral that is absorbed and retained by the animal species under study. Table 3 highlights some examples of zinc source bioavailability studies in poultry. It can be appreciated that in general organic zinc sources have greater bioavailability than their inorganic counterparts. Additionally, inter-study variability can be noted which is not only dependant on the measurement index used to assess bioavailability but also on the source used. In essence, even within groups of organic trace mineral (e.g. proteinates), differences in bioavailability can be observed.

Figure 1 www.international-animalhealth.com

Many factors affect bioavailability including (but not limited to) animal species, sex, physiological state, existing mineral International Animal Health Journal 39

FOOD & FEED Zn source

Zn indices

Relative bioavailability (%)

Reference

Zn sulphate (reagent grade) Zn sulphate (basic) Zn chloride (basic) Zinc oxide (feed-grade)

Bone Zn Bone Zn Bone Zn Bone Zn

100 101 107 49

Cao et al., 2000 Cao et al., 2000 Cao et al., 2000 Cao et al., 2000

Zn sulphate Zn proteinate

Weight gain Weight gain

100 183

Ao et al 2006 Ao et al 2006

Zn sulphate Zn proteinate

Tibia Zn Tibia Zn

100 157

Ao et al 2006 Ao et al 2006

Zn acetate Zn proteinate Zn methionine

Bone Zn Bone Zn Bone Zn

100 110–124 78–91

Cao et al., 2002 Cao et al., 2002 Cao et al., 2002

Zn acetate Zn proteinate Zn methionine

Mucosal MT Mucosal MT Mucosal MT

100 99–130 77–94

Cao et al., 2002 Cao et al., 2002 Cao et al., 2002

Zn sulphate TBZC

Weight gain Weight gain

100 110

Batal et al 2001 Batal et al 2001

Zn sulphate Zn Aa chelate Zn proteinate A

Bone Zn Bone Zn Bone Zn

100 83–104 116 –139

Cao et al., 2000 Cao et al., 2000 Cao et al., 2000

Zn sulphate Zn Aa chelate Zn proteinate A

Mucosa Zn Mucosa Zn Mucosa Zn

100 64–104 65–133

Cao et al., 2000 Cao et al., 2000 Cao et al., 2000

Zn sulphate Zinc-methionine

Bone Zn Bone Zn

100 117–177

Wedekind et al., 1992 Wedekind et al., 1992

Zinc oxide Zinc-methionine

Multiple parameters Multiple parameters

100 100

Pimental et al., 1991 Pimental et al., 1991

Table 3 Relative bioavailability of zinc sources in poultry

status, choice of response criteria, choice of standard source and chemical form and solubility of the mineral element. With respect to chemical form of the mineral, the chelation strength between the mineral and bonding group will define OTM stability and ultimately play a significant role in influencing relative bioavailability. A limited number of studies exist which compare OTM chelation strength with bioavailability. These studies are, however, very insightful and demonstrate a direct link between OTM stability and bioavailability. Table 4 highlights the key findings from a number of these studies in which it is apparent that as chelation strength increases, so too does the relative bioavailability of mineral source. To enhance OTM bioavailability, increasing the strength of bond between the mineral and the bonding group used, will therefore, prove to be a very effective strategy. Premix and feed antagonisms Increasingly, the agonistic and antagonistic effects of feed components have come under scrutiny, with choice of components gaining increasing importance in diet formulation. The possibility for negative interactions occurring between individual components within premixes and feeds is high and often overlooked as are the underlying effects at a cellular level following digestion and absorption of the mineral source. Recent studies have focused on assessing these potential antagonisms. The differential effects noted indicates that not all chelates are created equal; moreover, they all differ in terms of their stabilities, releasing mineral in a pH dependent fashion based on the pH in the local micro-environment. This instability results in some chelates having a negative impact on premix and feed components. Effect of minerals on enzyme activity Very little information is available comparing the potential antagonisms which can occur between different mineral 40 International Animal Health Journal

sources and enzymes within premixes as well as the repercussions that this might have in terms of losing enzyme efficacy. Santos et al., (2014) focused on assessing the potential in vitro interaction between inorganic and organic chelated sources of Fe, Zn and Cu with three commercially available phytase preparations. The study also investigated if the degree of enzyme inhibition was dependent of the type of OTM used as mineral source. The authors demonstrated that a highly significant relationship between phytase inhibition, trace mineral type as well as mineral source and concentration existed. Proteinates were consistently and significantly less inhibitory than the other mineral sources and this was shown in the case of the Escherichia coli and Peniophora lycii phytases for Fe and Zn, as well as for Cu with E. coli and Aspergillus niger phytases. The authors further highlighted the impact of mineral form on phytase activity by calculating the half-maximal inhibitory concentration (IC50), thereby allowing them to indicate how much of each mineral source was needed to inhibit phytase function by half, thus providing a measure of potency of individual mineral sources. Table 5 summarises the IC50 values for the individual Fe sources and phytase enzymes assessed, further illustrating that not all mineral sources are equivalent with respect to their inhibitory impact. Overall, different OTM sources displayed differential effects in their inhibition of exogenous phytase activity. This differential effect indicates that not all OTMs are created equal; moreover, they all differ in terms of their stabilities, releasing mineral in a pH dependant fashion based on the pH in the local micro-environment. The consequences that this Volume 8 Issue 4

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It may well be that the trend towards super-dosing of phytase activities in diets is an unintentional consequence of the negative interactions of premix components. Effect of minerals on Vitamin stability Vitamin oxidation and antioxidant function is primarily caused by autooxidation of fats (a phenomenon which can be selfpropagating) or by trace minerals through Fenton type oxidising reactions. In trace mineral premixes, oxidation-reduction reactions are the predominant cause of vitamin instability. The type of trace mineral will influence its reactivity; copper, iron and zinc being the most reactive and having the greatest potential for vitamin destruction. The form that the trace mineral is presented in however has an even more significant role to play in influencing vitamin stability.

Relative vitamin E acetate loss (%)

A recent study (Concarr et al., 2021) illustrates these effects nicely. The study, which examined Vitamin E stability following short term inclusion in mineral premixes containing inorganic sulphates or different forms of organic minerals, demonstrated that mineral form significantly influenced the stability of α-tocopherol (Figure 2). 0 -5 -10

Vitamin Control

Proteinate 1

Proteinate 2

a,b

Amino Acid Complex

Glycinate

losses than were found within the vitamin control and the proteinate source. This data demonstrates the importance of carefully choosing premix components. An additional study by Concarr et al., (2021b) further examined the destabilising impacts of mineral form on vitamins in premix. The author found that both retinol acetate and cholecalciferol stabilities significantly (P≤0.05) increased within vitamin-trace mineral premixes through the inclusion of chelated mineral. The data indicated that increased levels of trace mineral increased retinol acetate and cholecalciferol degradation in line with the duration of time in storage but that losses could be minimised by switching from inorganic to organic forms. This is nicely illustrated in Figure 3 where the impact of mineral form and level on Vitamin D3 stability is apparent. OTM TRT 30

ITM NRC

ITM Ind 12

0 -10

Week 2

Week 12

-20 -30 -40

-50

Cu Sulphate

Vitamin Premix (Mineral Source and Inclusion rate)

Figure 3

-15

b,c

-20

c,d

-25

-30 -35 -40

OTM NRC 12

-60

Cholecalciferol loss (%)

mineral induced inhibition of enzyme activity has for premix and feed formulation are tremendous and go some way towards explaining the variation noted in supplementation response.

Copper Source

Figure 2

Vitamin E stability in the premixes containing proteinated chelates were not significantly different when compared to the vitamin control. The two premixes that had the highest α-tocopherol acetate loss were those containing the amino acid complex (25.7% decrease) and the glycinate (31.9% decrease). Both these premixes were noted to have higher

Impact of mineral form on antioxidant efficacy Additional research by the same author assessed the effect of mineral form in reducing the efficacy of recognised feed antioxidants such as BHT (butylated hydroxytoluene). This study compared inorganic copper sulphate to different organic mineral sources of copper (glycinates, amino acid chelates and proteinates). The results further indicate that the efficacy of commonly used premix components such as antioxidants can be compromised by the use of inorganic trace elements (Figure 4). The data further indicates that in some cases, organic trace elements also had a significant destabilising impact on antioxidant function. Essentially, weakly bonded minerals may result in liberation of free

Mineral source

Chelation strength (Qf)

Relative bioavailability (%)

Reference

Zn proteinate C Zn proteinate B Zn polysaccharide

120 91 3.8

108 99 94

Cao et al., 2000 Cao et al., 2000 Cao et al., 2000

Zn proteinate A Zn proteinate B Zn Aa complex Zn glycinate Zn methionate

944.02 30.73 6.48 n/r n/r

111–196 107–187 106–162 108–157 109–145

Yu et al., 2010 Yu et al., 2010 Yu et al., 2010 Yu et al., 2010 Yu et al., 2010

Mn Proteinate Mn Aa chelate Mn Aa complex

147 61.9 2.35

169 141 112

Liao et al., 2019 Liao et al., 2019 Liao et al., 2019

Mn Aa chelate C Mn Aa chelate B Mn methionate E

115.4 45.3 3.2

93–114 96–133 95–110

Li et al., 2004 Li et al., 2004 Li et al., 2004

Fe proteinate ES Fe proteinate M Fe methionate

8590 43.6 1.37

174 143–164 102–129

Zhang et al., 2016 Zhang et al., 2016 Zhang et al., 2016

Table 4 Chelation strength is critical to relative bioavailability of OTMs www.international-animalhealth.com

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Conclusions Despite the confusion and often contradictory information that exists, mineral chelation is a relatively straightforward process governed by some fundamental chemistry basics. By carefully considering factors important in mineral chelation, one can begin to distinguish between the products on the basis of their biological stabilities and thus biological bioavailability.

Proteinate 1 Antioxidant activity loss(%)

mineral ions resulting in reactive oxygen species generation which leads to greater oxidation and a reduction in the efficacy of feed antioxidants such as BHT.

Proteinate 2

Amino acid Complex

Glycinate

Cu Sulphate

0 -5 -10

-15

-20 -25

-30 -35 -40

Copper Source

Figure 4

Phytase source

Proteinate

Glycinate

Amino acid complex

Polysaccharide complex

Sulphate

E. coli

6.7 ± 1.1 A 1

3.4 ± 0.3 B C 1

0.9 ± 0.1 C 1

3.7 ± 0.1 B 1

4.1 ± 0.4 B 1

P. lycii

16.7 ± 2.5 A 1

3.8 ± 0.8 B 1

1.4 ± 0.2 B 1

10.4 ± 1.3 C 1

2.9 ± 0.4 B 1

A. niger

10.9 ± 1.0 A 1

6.0 ± 0.9 B 1

1.1 ± 0.1 C 1

12.0 ± 1.3 A 1

8.5 ± 1.0 A B 1

Table 5 IC50 concentration values (ppm) of iron sources for inhibition of phytase (Adapted from Santos et al., 2014)

Richard Murphy Richard Murphy is the research director at Alltech’s European Bioscience Centre. He maintains strong links with numerous Universities and research institutes and has been appointed as an Adjunct Professor at the Faculty of Science and Health at Dublin City University. His research activities are diverse, including areas such as trace element and mineral bioavailability, gastrointestinal health, pathogen control and antimicrobial resistance. Email: rmurphy@alltech.com

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FOOD & FEED

The Value of Circularity in Sustainable Food Systems

The circular economy, or circularity of a system, is an intentional effort to design out waste and pollution, keep products and materials in use and regenerate natural systems. Waste reduction and food loss recovery via the utilisation of coproducts and by-products from other industrial processes has long been a component of the animal feed and pet food supply chain. As the animal food industry considers its role in climate neutrality and other sustainability outcomes, existing processes, as well as new technologies and innovations, are being explored. In this article, we address the role a circularity metric could have in assessing and valuing sustainability efforts for the animal food supply chain and consider measurement and assessment limitations that exist, which restrict the industry’s potential to accurately account for its environmental impact and role in a circular economy. Defining Circularity In the most basic sense, the transition toward a circular economy aims to close the loop on existing linear systems such that produced materials stay in use, waste and pollution are designed out and the regeneration of natural resources is pursued. A pure linear system follows a path of take-makeuse-waste;1 whereas a circular system is a cycle of makeuse-reuse-recycle-recover.

A pure linear system follows a path of take-make-use-waste. In this example, oranges are made into orange juice and the resulting citrus pulp is discarded.

Idealistically, circular systems could operate where waste no longer exists, material loops are closed, and products are recycled indefinitely, but in reality, materials degrade over time and some quantity of new materials and energy must be injected into any circular material loop to overcome these losses.2

As food and agricultural systems evolved in the last 100 years to the scale that currently allows us to feed 7.9 billion people, it generally evolved linearly with input dependencies, resource degradation (e.g., soil organic matter depletion) and environmental pressures generation.1 Increasing the scope of circularity into existing linear food and agricultural systems is paramount to our ability to achieve the desired outcome to sustainably feed the United Nations predicted population of 9.7 billion by 2050, while further reducing and enhancing agriculture’s impact on the environment. The linkage between circular economy and sustainable development is not generally noted in circular economy definitions; however, circular economy transitioning should parallel ongoing sustainability efforts.4 Food and agriculture consists of a complex system of systems. Identifying components to map and transition toward a circular economy within food and agricultural systems depends on where you draw a boundary and consider scale. Here, the animal food sector circularity is considered, and in particular, the role of coproduct and by-products. Scale is variable, but could ultimately be national, across a region or livestock type, or at a corporate level. Coproducts from arable product processing, which are not consumed by people as food or drink or used to produce biofuels or other industrial products, as well as by-products, generally rendered from inedible animal protein, recovered fats and oils, or waste materials, are important ingredient sources for the animal feed and pet food industry. Using these products, the animal feed and animal protein sector contribute to the circular economy.5 Waste reduction and food loss recovery via the utilisation of coproducts and by-products from other industrial processes have long been a component of the livestock feed and pet food supply chain. As the industry considers its role in climate neutrality and other sustainability outcomes, existing processes as well as new technologies and innovations are being explored.

A circular economy, or circularity, can be contemplated at multiple levels of scale across all industry sectors. While numerous definitions exist, Kirchherr et al.3 propose the definition:

Example Circular Components in Feed Production There are many examples in the feed industry where using coproducts or by-products allows the industry to further ‘close a cycle’ toward circularity. Although these materials are intentionally produced, the potential for animal feed use increases the value and sustainability of the production process.

“Circular economy describes an economic system that is based on business models which replace the ‘end-oflife’ concept with reducing, alternatively reusing, recycling and recovering materials in production/distribution and consumption processes, thus operating at the micro level (products, companies, consumers), meso level (ecoindustrial parks) and macro level (city, region, nation and beyond), with the aim to accomplish sustainable development, which implies creating environmental quality, economic prosperity and social equity, to the benefit of current and future generations.”

For example, rapeseed and sunflower production’s primary product is vegetable oil. The remaining coproducts, rapeseed and sunflower meals, concentrated in protein content, are used in feed production. Sugar extracted from sugar beets result in residual sugar beet pulp, which is an excellent ruminant feed. Molasses, for which no human consumption market can be found, is often diverted to feed manufacturing. Citrus pulp left over after juice fruit extraction is another example of a material that represents a significant biophysical share of the original raw material where animal feed provides a solution.

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FOOD & FEED highlighted in platforms of significance, such as the 2021 United Nations Food Systems Summit. The environmental benefits of coproduct and by-product use can be considered from multiple perspectives, each offering insights into the product use impact, though none singularly provide complete insight into the potential value. When used as feed and pet food, coproducts and by-products offer landfill avoidance, material upcycling, reduced environmental footprint, minimised food waste and water recovery. However, while the benefit concepts are identified, benefit quantification is limited. The following offers examples of quantified environmental benefits.

A circular system follows a make-use-reuse-recycle-recover cycle. In this example, oranges are made into orange juice, the resulting citrus pulp is fed to dairy cows, and the resulting organic matter becomes fresh fertilizer for the orange grove.

There are also examples of by-products where the materials destined for feed represent a smaller biophysical share compared to the main product, usually destined for human consumption. Wheat bran remains from bread production and brewers’ grains, and yeasts are left over from beer production. Many people are unaware of the by-products resulting from palm oil production that have a useful purpose in animal nutrition, namely palm kernel meal and palm fatty acid distillates. There are also examples in the slaughtering process of animal carcasses. Because of dietary preferences, a proportion of nutritious and safe animal protein is not destined for human consumption. In meat production, the processed animal protein resulting from the rendering process is a highly digestible protein source, which is very suitable for animal nutrition. In fish processing sites, trimmings remain, which are suitable for carnivorous fish species raised in aquaculture. Another important diversion to animal food is surplus food, which is sometimes referred to as “former foodstuffs.” These are foodstuffs clearly intended for human consumption, but due to a production error are no longer considered suitable. Examples include bread, cookies, breakfast cereals and confectionery. The use of these materials in feed is the most direct contribution of feed production to food waste prevention, as these products obtained a food status and are not downgraded to waste, thanks to their feed use. Due to the use of food-grade ingredients in this case, former foodstuffs represent a source of high-quality nutrients that in principle are not affordable for the feed production sector. Often these processed former foodstuffs can be found on the feed market as bread meal or cookie meal. The former foodstuff processing sector is increasingly able to source materials beyond their traditional suppliers. Coproduct and By-product Environmental Benefits ‘Embracing’ livestock sector circularity potential has been www.international-animalhealth.com

Ondarza and Tricarico6 recently assessed methane emissions from human-inedible, coproduct and by-product feeds based on product type and use within U.S. dairy cattle rations. Accounting for both enteric and manurebased methane production, by-product and coproduct use generated significantly lower emissions than if the material had instead gone to a landfill, 68 versus 3,448 CO2equivalent, g/kg product dry matter. A similar quantification can be made across the U.S. feed supply chain. Each year, U.S. domestic livestock consume 258 million tonnes of feed and approximately 40 percent of those ingredients are upcycled from other industries.7 Because of its use as animal feed, 103 million tonnes are diverted from the landfill and 61 million tonnes of CO2-eq loss are avoided. Rendering is the cooking and drying of meat and/or other animal by-products not used for human consumption in order to recover fats and protein. There are 28.1 million tonnes of materials that can be rendered and produced annually in the U.S. and Canada; without rendering, that volume is sufficient to fill all U.S. landfills in four years.8 In the process of upcycling renderable materials, Gooding9 estimated that the average facility directly emits 20,000 tonnes of CO2 through internal operations and indirectly emits 4,000 tonnes of CO2 via their electrical utility. However, the CO2 emitted through the total direct and indirect emissions is still only 30 percent of the emissions that would result from material decomposition in landfills. Water is also recovered, treated and returned to the environment during rendering. The North American Renderers Association estimates 3.7 billion gallons of clean water are reclaimed and returned to rivers, lakes and streams annually. The above examples illustrate that for the initial owner of coproducts and by-products, the feed and pet food outlet is more sustainable than alternative disposal options. For a feed manufacturer, the use of coproducts and by-products also offer a more sustainable choice when compared to alternative, land-requiring arable sources. Data from the European Former Foodstuff Processors Association (EFFPA) estimates that the use of 3.5 million tonnes of processed former foodstuffs in European countries is equal to over 400.000 hectares of corn, thereby reducing the pressure feed demand puts on arable land. And those processed former foodstuffs have nutritional qualities that make them a good alternative feed ingredient to arable sources such as corn, wheat or barley. A study by Giromini et al.10determined that processed former foodstuffs can be considered a “fat-fortified version of common cereal grains” thanks to the generally higher fat content while being comparable in starch content. From a biochemical point of view, lipids contain carbon and hydrogen in a more reduced state compared with other nutrients (e.g., carbohydrates and proteins). The better potential for oxidisation therefore provides a greater energy yield. Given the proven difficulties in handling fats International Animal Health Journal 45

FOOD & FEED

and mixing them with other ingredients for the formulation of complete feeds, former foodstuffs represent a valuable processing advantage in compound feed production given that the lipids are already part of the matrix. The previously heat-treated (or cooked) starch can be speculated to be of relatively high digestibility quality, which is of particular interest due to the limited capacity of piglets to digest raw starch. While the aforementioned data points to the environmental benefit potential of coproduct and by-product use, the data set is limited. As the value of circular economy in food and agricultural systems gains prominence, the quantification of outcomes and impacts must expand. Potential Circular Economy Metrics The circularity of a system is ultimately defined by how completely the loop can be closed. And while it can be helpful to assess the circularity of each input singularly (i.e., flow of a particular material within the system), ultimately all inputs and losses must be considered collectively. Therefore, energy or water requirements needed to upcycle a coproduct or by-product, as well as carbon or nitrogen loss following animal consumption, are as important to assessing the circular economy as tracking the flow of the materials within the system. Circular economy can be used as a benchmark to measure progress on a scale ranging from linear at one end to perfectly circular at the other. Therefore, we could ask: what is the current degree of circularity and how far could we realistically move toward perfect circularity within any food system? With this as a measure, a ratio (α) of total recovered material to total material demand could be performed where perfect circularity equals one.2 Unfortunately, material “upcycling” (i.e., transforming unwanted by-product into a higher quality or higher valued item) is generally only possible with the addition of energy to the system; therefore, additional questions and metrics should be considered. How much energy is needed to upcycle or restore the material to the desired product, and how does the required energy needed for upcycling compare to obtaining the desired equivalent from a virgin source?2 Cullen proposes, calculating energy considerations (β) as one minus the ratio of energy required to recover or upcycle 46 International Animal Health Journal

material (e.g., coproduct or by-product) to energy required to create an equivalent input from a virgin source, where, again, the resulting value equals one for perfect circularity. For animal feed coproduct and by-product use, this could mean quantifying energy for the virgin-sourced material based on nutritional equivalents from other sources or raw commodities. Proponents of circular economy do not always consider the energy input needed to sustain circularity, but the benefit of material handling should not be considered without addressing the impact in conjunction with energy. Therefore, cojoining the two means a circularity index could be quantified by multiplying α and β, again where perfect circularity equals one.2 Two other circularity metric considerations include: 1) identifying the number of times a resource is used in a product system along with longevity, the length of time a resource is used, as an indicator of resource efficiency in the circular economy. Where the higher the number the indicators show, the higher the contribution to circularity.11 And 2) accounting for a combination of material mass flow and product utilisation rate, where the greater the material recirculation and higher the utilisation rate of a product, the better the circularity. Assessing circular economy within food and agricultural systems is challenging because both material and energy flows must be coupled with production, processing, distribution and consumption. 1 The Ellen MacArthur Foundation12 revised methodology for circularity indicators includes considerations for biological based materials, and it acknowledges the challenge associated with ‘material loss’ when typical downstream product recovery (e.g., the recovery of plastic for continued reuse) is not possible in agricultural systems. This complexity inherent to food and agricultural systems deserves additional focus and standardisation to accurately assess their value and benefits. Market Recognition and Acceptance of Circularity’s Value Circularity in feed production has clear sustainability merit. It allows for a different kind of resource use, while contributing to food production. In the animal production value chain, the circularity of feed, i.e., the use of coproducts and by-products, deserves to be a key indicator of sustainable feed production. Volume 8 Issue 4

FOOD & FEED Increasingly, there are production specifications laid down by upstream value chain partners that could restrict feed producers from optimising circularity in feed formulation. A product specification that surfaces and is often part of a claim on the animal product labelling, is the demand for plant-based animal feed. In Europe, market development for animal protein produced with plant-based feed has increased with the reapproval of certain processed animal proteins as feedstuffs. In Europe, former foodstuffs, where often animal by-products, such as milk, eggs or honey, are constituents, are then also excluded. In the U.S., examples have arisen where products labelled as natural beef require vegan-based animal feed sources, thus preventing use of the key coproduct, whey, from milk processing. The by-products resulting from palm oil production are sometimes even specifically targeted. Due to the negative perception of palm oil, feed manufacturers are sometimes asked to exclude all ‘palm oil products’ from the feed formulation, although it can hardly be argued that the feed use of palm kernel meal or palm fatty acid distillates are a factor of importance for palm tree expansion. At the same time, food manufacturers themselves are suppliers to the feed industry through the supply of former foodstuffs. In this position as supplier, it is important that a circular economy mentality is adopted. Selling resources to the feed sector should never be seen as a disposal route, but very much the placing on the market of so-called “secondary raw materials.” Future Opportunities for Circularity in Animal Feed and Pet Food Circularity will continue to evolve as an indicator within the broader sustainability discussion. Relative to the biologic, interconnected and complex nature of food and agricultural systems, advancing our ability to measure and assess those systems’ circularity is paramount to gaining sustainability benefits of a circular economy. The use of coproducts and by-products in feed and pet food is just one component of circularity opportunities in food and agricultural systems. As the feed and pet food industry strives to offer and communicate benefits and solutions available to the animal and pet food consumers, the industry must be able to accurately reflect economic and environmental opportunities available through greater circularity. This is important in demonstrating reduced environmental impact as well as need to access and use of currently available ‘waste’ from other industries. Due to the potential scope and scale of system boundaries, the industry would benefit from methodologies capable of assessing the circularity of complex food and agricultural systems. As a methodology for complex biological systems becomes standardised and normalised by use, feedstuff circularity could become commonplace, thereby providing feed manufacturers with a clear incentive to maximise the content of ‘circular feed’ in their formulations. REFERENCES 1. 2. 3. 4.

Basso, B., Jones, J., Antle, J., Martinez-Feria, R., Verma, B. Enabling circularity in grain production systems with novel technologies. Agricultural Systems. 193, 103244 (2021) Cullen, J. Circular Economy – theoretical benchmark or perpetual motion machine. Journal of Industrial Ecology. 21 (3) 483-486 (2017) Kirchherr, J., Reike, D., Hekkert, M. Conceptualizing the Circular Economy: an analysis of 114 definitions. Resources, Conservation & Recycling 127, 221-232 (2017). Pauliuk, S. Critical appraisal of the circular economy standard

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9. 10.

11.

12.

BS 8001:2017 and a dashboard of quantitative system indicators for its implementation in organizations. Resources, Conservation & Recycling 129, 81-92 (2018) European Feed Manufacturer’s Federation. Resource efficiency champions: co-products, an essential part of animal nutrition. https://fefac.eu/wp-content/uploads/2020/07/05362_coproducts_brochure_003.pdf (2019) de Ondarza, M.B., Tricarico, J.M. Nutritional contributions and non-CO2 greenhouse gas emissions from human-inedible byproduct feeds consumed by dairy cows in the United States. Journal of Cleaner Production 315, 128125 (2021). Institute for Feed Education and Research. Animal feed/food consumption and COVID-19 impact analysis. Prepared by Decision Innovation Solutions. http://ifeeder.org/wp-content/ uploads/210301-FINAL-REPORT-IFEEDER-Animal-Feed-FoodConsumption-COVID-19.pdf (2020) Wilkinson, A.D. and Meeker, D.L. How agriculture rendering supports sustainability and assists livestock’s ability to contribute more than just food. Animal Frontiers 11(2) 24-34 (2021). Gooding, C.H. Data for the carbon footprinting of rendering operations. Journal of Industrial Ecology. 16(2) 223-230 (2012). Giromini, C., Ottoboni, M., Tretola, M., Marchis, D., Gottardo, D., Caparulo, V. Nutritional evaluation of former food products (ex-food) intended for pig nutrition. Food Additives and Contaminants: Part A. 34(8) pp. 1436-1445 (2017). Figge, F., Thorpe, A.S., Givry, P, Canning, L., Franklin-Johnson, E. Longevity and circularity as indicators of eco-efficient resource use in the circular economy. Ecological Economics. 150, 297306 (2018). Ellen MacArthor Foundaton Circularity Indicators: An approach to measuring circularity. https://ellenmacarthurfoundation. org/material-circularity-indicator (2019)

Lara Moody Lara Moody is the executive director for the Institute for Feed Education and Research (IFEEDER), where she provides visionary leadership to the U.S.-based public charity’s activities, including program development, strategic partnerships and collaborative resourcing. Most recently, she has been working to promote awareness of IFEEDER’s Sustainability Road Map project, which will help the U.S. animal food industry lower its environmental footprint and advance industry solutions to the global climate change challenge. Email: lmoody@afia.org

Anton van den Brink Anton van den Brink is the senior policy and communication manager for the European Compound Feed & Premixes Association (FEFAC), where he is engaged on sustainabilityrelated topics connected to European compound feed manufacturing, such as responsible soy sourcing, environmental foot printing, circular economy, nutrient efficiency and food waste. He played a leading role developing FEFAC’s Feed Sustainability Charter 2030 and revised Soy Sourcing Guidelines. Van den Brink also serves as executive director of the European Former Foodstuff Processors Association. Email: avandenbrink@fefac.eu

International Animal Health Journal 47

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Trace minerals –

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With more seemingly “organic” trace mineral sources being produced, nutritionists need to pay close attention to what they are using — right down to biological structure and stability — to get the most nutritional and economic value from their feed formulation. The newly launched white paper review from Dr. Richard Murphy comprehensively surveys all considerable aspects when selecting a trace mineral source for your business.

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