The effect of the fat / starch ratio in young

horses´ diet on muscle endurance and

temperament

M.Sc. Thesis by

Carina Beblein

Aarhus University

June 2017

The effect of the fat / starch ratio in young

horses´ diet on muscle endurance and tem-

perament

M. Sc. Thesis by

Carina Beblein Au339496

Department of Animal Science, Faculty of Science and Technology Aarhus University

June 2017

Supervisors

Søren Krogh Jensen, Dept. of Animal Science, Faculty of Science and Technology, Aar-hus University, Blichers Allé 20, 8800 Tjele, Denmark Janne Winther Christensen, Dept. of Animal Science, Faculty of Science and Technol-ogy, Aarhus University, Blichers Allé 20, 8800 Tjele, Denmark Lone Hymøller, Dept. of Animal Science, Faculty of Science and Technology, Aarhus University, Blichers Allé 20, 8800 Tjele, Denmark

Preface

3

Preface This thesis is written as a completion of my Master of Science in Agrobiology at Department

of Animal Science, Aarhus University. The thesis corresponds to 45 ECTS points. The horses

used for the study had only experienced minimal handling. Therefore, habituation to han-

dling was necessary. Habituation to handling was initiated in august 2016, in September 2016

baseline fear tests were made and the following months until 20th, of February, the horses

were trained to enter and walk on a treadmill. Subsequently there was a training and data

collection period of 9 weeks. Analysis conducted in the study are on both whole blood and

plasma samples, but due to limited time, this thesis only contains the analysis on whole blood

samples.

This thesis contains a main part and three appendixes. The main part consists of a manuscript,

based on the experiments performed for this master thesis. This part will be submitted to a

scientific journal and is therefore intended for people with scientific interest in behaviour and

muscle endurance related to the fat / starch ratio in diets.

The appendix A consists of additional illustrations, habituation protocols, training protocol

and an overview of the training and data collection period. Appendix B contains additional

illustration of results and Appendix C contains a summary of a previous literary study on the

dietary effect on inflammation in riding horses.

I would like to thank my supervisors Søren Krogh Jensen, Janne Winther Christensen and

Lone Hymøller for guidance, inspiration and support during the whole process of conducting

this thesis. Furthermore, I would very much like to thank Nanna Eline Uhd Weldingh, Isabella

Hansen, Mette Dam Madsen, Rikke Bundgaard Tolstrup, Inuk Kathrine Melgaard and Anne

Cathrine Jensen for the practical help during the training periods. For making this study pos-

sible, I would like to thank St. Hippolyt and the Toosbuy foundation for their financial contri-

bution. Also thanks to all the participating horses and their owner, without the horses the

study would not have been possible.

Finally, I would like to thank Anders Fjordside Madsen, my family and friends for proofread-

ing and general support throughout the process.

Aarhus, June 2017

_______________________________________________

Carina Beblein

Abstract

4

Abstract Horses are often fed large amounts of starch to accommodate the energy requirements of mus-

cles during exercise. Feeding large amounts of starch has resulted in negative health issues

for horses. To reduce starch intake, fat has been used as substitute energy source. The aim of

this study was to investigate how the fat/starch ratio in feed affects young horses’ muscle

endurance and temperament. Twenty Danish Warmblood stallions were randomly allocated

to two treatments: Low Starch/High Fat (LS_HF) (n=10) and High Starch/Low Fat (HS_LF)

(n=10) for nine weeks. During the two last weeks, two exercise tests were performed. An in-

cremental exercise test (ET-1) and a single step exercise test (ET-2). The intensity of the exercise

tests, ensured that horses remained on aerobic metabolism during exercise. Additionally, two

fear tests were conducted to examine if the treatments used affected fearfulness. The fear tests

were a novel object test (T1) and object test (T2). No significant difference was found between

treatments with respect to whole blood glucose levels, though horses fed LS_HF decreased

significantly in whole blood glucose as a consequence of exercise in ET-2 (P=0.045). Horses

fed LS_HF had significantly higher whole blood calcium levels compared to horses fed HS_LF

in ET-2 (pre: P=0.04 and post: P=0.04). Whole blood lactate levels increased slightly when

horses were fed HS_LF, whereas whole blood lactate levels decreased slightly in horses fed

LS_HF (Lac∆: P=0.10). Results indicated that horses fed LS_HF might be able to postpone

muscle fatigue because of a later shift to anaerobic metabolism and higher post-exercise whole

blood calcium levels. No difference was found in T1 between treatments, though horses fed

LS_HF showed more investigative behaviour in T2 (P=0.07).

Resumé

5

Resumé Heste bliver ofte fodret med store mængder stivelse for at imødekomme det energikrav som

muskler har under træning. Den store mængde af tildelt stivelse har vist sig at påvirke hestens

sundhed negativt. For at reducere stivelsesmængden, er fedt blevet brugt som supplement

eller substitut til energiforsyningen. Formålet med dette studie var at undersøge hvordan sti-

velses/fedt forholdet i unge hestes foder påvirker deres muskeludholdenhed og tempera-

ment. I forsøget blev tyve Dansk Varmblods hingste fordelt tilfældigt på to diæter: Lav Sti-

velse/Høj Fedt (LS_HF) (n=10) og Høj Stivelse/Lav Fedt (HS_LF) (n=10) i ni uger. I de to

sidste uger af behandlingsperioden blev der foretaget to træningstests. Den første trænings-

test (ET-1) steg gradvist i intensitet igennem 12 minutter, mens den sekundære test (ET-1)

hurtigt steg til et forud bestemt niveau og efterfølgende gik hesten seks minutter på dette

niveau (niveau 6). Intensiteten i træningstestene sikrede at hestene generede deres energi gen-

nem aerob forbrænding. Desuden blev to frygttests gennemført for at se om diæterne havde

indflydelse på hestenes frygtsomhed. Den første var en objekt test (T1) hvor et ukendt objekt

blev benyttet og den sekundære objekt test (T2) blev et kendt objekt benyttet. Der blev ikke

fundet signifikant forskel mellem behandlingerne mht. fuldblod glukose koncentration, dog

faldt heste fodret med LS_HF signifikant i fuldblod glukose koncentration som konsekvens af

træning. Heste fodret med LS_HF havde signifikant højere kalcium koncentrationer i blodet

sammenlignet med heste fodret med HS_LF. Der var en tendens til at heste fodret med HS_LF

steg i laktat koncentration i blodet, mens heste fodret med LS_HF faldt i laktat koncentratio-

nen i blodet, som konsekvens af træning. Resultaterne indikerer at heste fodret med LS_HF

muligvis er i stand til at udsætte muskeltræthed, pga. det senere skift fra aerob forbrænding

til anaerob forbrænding. Dette begrundes med at heste fodret med LS_HF havde et fald i

laktat niveauet og højere kalcium koncentrationer i blodet. Ingen signifikante forskelle mellem

behandlingerne blev fundet i objekt testene, dog var der en tendens til at heste fodret med

LS_HF udviste mere udforskende adfærd i T2.

List of abbreviations

6

List of abbreviations Accu.: Accumulated

ADP: Adenosine diphosphate

ATP: Adenosine Triphosphate

Avg: Average

Bwt: Body weight

CoA: Acetyl Coenzyme A

CO2: Carbon Dioxide

CAT-1/CAT-2: Carnitine Translocase 1 and 2

ET-1: Exercise Test 1

ET-2: Exercise Test 2

ETs: Exercise Tests

FA: Fatty Acids

FACS: Fatty Acid Synthase

FADH: Flavin adenine dinucleotide

FADH2: Flavin adenine dinucleotide (reduced)

GLUT: Glucose Transporter

h: Height

HR: Heart Rate

HS_LF: High Starch / Low Fat

LDH: Lactate Dehydrogenase

l: Length

LS_HF: Low Starch / High Fat

m: Meter

Max: Maximum

NA: Not Available

NAD: Nicotinamide adenine dinucleotide

NADH: Nicotinamide adenine dinucleotide (reduced)

O2: Oxygen

Pi: Phosphate group

Sec: Seconds

SEM: Standard Error Mean

TAG: Triacylglycerol

THRM-1: First Training Heart Rate Measurements

THRM-2: Second Training Heart Rate Measurements

T1: Object Test 1

T2: Object Test 2

VFA: Volatile Fatty Acids

w: Width

Content

4

Content

Preface .................................................................................................................................................... 3

Abstract.................................................................................................................................................. 4

Resumé .................................................................................................................................................. 5

List of abbreviations ............................................................................................................................ 6

List of figures ........................................................................................................................................ 9

List of tables ........................................................................................................................................ 10

1. Introduction ................................................................................................................................ 12

1.1. Digestion ............................................................................................................................. 13

1.2. ATP generation ................................................................................................................... 14

1.3. The exercising horse .......................................................................................................... 18

1.4. Behaviour ............................................................................................................................ 19

2. Materials and methods .............................................................................................................. 21

2.1. Animals and diets .............................................................................................................. 21

2.2. Physical performance ........................................................................................................ 22

2.2.1. Exercise tests ............................................................................................................... 23

2.3. Behaviour ............................................................................................................................ 24

2.3.1. Baseline fear tests ....................................................................................................... 24

2.3.2. Final fear tests ............................................................................................................. 24

2.3.3. Feed motivation test................................................................................................... 26

2.4. Statistics ............................................................................................................................... 26

2.4.1. Exercise tests ............................................................................................................... 26

2.4.2. Final fear test ............................................................................................................... 26

3. Results .......................................................................................................................................... 28

3.1. Exercise tests ....................................................................................................................... 28

3.2. Final fear tests ..................................................................................................................... 33

4. Discussion ................................................................................................................................... 35

4.1. Glucose ................................................................................................................................. 35

4.2. Muscle fatigue .................................................................................................................... 38

4.3. Behaviour ............................................................................................................................ 42

5. Conclusion .................................................................................................................................. 46

6. Perspectives................................................................................................................................. 47

7. Literature ..................................................................................................................................... 48

Appendix A ........................................................................................................................................... A

Content

8

Appendix B ........................................................................................................................................... B

Appendix C ........................................................................................................................................... C

List of figures

9

List of figures Figure 1: Glucose metabolic pathways for both aerobic and anaerobic conditions (p. 14)

Figure 2: Citric acid cycle and the electron transport chain (p. 15)

Figure 3: Cori cycle (p. 15)

Figure 4: Fatty acid metabolic pathway to generate ATP during aerobic condition (p. 16)

Figure 5: Oxidation of fatty acids (p. 16)

Figure 6: Muscle contraction (p. 17)

Figure 7: Insulin sensitivity (p. 19)

Figure 8: Overview of the settings used in this study (p. 20)

Figure 9: Overview of the timeframe and structure of the study (p. 21)

Figure 10: Illustrations of the object test (p. 24)

Figure 11: Positive correlation pre-exercise whole blood lactate and glucose levels for horses

fed HS_LF in ET-1 (p. 31)

Figure 12: Positive correlation post-exercise whole blood lactate and glucose levels for

horses fed HS_LF in ET-2 (p. 32)

Figure 13: Decrease in whole blood glucose levels during test days (p. 32)

List of tables

10

List of tables Table 1: Speed levels used on treadmill (p. 22)

Table 2: The time horses spent on the different speed levels in ET-2 (p. 22)

Table 2: Ethogram of the recorded behaviours in object tests (p. 25)

Table 4: Whole blood levels for oxygen, carbon dioxide, glucose, lactate and calcium pre-

and post-exercise in ET-1 (p. 28)

Table 5: Whole blood levels for oxygen, carbon dioxide, glucose, lactate and calcium pre-

and post-exercise in ET-2 (p. 29)

Table 6: Difference between pre-exercise and post exercise for whole blood levels for

oxygen, carbon dioxide, glucose, lactate and calcium (p. 29)

Table 7: Maximum heart rate, average heart rate and accumulated heart rate for ET-1, ET-2,

THRM-1 and THRM-2 (p. 30)

Table 8: Duration of behavioural displays and heart rate during the two object tests (p. 33)

Table 9: Comparison between the different studies used in physiological discussion (p. 40)

Table 10: Comparison between the different studies used in the behavioural discussion (p.

44)

Table 11: Habituation of protocol to handling and object test (p. A1)

Table 12: Treadmill habituation protocol (p. A2)

Table 13: Training programs (p. A3)

Table 14: Overview over the nine weeks of study (p. A4)

Table 15: Correlations between whole blood levels of carbon dioxide, oxygen, glucose,

lactate, Max heart rate and average heart rate in ET-1, part 1 (p. B1)

Table 16: Correlations between whole blood levels of carbon dioxide, oxygen, glucose,

lactate, Max heart rate and average heart rate in ET-1, part 2 (p. B1)

Table 17: Correlations between whole blood levels of carbon dioxide, oxygen, glucose,

lactate, Max heart rate and average heart rate in ET-1, part 3 (p. B2)

List of tables

11

Table 18: Correlations between whole blood levels of oxygen, glucose, lactate, Max heart

rate and average heart rate in ET-2, part 1 (p. B3)

Table 19: Correlations between whole blood levels of oxygen, glucose, lactate, Max heart

rate and average heart rate in ET-2, part 2 (p. B4)

Table 20: Correlations between latency, average heart rate, maximum heart rate, object

focus, touch, sniff and feed motivation in T1 (p. B5)

Table 21: Correlations between latency, average heart rate, maximum heart rate, object

focus, touch, sniff and feed motivation in T2 (p. B5)

Introduction

12

1. Introduction Horses have evolved on scarce grasslands. The adaptation to these grasslands enables horses

to generate energy from extraordinarily poor forage by bacterial fermentation in the hindgut

(Kuntz et al., 2006). Today, most sports horses are fed two - three times per. day with an energy

dense diet, to accommodate the energy demand from the muscles during exercise. Horses are

often fed diets based on cereals (barley, wheat or maize) (Vervuert et al., 2009), which mainly

consist of starch in order to cover the energy demand for exercising. Starch is broken down

by -amylase and digestive enzymes in the small intestine. If large amounts of starch are fed

to horses, it may exceed the degradation capacity of digestive enzymes in the small intestine.

This causes undigested starch to pass to the hindgut where it is rapidly fermented by bacteria.

Fermentation of starch causes an imbalance between the volatile fatty acids which lowers the

pH in the hindgut (Geor et al., 2013). Low pH in the hindgut leads to negative effects on the

horse´s health such as; overgrowth of undesired bacterial populations and lysis of desired

bacterial populations and laminitis (Hoffman, 2009). Vervuert et al. (2009) found that increas-

ing amounts of starch, increased post-prandial blood glucose and insulin levels. Similar re-

sults have been found by Jansson and Lindberg (2012), who examined the effect of allocation

of only haylage or haylage and concentrate. Treiber et al. (2005) suggested that starch-rich

diets may promote insulin resistance in the horse, when exposed to high blood glucose levels

for a long period of time. Insulin resistance is a potential risk factor associated with osteochon-

dritis, Cushing´s disease, colic and laminitis (Hoffman, 2009). Meyers et al. (1989) reported

that adding fat to a diet could allow a reduction in feed intake, thereby reducing the risk of

the above-mentioned health issues (Delobel and Cuvelier, 2008). Fats are hydrophobic and

therefore cluster in the aquarious environment of the digesta. For lipases to be able to break

down fat into fatty acids, fat is emulsified by bile (Geor et al., 2013). Adding fat to diets has

been found to maintain a high-energy level in the feed, constant body weight and condition

for the horse (Meyers et al., 1989). The energy density is higher in fatty acids compared to

glucose. One molecule of glucose with a molecule weight of 180.2 g/mol results in 38 ATP

(Berg et al., 2012), whereas one molecule of fatty acid (C18) with a molecule weight of 216.2

g/mol can generate 120 ATP (Berg et al., 2012). I.e. less fat is needed to maintain energy levels,

therefore a reduction in feed intake is possible.

Studies have showed ambiguous results regarding fats effect on blood glucose. Addition of

10% added fat to the diet of horses reduced blood glucose levels (Meyers et al., 1989), whereas

Hambleton et al. (1980) found that high levels (16%) of added fat increased blood glucose

levels.

Besides the energy density being different between fat and starch, so are the metabolic path-

ways of fat and starch. The horse as a biological system is able to use different pathways to

generate energy for exercising, depending on the substrate available (Bosma, 2016). When

consuming starch-rich diets, the biological system enhances metabolic pathways relevant to

glucose, whereas when horses digest fat-rich diets, fat metabolism pathways are upregulated

(Noland, 2015). Besides the substrate availability affecting metabolic pathways, the intensity

Introduction

13

and type of exercise will also influence the metabolic pathways during exercise. High intensity

exercise favours anaerobic metabolism, whereas low intensity exercise favours aerobic metab-

olism. The shift between aerobic and anaerobic metabolism, together with lactate accumula-

tion in muscles, has been used as a measure of fitness in horses (Davie and Evans, 2000). Lac-

tate accumulation is initiated during anaerobic metabolism and has been associated with mus-

cle fatigue (Andrade and McMullen, 2006). To examine the shift between aerobic and anaero-

bic metabolism during exercise and when lactate accumulation occurs, blood lactate response

to exercise at different intensities has been studied in horses (Andrade and McMullen, 2006;

Roneus et al., 1994; Schuback and Essen-Gustavsson, 1998). None of these studies had consid-

ered feed as a potential factor influencing the exercise capacity of the horse. Meyers et al.

(1989) found that blood lactate levels tended to be lower, when fat was added to the horses´

diets and Jansson and Lindberg (2012) found that the absence of starch in the feed lowered

blood lactate levels. Generally, the current knowledge about how starch and fat influence the

horse during exercise is limited.

Additionally, it has been suggested that diet may not only affect physical performance in

horses, but may also affect behaviour. The effect of feed has been examined in relation to ab-

normal behaviour and it has been suggested that feed influences abnormal behaviour, though

the mechanism is not clear (Hemmann et al., 2013). Nicol et al. (2002) found that foals were

more likely to develop crib-biting when fed a high concentrate feed. Furthermore, Freire et al.

(2009) found that horses fed a starch-rich diet explored their barn less. In addition, foals fed

fat-rich diets have been shown to investigate more compared to foals fed starch-rich diets

(Nicol et al., 2005). Finally, Redondo et al. (2009) found that horses fed a fat-rich diet were less

startled by an umbrella opening compared to horses fed a starch-rich diet.

Knowledge about how fat and starch affects muscle endurance and behaviour is limited.

Therefore, the aim of this study was to examine how two diets with High Starch/Low Fat and

Low Starch/High Fat, respectively, affected muscle endurance and behaviour in young

horses. The hypothesis was, that horses fed a Low Starch/High Fat diet, would have better

muscle endurance, by having lower whole blood lactate levels, compared to horses fed a High

Starch/Low Fat diet. Furthermore, it was hypothesised that horses fed a High Starch/Low

Fat diet would have higher whole blood glucose levels, compared to horses fed a Low

Starch/High Fat diet. Finally, it was hypothesised that horses fed a Low Starch/High Fat diet

would show reduced fear reactions, compared to horses fed a High Starch/Low Fat diet.

1.1. Digestion

Starch

Starch is a sub classification of carbohydrates which can be subject to hydrolysation or may

undergo fermentation in the gastrointestinal tract (Geor et al., 2013). Starch is hydrolysable

Introduction

14

by α-amylase, because of the α-1,4 linkages between the glucose molecules, whereas carbohy-

drates containing β-1,4 linkages e.g. β-glucans, hemicellulose and cellulose must be fer-

mented.

In humans, hydrolysation of starch is initiated in the mouth by α-amylase present in the saliva.

Relatively little α-amylase is present in the saliva of horses, therefore the degree of hydroly-

sation in the mouth is limited in horses (Geor et al., 2013). Furthermore, the digestion of starch

is partial in the stomach, due to α-amylase being non-functioning in the acidic environment

of the stomach. When digesta reaches the small intestine, the pancreas releases α-amylase,

which hydrolyses starch into disaccharides and oligosaccharides. Additionally, enterocytes

lining the small intestine have brush border enzymes, such as maltase and isomalactase,

which complete the hydrolysation of disaccharides and oligosaccharides into glucose (Geor

et al., 2013). Glucose is able to enter the enterocytes through a transport protein, with sodium

functioning as an osmotic gradient. Once glucose is inside the enterocyte, it can enter the blood

stream through the glucose transporter protein GLUT 2, consequently raising blood glucose

levels. At this point glucose is available as energy for the cells or for storage as glycogen in

muscles or the liver.

Fat

Dietary fat and fat added to horses´ diets is mainly composed of triacylglycerol (TAG) (Geor

et al., 2013). TAG is an organic structure containing a glycerol backbone and three fatty acids

(FAs). FAs are hydrocarbon chains, which can vary in length from 2 to 28 carbons atoms.

Digestion of TAG starts in the mouth, where the teeth mechanically disrupt the TAG, which

cluster in lipid droplets. Subsequently the partially disrupted TAG continues to the stomach

where especially the contractions of the antrum, when the pylorus closes, initiates the emul-

sification of TAG (Phan and Tso, 2001). Besides from the mechanical disruption of TAG in the

stomach, there is also some degree of enzymatic hydrolysation. Subsequently, the partially

disrupted TAG enters the upper part of the small intestine (duodenum). Here bile, which is

released continuously because horses lack of a gall bladder (Geor et al., 2013), emulsifies the

lipid droplets once again. This allows lipase, which is positioned in the mucous of the entero-

cytes, to hydrolyse TAG into a glycerol back bone and three free FAs. FAs diffuse across the

cell membrane into the cytoplasm of the cell. Inside the cell, FAs are reconverted into TAGs

which pack together with lipoprotein surrounding the cluster of TAGs, forming a chylomi-

cron. This is a necessary process, because TAGs are hydrophobic and therefore cannot be

transported in aquarious environments, unless they are covered with hydrophilic lipopro-

teins. Chylomicrons are transported to the lymphatic vessels, from where the chylomicrons

are later released to the blood stream. When chylomicrons enter the bloodstream, they are

hydrolysed to FAs by lipoprotein lipases. FAs are at this point available for absorption by the

cells to generate energy or for storage as TAG in adipose tissue.

1.2. ATP generation For the exercising horse, there is primarily two substrates from where energy (ATP) can be

generated: Glucose or fatty acids. Muscles can obtain glucose from the blood stream or from

Introduction

15

intracellular glycogen. Glucose crosses the sarcolemma by facilitated diffusion and enters into

the cytoplasm of the muscle cells. Facilitated diffusion, is dependent on the presence and ac-

tivation of a transport protein complex named GLUT4 and a diffusion gradient (Na+) (Richter

and Hargreaves, 2013; Rivero and Hill, 2016). Through glycolysis, glucose generates pyruvate

(fig. 1). The following process is dependent on whether the environment in the cell is anaero-

bic or aerobic (Michalsik, 2007a, b). Under aerobic conditions pyruvate is transported into the

mitochondria matrix where it is oxidised to Acetyl CoA (equation 1). Subsequently Acetyl

CoA enters the Citric Acid Cycle, where high energy electrons (NADH and FADH2) are re-

moved from the carbon chain (fig 2.a). NADH and FADH2 enter the Electron Transport Chain

(fig. 2.b), positioned in the inner mitochondrial membrane, where they reduce O2 into H2O,

which creates a proton gradient (H+), which is then used to generate ATP through the protein

complex called “ATP-synthase” (Fig 2.b).

(1) 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷+ + 𝐶𝑜𝐴 → 𝑎𝑐𝑒𝑡𝑦𝑙 𝐶𝑜𝐴 + 𝐶𝑂2 + 𝑁𝐴𝐷𝐻 + 𝐻+

Figure 1. Glucose enters the cell through facilitated transport. In the cytoplasm glucose undergoes glycolysis and generates pyruvate. The following pathway is dependent on whether oxygen (O2) is present or absent. When O2 is present (aerobic) pyruvate enters the mitochondria, where it is oxidised to Acetyl CoA and subsequently enters the Citric acid cycle and the electron transport chain to generate energy. When O2 is absent (anaerobic) pyruvate is fermented to lactate to generate ATP.

Introduction

16

During anaerobic conditions pyruvate is fermented into lactate to generate ATP (fig. 1, equa-

tion 2). This takes place in the cytoplasm of the muscle cell, instead of entering the mitochon-

dria as during aerobic conditions.

(2) 𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 3𝐴𝐷𝑃 + 3𝑃 → 2 𝑙𝑎𝑐𝑡𝑎𝑡𝑒 + 𝐴𝑇𝑃

Lactate is converted back into pyruvate by lactate dehydrogenase (LDH) in the liver (fig. 3).

Imbalance between lactate production and lactate removal, results in lactate accumulated in

the cells during anaerobic conditions (Andrade and McMullen, 2006). Lactate accumulation

Figure 2. A shows the Citric Acid Cycle and B shows the Electron Transport Chain

Figure 3. Cori cycle, where Lactate dehydrogenase (LDH) is the key enzyme in fer-menting pyruvate to lactate and in the reverse reaction.

Introduction

17

has been associated with muscle fatigue and lactate response to exercise is often used as a

measure of the horses performance capacity (Lindner et al., 1992).

Fatty acids

The other substrate from where horses can generate ATP is

FAs. FAs are oxidised in the mitochondria matrix to generate

energy and this can only proceed under aerobic conditions. Be-

fore FAs can function as fuel for muscle cells during exercise,

FAs need to be activated. Fatty acid synthase (FACS) activates

FAs by adding a Coenzyme A (CoA), thereby converting the

FAs to Acyl CoAs (fig. 4). Subsequently the Acyl CoAs are to

be transported into the mitochondrial matrix aided by the en-

zyme Carnitine Translocase 1 (CAT-1). CAT-1 adds a carnitine

to the Acyl CoA, forming an Acyl Carnitine (fig. 4). The Acyl

Carnitine is able to cross the inner mitochondria membrane

through a transport protein “Acyl Carnitine Translocase”

(Akers and Denbow, 2008). Inside the mitochondrial matrix, an

enzyme “Carnitine Translocase 2” (CAT2), detaches the car-

nitine, reconverting the Acyl CoA (fig. 4). Acyl CoA is then ox-

idized and the end products (NADH, FADH2 and Acetyl CoA)

Figure 4. Fatty acid pathway to generate ATP under aerobe conditions. Fatty acids enter the cell, where they are activated by Fatty acid synthase (FACS) converting the fatty acids into Acyl CoA. Subsequently Carnitine translocase 1 (CAT-1) adds a carnitine to the Acyl CoA, which allows it to cross the inner mitochondrial matrix through Acyl carnitine translocase (blue). Inside the mitochondria the carnitine is detached by Carnitine translocase 2 (CAT-2) and the Acyl is oxidised into Acetyl CoA, which can enter the Citric acid cycle.

Figure 5. β-oxidation of a fatty acid.

Introduction

18

(fig. 5) enter the Citric Acid Cycle and subsequently the electron transport chain to produce

ATP by oxidation (equation 3, fig. 4) (Akers and Denbow, 2008)

(3) 𝐹𝑎𝑡 + 𝑂2 + 𝐴𝐷𝑃 + 𝑃 → 𝐶𝑂2 + 𝐻2𝑂 + 𝐴𝑇𝑃

1.3. The exercising horse Horses are used in a variety of disciplines (Riding competitions, racing etc.), where the per-

formance is dependent on the muscles being supplied with the needed ATP and postpone-

ment of muscle fatigue is favourable. As described above, ATP can be generated from glucose

and FAs, nonetheless the mechanisms behind muscle movements are the same. To create

movement, skeletal muscles need to contract. Skeletal muscles are made up of muscle fibres,

which contain myofibrils. Myofi-

brils consists of sarcomeres, con-

taining thin and thick filaments,

which have an important role in

muscle contraction (Akers and

Denbow, 2008). Muscle contrac-

tion is (1) initially generated by

the release of an action potential,

which is triggered by signalling

from motor neurons. (2) The re-

lease of an action potential acti-

vates the release of calcium from

the sarcoplasmic reticulum.

(3) Subsequently calcium binds to

troponin (fig. 6, “T”), a protein

found in the thin filament (fig. 6,

a). This uncovers a myosin bind-

ing site on the thin filament, which

causes (4) a myosin head to bind

to the myosin binding site. As a re-

sult, Adenosine diphosphate

(ADP) and phosphate (Pi) are re-

leased from the myosin head (fig.

6, b), which initiates the power

stroke.

For the myosin head to detach,

enabling a contraction of the

muscle, Adenosine Triphosphate (ATP) is needed. (5) ATP binds to the myosin head to detach

it from the myosin binding site (fig. 6, c). While detaching, the myosin head hydrolyses ATP,

Figure 6. Muscle contraction. (A) illustrates the state before an action potential is stimulated by a motor neuron. The action potential initiates the release of calcium, enabling (B) the myosin head (MH) is able to bind to the myosin bind-ing site, when calcium (Ca+) binds to troponin (T). (C) ATP binds to the MH to detach the MH from the myosin binding site, which allows the thin filament to move and MH can bind to the next binding site. This movement enables the muscle to contract.

Introduction

19

enabling the return to stage four, resulting in a cyclic process generating motion. Muscle con-

traction is dependent on calcium to initiate the uncovering of the myosin binding site and

phosphorylation (Kuo and Ehrlich, 2015).

ATP can be generated from different substrates, depending on exercise intensity, duration,

and substrate availability (Bosma, 2016). Exercise intensity and duration creates different en-

vironments, dependent on whether O2 is present or absent, which influences the metabolism

during exercise. When O2 is present, aerobic metabolism is favoured. Aerobic metabolism has

a higher ATP production compared to anaerobic metabolism (Michalsik, 2007a). If O2 is absent

or insufficient to supply aerobic metabolism for generation of ATP, the muscle can supply

itself with ATP through anaerobe metabolism (Michalsik, 2007b). If exercise intensity in-

creases beyond 45-65% of O2 max., glycogen and glucose become primary fuels, whereas fatty

acid utilisation decreases (Horowitz and Klein, 2000).

1.4. Behaviour Besides fat and starch influencing how ATP is generated during exercise, the available dietary

sources also determine the fuel to maintain normal biological function (Treiber et al., 2008).

Studies (Hemmann et al., 2013; McGreevy et al., 1995; Nicol et al., 2002) have shown links

between high starch diets and abnormal behaviour, but little research has been done on die-

tary influence on normal behaviour, reactivity or stress responses in horses. Haagensen et al.

(2014) found that mini-pigs fed a high fat / low carbohydrate diet, showed less agonistic be-

haviour and more social contact towards other mini-pigs. Recordings of the number and se-

verity of lesions similarly showed that mini-pigs on the high fat/low carbohydrate diet had

fewer and less serious lesions. In horses, exclusion of starch from the diet has resulted in de-

creased nervousness and lower resting heart rates (Wilson et al., 2007). Nicol et al. (2005) re-

ported that foals fed a fat-rich diet had reduced locomotion in response to weaning and were

calmer and more investigative during temperament testing compared to foals fed a starch-

rich feed. Different theories exist about the dietary effect on behaviour. One theory regards

insulin regulation. When insulin regulation functions optimal (high insulin sensitivity), then

insulin binds to an insulin receptor on the cell, when glucose levels are high. This results in

an opening for the glucose to enter the cell through a transport protein and thereby lowering

the glucose levels in the blood (Fig. 7). When insulin sensitivity is low, the insulin receptors

do not react on the insulin wanting to bind, therefore glucose is not able to enter the cell,

resulting in the blood glucose levels remaining. Even though the horses are not exercising, the

cells still needs energy for maintenance. Therefore, adrenalin is released to stimulate the

Introduction

20

breakdown of glycogen. Higher amounts of adrenalin in the body creates a condition of stress

and as a result more responsiveness and anxiety (Wilson et al., 2007).

Figure 7. A illustrates when insulin sensitivity is high, then insulin receptors capture the insulin, which activates the transport protein (TP) GLUT4 and glucose is able to enter the cell. B illustrates when inulin sensitivity is low, insulin recep-tors do not recognise the insulin and therefore TP is not activated and glucose cannot enter the cell. As a result, the cell is only able to generate energy by the breakdown of glycogen, which is initiated by adrenalin.

Materials and methods

21

2. Materials and methods The study complied with the Danish Ministry of Justice law no. 1306 (25th November 2007)

concerning experiments with animals and care of animals used for experimental purposes

and conducted under the approval of the Danish Veterinary and Food Administration under

the Danish Ministry of Environment and Food (2016-15-0201-01035). The study was carried

out a private stud.

2.1. Animals and diets

Twenty, three years old Danish Warmblood stallions were used in the study. All horses were

owned by and born at the same private stud and grew up under the same conditions. The

horses had only been halter and lead trained for a previous study conducted in 2014 where

all horses received the same amount of handling (Christensen et al., 2017).

The horses were housed in four group

pens (n=6 per pen) without access to pas-

ture. Twenty-four horses were initially in-

cluded into the study, but one had balance

and leg coordination issues, which made

the ramp on a treadmill an obstacle and a

few showed dangerous behaviour during

the initial habituation to a treadmill.

Therefore, it was decided to use only five

of the six horses per pen for the study. The

study was carried out in a test arena (fig. 8)

in a separate building that was connected

to the barn. The horses were randomly al-

located to two treatment groups (LS_HF

and HS_LF, i.e. treatment groups LS_HF in

pen A1 and A2 and treatment groups

HS_LF in pen B1 and B2, fig. 8), balanced

according to sire and reactions in a base-

line fear test (described below). There were

only two different sires to the horses used

in the study. There was no significant dif-

ference in average bodyweight between the

treatment groups (LS_HF: 522.2 ± 30.2 kg

and HS_LF: 510.6 ± 55.2 kg, P=0.58). The

horses were assigned numbers together with a physical description, making identification

possible. A fifth pen contained horses that were not part of the study (fig. 8, “Pen”). These

horses ensured that the test horses had visual contact to other horses during all training and

testing. The treatments consisted of two different types of diets; a low starch (6.0%) and high

fat (8.5%) diet (diet LS_HF), and a high starch (30.0%) and low fat (4.3%) (diet HS_LF). The

pens alternated in treatment (LS_HF and HS_LF) to minimize the effect of pen placement in

the barn. The feed type was known only by the project leader, whereas other staff remained

blind regarding the treatments.

Figure 8. Overview of the stable and experimantal area. The grey area is where all training and tests (object tests and treadmill training) were carried out. The yellow bar demonstrates the en-trance and exit of the test arena. The corridor was used for the initial handling of horses as well as for fitting of heart rate equip-ment etc. for the fear tests. Pens A and B contain the horses for this study (n5/pen). Horses in the last ‘pen’ were not used for this study.

Materials and methods

22

Feeding

The horses were fed three kg of feed in both treatments (LS_HF and HS_LS). On training days,

the horses were fed three times per day: in the morning: during training and in the evening.

On days without training, the horses were fed twice a day (morning and afternoon). The

horses were fed individually in buckets at least once a day, typically in the morning and on

training days also during training. This was to ensure that all horses at least consumed 1.5 kg

of feed. The horses were also monitored during group feeding to ensure that all horses were

allowed to eat. An overview of the timeframe and structure of the study is shown in figure 9.

2.2. Physical performance

Besides testing the behavioural effects of the two diets (LS_HF and HS_LF) in the final fear

tests, the horses’ performance on a treadmill (Hoffmann, Oldenburger Laufbänder, Germany)

was also tested. Prior to initiating the treadmill training, the horses were habituated to enter

and walk on the treadmill. The habituation to the treadmill was divided into 11 stages (table

12, appendix A2) and when a horse had completed the 11 stages it was considered ready for

the treadmill training. Treadmill habituation training took place two-three days/week for

four months. The horses met the criteria for the habituation at different times, therefore, the

horses which had met the criteria for the habituation to the treadmill, maintained the training

once a week until testing.

The horses have a total of 22 training days distributed four times a week for five weeks and

two times in the sixth week. Treadmill training was divided into six sub programs (table 13,

appendix A2), which was adjusted to the fact that the horses had not received any previous

physical training. During training the handler had a bag around the waist with one kg of feed

in, from which the handler offered the horses feed while exercising. This was to keep the mo-

tivation for entering the treadmill. During a training session, most horse ate half a kilo of the

Figure 9. Overview of the time frame and structure of the study. Handling and habituation of the horses was carried out in September 2016. The baseline fear tests were conducted as soon as the horses met the habituation criterion for the test. The initial treadmill habituation started in October and the horses were ready for starting their six weeks of treadmill training by the end of February 2017. The 14 days of feed adaptation started on February the sixth, ensuring that the horses had adapted to the feed for the start of the training period. After the treadmill training period, the final fear tests took place in the following week. ET-1 the first exercise test and ET-2 is the second exercise test.

Materials and methods

23

feed assigned to them (LS_HF or HS_LF). They ate the remaining feed after finished exercise.

During the six weeks of treadmill training, the performance of each horse was tested by meas-

uring its heart rate during training twice (table 14, appendix A4). Due to is was time-consum-

ing equipping the horses with the heart rate monitoring equipment, heart rates measurements

were only done on ten horses per day, even though 20 were trained. Therefore, the heart rate

measurements were divided over two days. The first training heart rate measurement

(THRM-1) was in the two last days of program one (table 14, appendix A4) and second heart

rate measurement (THRM-2) in the last two days of the third program (table 14, appendix A4).

2.2.1. Exercise tests

ET-1

The first exercise test (ET-1) was an “incremental” exercise test with a 6.0% incline. The horses

had a four-minute warm-up on the treadmill at level 0 (table 1). Subsequently the horses were

exercised for one minute at each level 1-8 (table 1). The test order alternated between the treat-

ment groups (LS_FH and HS_LF).

Table 1 Speed levels used on the treadmill

Level 0 1 2 3 4 5 6 7 8

Speed (km/h) 4.8 5.2 6.2 7.8 8.6 9.5 10.4 11.2 12.5

ET-2

The second exercise test (ET-2) was a “single step” exercise test with a 6.0% incline, based

upon the heart rate measurements from ET-1. An assumption about fat oxidation peaking at

45-65% of maximum heart rate was used to design ET-2, to test the horses at this specific level

for a longer time. For ET-2, the horses were walked around the corridor six times (fig. 8), be-

fore entering the treadmill. After the horses had entered the treadmill the horses walked 15

sec on levels 0-3, followed by 30 sec on levels 4 and 5 and then 360 sec on level 6 as presented

in table 2.

Table 2. The time horses spent on the different levels in ET-2

Before initiating the ETs, horses were equipped with heart rate monitoring equipment (Polar

Equine RS800CX G3), measuring their heart rate during exercise. Immediately after the horse

finished the ETs and had exited the treadmill, blood samples were taken. Differences in blood

sampling protocol occurred prior to treadmill exercise. In ET-1 the blood samples were taken

before the horse entered the treadmill and before warm-up. In ET-2 blood samples were taken

after the horse had walked six rounds in the corridor and therefore the horse had experienced

warm-up before the blood sample was taken. Blood samples were taken with syringes (Sie-

mens healthcare diagnostics, USA) which contained heparin. Whole blood analysis occurred

directly after extraction on a RapidPoint 500 system blood gas analyser (Siemens healthcare

Level 0 1 2 3 4 5 6

Time (sec) 15 15 15 15 30 30 360

Materials and methods

24

diagnostics, USA). The following analyses were conducted on the whole blood sample; glu-

cose, lactate, O2, CO2, calcium.

2.3. Behaviour

As already mentioned, the horses were minimally handled and had been handled only for

necessary veterinary or farrier treatment since taking part in a behavioural study as yearlings.

Initial handling and habituation was therefore necessary before the baseline fear tests could

be conducted. The habituation to handling was divided into five stages (fig. 11, appendix A1)

and when a horse had completed the five stages, it was considered ready for the baseline fear

tests.

2.3.1. Baseline fear tests

The horses were initially tested in two object tests (fig. 10 a and b). In the first object test (T1),

the novel object was a green basket (0.7 m high) with a pink pilates ball (0.6 m) on top (fig.

10.a). In the second object test (T2), white, shiny plastic (1 x 4 m) was placed on the floor

between the entrance and the feed container and four boxes (two pink and two blue (w;h;l:

0.3;0.3;0.4 m) were placed on the corners (fig. 10.b).The second object test was a repetition of

a previous test, which the horses had been tested in at five months and one year of age

(Christensen et al., 2012). The object used in T2 was therefore a familiar object. Before the start

of the first object test, the horse was equipped with heart rate monitoring equipment (Polar

Equine RS800CX G3) in the stable corridor. Subsequently led to the feed container where it

was allowed to eat for ten seconds (sec) after which it was led back to the corridor. The novel

object was then placed in the test arena (fig. 10.6). The test time (120 sec) started when the

horse was released at the entrance of the test arena (i.e. immediately after the object became

visible). After the T1, the horse was led back into the corridor and the object was removed

from the arena. The horse was then led back to the arena and allowed to eat for approximate

ten secs before returning to the corridor. The test arena was subsequently prepared for the

second test. Again, the test time (120 sec) started when the horse was released at the entrance.

Both tests were recorded on video for later behavioural analysis (table 3).

2.3.2. Final fear tests

Like in the baseline fear test the horses were tested in two object tests (fig. 10.c) The first test

(T1) was a novel object test (two umbrellas placed 0.5 m from the feed container (fig. 10.c).

The second test (T2) was equal to the second test in the baseline fear test (fig. 10.b). The pro-

cedure and test time was equal to the baseline fear tests. Both tests were recorded on video

for later behavioural analysis (table 3).

Materials and methods

25

Figure 10. Illustration of the different object tests performed. A is the novel object test used in baseline fear test. C is the novel object test used in the final fear test. B is an object test, which is performed in both baseline and final fear test.

A

B

C

Materials and methods

26

Table 3. Ethogram of the recorded behaviours in the object test.

2.3.3. Feed motivation test

In order to investigate whether the test groups differed in feed motivation, the horses were

allowed to eat one kg of their usual test feed. The test was carried out in the stable corridor

where the food was offered in a bucket and the latency for the horse to empty the bucket was

measured.

2.4. Statistics

All statistics were performed in Sigmaplot13, SyStat, www.systat.com.

2.4.1. Exercise tests

A paired t-test was used to analyse the effect of treatment on the response variables (glucose,

lactate, calcium, O2 and CO2) before and after the horse had performed ET-1 and ET-2. If data

did not meet the assumptions for the model (normal distribution and variance homogeneity),

a MannWhitney test was used. Normal distribution was assessed from plots and the Shapiro

Wilks test.

An unpaired t-test was used to analyse for the effect of feed type (LS_HF or HS_LF) on glu-

cose, lactate, calcium, O2, CO2, maximum heart rate (Max HR), average heart rate (Avg HR)

and accumulated heart rate (Accu. HR) in both ET-1 and ET-2. Furthermore, a t-test was per-

formed on the difference between pre- and post-exercise levels of glucose, lactate, calcium, O2

and CO2.

To analyse the correlation between the different response variables a Spearman test was used.

2.4.2. Final fear test

The response variables (Avg HR, Max HR, latency, object focus, touch, sniffing, feed motiva-

tion) were analysed for an effect of treatment (LS_HF or HS_LF) in an unpaired t-test. The

video analysis and reading of heart rate data was performed by a person who was blind re-

garding the treatment (LS_HF or HS_LF). If data did not meet the assumptions for the model

(normal distribution and variance homogeneity), the MannWhitney test was used.

To analyse the correlation between the different response variables a Spearman test was used.

Behaviour Definition

Object focus Vigilant with either neck raised over or below horizontal position, head and

ears oriented towards the object.

Touch Touching or manipulating the object (either umbrella, white plastic or buck-

ets)

Sniff Head within 20 cm of food container, neck horizontal or lower, clear exhala-

tions from nostrils

Materials and methods

27

Data was presented as; “mean ± SEM” or “median [25;75% quartiles]” for data that did not

meet the normal distribution criteria. P<0.05 was considered significant and P<0.10 was con-

sidered a trend.

Results

28

3. Results

3.1. Exercise tests

Glucose

No significant differences in whole blood glucose levels were found between treatments

(LS_HF and HS_LF) in either of the exercise tests (ET-1 and ET-2) (table 4 and 5). In ET-1,

horses fed LS_HF showed a tendency to have decreased in whole blood glucose levels as a

consequence of exercise (table 4) and in ET-2 horses fed LS_HF decreased significantly in

whole blood glucose levels (table 5). In ET-1 no difference in whole blood glucose levels as a

consequence of exercise, was found for horses fed diet HS_LF, whereas in ET-2 whole blood

glucose levels tended to decrease as a consequence of exercise (table 5).

Lactate

No significant differences in whole blood lactate levels were found between treatments

(LS_HF and HS_LF) in either of the exercise tests (ET-1 and ET-2) (table 4 and 5). Also, no

significant differences from pre- to post-exercise were found in either of the ETs, though

horses fed HS_LF tended to increase in whole blood lactate levels as a consequence of exercise

in ET-1 (table 4). In ET-2, whole blood lactate levels decreased numerically during exercise in

horses fed LS_HF, while whole blood lactate levels increased numerically in horses fed HS_LF

(table 5). The difference in whole blood lactate levels from pre-to post-exercise between the

treatments (LS_HF and HS_LF) in ET-2, tended to be different (table 6).

Calcium

Significant differences in whole blood calcium levels were found between the treatments in

both exercise tests (ET-1 and ET-2) (table 4 and 5). In ET-1, horses fed HS_LF had significantly

higher whole blood calcium levels pre-exercise compared to pre-exercise whole blood calcium

levels in horses fed diet LS_HF (table 4). In ET-2, horses fed diet LS_HF had significantly

higher whole blood calcium levels in both pre-and post-exercise compared to horses fed diet

HS_LF (table 5). In ET-1, no difference between pre-and post- exercise was found in horses

fed diet LS_HF (table 4), whereas horses fed HS_LF decreased significantly in whole blood

calcium levels as a consequence of exercise (table 4).

Respiration

No significant difference in whole blood O2 levels were found between the treatments (LS_HF

and HS_LF) in either of the exercise tests (table 4 and 5).

Whole blood O2 levels increased significantly as a consequence of exercise for horses fed

HS_LF in ET-2, whilst horses fed LS_FH only showed a tendency to increase in whole blood

O2 levels in both ETs (table 4 and 5). In ET-1, horses fed HS_LF tended to increase more in

whole blood O2 levels as a consequence of exercise compared to horses fed LS_HF (table 6).

A significant difference in pre-exercise whole blood CO2 levels between treatments (LS_HF

and HS_LF) was found in ET-1, were horses fed HS_LF had significantly higher whole blood

CO2 levels compared to horses fed LS_HF (table 4). Unfortunately, whole blood CO2 levels

was not measured in ET-2.

Results

29

Whole blood levels of CO2 tended to decrease for horses fed HS_LF as a consequence of exer-

cise (table 4), whereas whole blood CO2 levels increased for LS_HF horses in ET-1, although

this was not significant. The difference between pre- and post-exercise tended to be different

between the treatments (table 6).

Table 4. Whole blood levels of for oxygen (O2), carbon dioxide (CO2), glucose, lactate and calcium pre- and post-exercise for

the two treatments: Low Starch / High Fat (LS_HF) and High Starch / Low Fat (HS_LF) in exercise test 1 (ET-1). Values are

presented as mean ± SEM.

ET-1

LS_FH HS_LF P value

Pre Post P

value Pre Post

P value

Pre-LS_FH

vs. Pre-HSLF

Post-LS_FH vs. Post-HS_LF

Glucose (mmol/L)

4.73 ± 0.1 (n=8)

4.56 ± 0.01 (n=9)

0.08 5.13 ±0.3

(n=8) 4.72 ± 0.2

(n=9) 0.2 0.3 0.4

Lactate (mmol/L)

0.54 ± 0.2 (n=8)

0.65 ± 0.2 (n=9)

0.2 0.50 ± 0.1

(n=8) 0.68 ± 0.2

(n=8) 0.1 0.5 0.8

Calcium (mmol/L)

1.59 ± 0.02 (n=8)

1.59 ± 0.02 (n=9)

0.7 1.64 ± 0.01

(n=8) 1.59 ± 0.02

(n=9) 0.0007 0.05 0.8

O2 (%)

29.7 ± 0.6

(n=8) 31.3 ± 0.9

(n=9) 0.07

28.1 ± 0.7 (n=8)

33.4 ± 0.9 (n=8)

0.0004 0.11 0.13

CO2 (%)

45.4 ± 1.6 (n=8)

47.2 ± 1.9 (n=9)

0.8 50.9 ± 1.9

(n=8)

45.4 ± 1.1 (n=8)

0.08 0.04 0.4

Results

30

Table 5. Whole blood levels of for oxygen (O2), glucose, lactate and calcium pre-and post-exercise for the two treatments: Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) in exercise test 2 (ET-2). Values are presented as mean ± SEM.

Table 6. Difference between pre-exercise to post-exercise (Δ= post-pre) for whole blood levels for glucose, lactate, calcium, oxygen (O2) and carbon dioxide (CO2) for two treatments: Low Starch / High Fat (LS_HF) and High Starch / Low Fat (HS_LF) in exercise test 1 (ET-1) and exercise test 2 (ET-2). Values are presented as mean ± SEM.

ET-2

LS_FH HS_LF P value

Pre Post

P value

Pre Post P

value

Pre-LS_FH vs. Pre-HSLF

Post-LS_FH vs. Post-HS_LF

Glucose (mmol/L)

4.63 ± 0.2 (n=9)

4.09 ± 0.1 (n=8)

0.045 4.96 ± 0.1

(n=8) 4.37 ± 0.2

(n=10) 0.07 0.2 0.2

Lactate (mmol/L)

0.66 ± 0.06 (n=8)

0.62 ± 0.04

(n=9)

0.4

0.60 ± 0.04

(n=8)

0.64 ± 0.04

(n=8)

0.2 0.4 0.7

Calcium (mmol/L)

1.64 ± 0.02 (n=8)

1.62 ± 0.02 (n=8)

0.7 1.58 ± 0.01

(n=8) 1.56 ± 0.01

(n=9) 0.15 0.04 0.03

O2 (%)

29.2 ± 1.1 (n=9)

34.8 ± 1.2 (n=8)

0.07 29.3 ± 0.8

(n=8)

32.9 ± 1.7 (n=9)

0.008 0.95 0.4

ET-1 ET-2

LS_HF HS_LF P

value LS_HF HS_LF

P

value

Δ Glucose

(mmol/L)

- 0.25 ± 0.1

(n=8)

- 0.26 ± 0.2

(n=8)

0.96

- 0.59 ± 0.2

(n=7)

- 0.63 ± 0.4

(n=8)

0.9

Δ Lactate

(mmol/L)

0.15 ± 0.10

(n=8)

0.18 ± 0.09

(n=7) 0.8

- 0.02 ± 0.02

(n=7)

0.06 ± 0.04

(n=6) 0.10

Δ Calcium

(mmol/L)

- 0.01 ± 0.02

(n=8)

- 0.05 ± 0.008

(n=7) 0.13

- 0.006 ± 0.01

(n=7)

- 0.03 ± 0.02

(n=7) 0.3

ΔO2 (%) 2.7 ± 1.2

(n=7)

5.3 ± 0.8

(n=7) 0.09

5.09 ± 2.32

(n=7)

4.51 ± 1.14

(n=7) 0.8

ΔCO2 (%) 0.6 ± 2.1

(n=8)

- 4.9 ± 2.4

(n=7) 0.10 - - -

Results

31

Heart rate

No significant differences between the diets were found in Max HR, Avg HR or Accu. HR in

the two ETs (ET-1 and ET-2) or in first training heart rate measurement (THRM-1, table 7).

During second training heart rate measurement (THRM -2) horses fed diet LS_HF had a sig-

nificantly higher Max HR compared to horses fed diet HS_LF (table 7).

Table 7. Maximum heart rate (Max HR), average heart rate (Avg HR), accumulated heart rate (Accu. HR) for both treatments: Low Starch / High Fat (LS_HF) and High Starch / Low Fat (HS_LF) in exercise tests (ET-1 and ET-2) and two training sessions, where heart rate was measured (THRM – 1 and THRM -2). Values are presented as mean ± SEM.

Correlations

In ET-1 there was a negative correlation between whole blood CO2 levels and whole blood

lactate levels for horses fed LS_HF pre -exercise (rs= - 0.76, P= 0.04, table 15, appendix B1).

Post-exercise in ET-1 horses fed LS_HF tended have a positive correlation between CO2 and

high Avg HR (rs= 0.61, P= 0.08, table 15, appendix B1). A similar tendency was seen between

CO2 and Max HR in ET-1 (rs= 0.58, P= 0.09, table 15, appendix B1) and for horses fed HS_LF

post-exercise between CO2 and Max HR in ET-1 (rs= 0.67, P= 0.06, table 15, appendix B1).

Additionally, a significant positive correlation between CO2 and AVG HR was found in ET-1

(rs= 0.74, P= 0.03, table 15, appendix B1). In ET-1, O2 was negatively correlated with Max HR

regardless of the diet consumed (LS_HF: rs= - 0.69, P= 0.05 and HS_LF: rs= - 0.71, P= 0.04, table

16, appendix B1). A similar tendency was seen in ET-2 in horses fed HS_LF (rs= - 0.60, P= 0.10,

table 18, appendix B3). In ET-2, a positive correlation between whole blood O2 levels and

whole blood calcium levels was found in horses fed LS_HF pre-exercise (rs=0.62 P=0.09, table

18, appendix B3). Horses fed HS_LF in ET-1, showed a negative correlation between Avg HR

and O2 post-exercise (rs= - 0.61, P=0.09, table 16, appendix B1). Horses fed LS_HF in ET-2

tended to have a positive correlation between O2 and Max HR post-exercise (rs= 0.64, P=0.07,

table 18, appendix B3). In ET-2 horses fed LS_HF had a negative correlation between glucose

AVG HR

(bpm)

MAX HR

(bpm)

ACCU HR

(bpm)

LS_HF HS_LF P

value

LS_HF HS_LF P

value

LS_HF HS_LF P

value

ET-1 92 ± 4

(n=10)

91 ± 3 (n =10)

0.4 151 ± 8

(n=10)

153 ± 10

(n=10) 0.9

1118 ±46

(n=9)

1092 ± 29

(n=10) 0.6

ET-2 109 ± 3

(n=10)

121 ± 8

(n=10) 0.2

140 ± 4

(n=10)

143 ± 7

(n=10) 0.7

906 ± 24

(n=10)

956 ± 36

(n=10) 0.3

THRM-1 63 ± 5

(n=10)

72 ± 9

(n=8) 0.3

128 ± 16

(n=10)

142 ± 18

(n=8) 0.5 - - -

THRM-2 74 ± 6

(n=9)

70 ± 5

(n=9) 0.8

177 ± 10

(n=9)

125 ± 11

(n=9) 0.008 - - -

Results

32

levels and Avg HR (rs= - 0.58, P= 0.09, table 19, appendix B4) and Max HR (rs= - 0.58, P=0.09,

table 19, appendix B4) pre-exercise. In ET-1 horses fed diet LS_HF had high glucose levels in

relation to high Avg HR (rs= 0.93, P<0.0001, table 17, appendix B2) and high Max HR (rs=0.89,

P<0.0001, table 17, appendix B2) post-exercise. In ET-1 horses fed HS_LF had a positive cor-

relation between glucose levels and lactate levels pre- and post -exercise (fig. 11 and 12, Pre:

rs= 0.94, P<0.001, table 17, appendix B2. Post: rs= - 0.74*, P= 0.04, table 17, appendix B2). In ET-

1 calcium levels negatively correlated with glucose for horses fed HS_LF post-exercise (rs= -

0.81, P=0.01, table 17, appendix B2). Furthermore, for horses fed diet HS_LF in ET-1, high

lactate levels were seen in relation to high Avg HR (rs= 0.68, P= 0.05, table 17, appendix B2)

and likewise a positive correlation was seen between lactate and Max HR post-exercise (rs=

0.74, P= 0.03, table 17, appendix B2). There was a significant positive correlation between Avg

HR and Max HR in all pre- to post-exercise in both ET-1 and ET-2.

Figure 11. Positive correlation between pre-exercise whole blood lactate and glucose levels for horses fed High starch/Low Fat (HS_LF) in an incremental exercise test (ET-1).

y = 0.15x - 0.22

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

3,5 4 4,5 5 5,5 6 6,5 7

Lact

ate

mm

ol/

L

Glucose mmol/L

Correlation between lactate and glucose pre-exercise for HS_LF

Results

33

Figure 12. Positive correlation between post-exercise whole blood lactate and glucose levels for horses fed High Starch/ Low Fat (HS_LF) in an incremental exercise test (ET-1)

Effect of feeding time

Glucose levels in horses declined as time from feeding increased for both ETs (fig. 13). In ET-

1, 16 horses were tested on the second day and only four the first day, where two glucose

values are not available, therefore glucose levels from feeding for the four horses was not

illustrated.

3.2. Final fear tests No significant differences were found between the treatment groups in the fear tests. How-

ever, in T2 horses fed diet LS_HF had a slightly longer latency to return to eat feed compared

to horses fed diet LS_HF (table 8). In addition, horses in the LS_HF group had a slightly longer

duration of sniffing compared to horses fed diet HS_LF (table 8).

In T1, horses that had a longer duration of sniffing had lower Max HR (rs = - 0.49 P = 0.03,

table 20, appendix B5). Furthermore, the duration of touching the object was positively corre-

lated to the duration of sniffing (rs = 0.47, P= 0.04, table 20, appendix B5).

y = 0.15x - 0.03

0

0,2

0,4

0,6

0,8

1

1,2

3,5 4 4,5 5 5,5 6

La

cta

te m

mo

l/L

Glucose mmol/L

Correlation between lactate and glucose post-exercise for HS_LF

Figure 13. A shows the decrease in whole blood glucose levels in ET-1, whereas B shows the decrease in ET-2 on the first day of test-ing and C shows the decrease in whole blood glucose levels for the horses tested on the second day of testing in ET—2.

A B C

Results

34

In T2 there was a negative correlation between the duration of “object focus” and the duration

of touching the object (rs = - 0.42, P=0.05, table 21, appendix B5).

In both T1 and T2 latency was positive correlated with Max HR and Avg HR, and “object

focus” (P<0.05, table 20 and 21, appendix B5).

In the feed motivation test, there was a tendency that horses fed LS_HF consumed their feed

slower than horses fed HS_LF (LS_HF = 8.7 ± 0.4 and HS_LF = 7.4 ± 0.5, P=0.06)

Table 8. Duration of behavioural displays and heart rate (mean ± SEM or mean [25%; 75%]) of horses fed either a Low Starch/High Fat diet (LS_HF) (n=10) or a High Starch/ Low Fat diet (HS_LF) (n=10) in two object tests (T1 and T2, respectively) after 8 weeks of feeding.

Object test 1 Object test 2

LS_HF HS_LF P

value

LS_HF HS_LF Pvalu

e

Latency (sec)

43.2 ± 13.1 34.9 ± 10.4 0.6 30.5 [19.8; 45.8] 16.0 [9.8; 30.3] 0.09

AVG HR (bpm)

60.1 ± 4.6 59.0 ± 3.0 0.8 53.8 ± 2.5 52.8 ± 2.2 0.8

MAX HR (bpm)

90.3 ± 6.0 99.7 ± 8.1 0.4 82.5 ± 4.9 84.4 ± 7.0 0.8

Object fo-cus (sec)

21.4 ± 6.9 18.2 ± 3.7 0.3 8.0 [2.0; 18.0] 3.5 [2.0; 10.0] 0.4

Touch (sec)

0.0 ± 0.0 0.4 ± 0.3 0.2 1.9 ± 1.3 1.3 ± 0.9 0.7

Sniff (sec)

5.5 [2.8; 7.0] 3.5 [1.5; 8.5] 0.7 2.0 [0.8; 2.3] 0 [0.0; 2.0] 0.07

Discussion

35

4. Discussion It has been recommended that horses should not be fed more than 2 g/kg bwt starch in one

meal, due to the risk of undigested starch reaching the hindgut (Geor et al., 2013; Vervuert et

al., 2009). Furthermore, it has been shown that horses which had ingested more than 1 g/kg

bwt starch, 20% of the ingested starch still reached the hindgut (Geor et al., 2013). The alloca-

tion of starch in this study met the recommendations of the National research council

(Lawrence et al., 2015), due to the horses fed HS_LF received 0.9 g/kg bwt starch per meal,

therefore the ingested starch was not likely to reach the hindgut of the horses. The levels of

fat given did not exceed the fat levels recommended by National research council (Lawrence

et al., 2015), therefore the risk of fat reducing fibre digestibility was not a factor. This study

tried to resemble the amounts of fat and starch the average horse would receive in “real life”.

4.1. Glucose No significant differences between the treatments (LS_HF and HS_LF) regarding whole blood

glucose was found in this study. Similarity in whole blood glucose levels between starch-rich

and fat-rich treatments had also been found in other studies (Hoffman et al., 2003; Treiber et

al., 2005; Treiber et al., 2008). Hoffman et al. (2003) studied the effect of starch-rich and fat-rich

diets in obese horses without an exercise test and no effect was found of treatment (table 9).

The body condition of the horses was suggested to be the reason for the lack of difference

between the treatments, due to the horses might already have been insulin resistant.

Treiber et al. (2008) showed no difference regarding plasma glucose between treatments (table

9) as an effect of exercise in Arabian horses, though the horses fed a starch-rich diet had higher

plasma glucose levels in the overall feeding period. This indicated that a difference in glucose

levels might be detectable when horses rested and not when exercised, where muscle contrac-

tions increased glucose uptake. In this study, the whole blood glucose measurements were

done pre- and post-exercise and this could be the reason for no significant differences between

treatments. Treiber et al. (2005) conducted a study on foals (table 9) and though no significant

difference in blood glucose levels was found between a starch-rich and fat-rich diet, they

found that insulin levels tended to be higher when foals were fed a starch-rich diet. This indi-

cated, that foals fed a starch-rich diet had a slightly lower insulin sensitivity and that horses

fed a fat-rich diet had a slightly higher insulin sensitivity. Higher insulin sensitivity resulted

in quicker glucose uptake by cells for horses fed a fat-rich diet, compared to horses fed a

starch-rich diet. So even though horses fed LS_HF only had numerically lower whole blood

glucose levels compared to horses fed diet HS_LF, there might be an effect on insulin. A higher

insulin sensitivity for horses fed fat-rich diet could also explain why horses fed LS_HF in ET-

2, had a significant decrease in whole blood glucose levels as a consequence of exercise,

whereas horses fed HS_LF did not. Insulin sensitivity being higher for horses fed a fat-rich

diet, has also been suggested by Wilson et al. (2007) (table 9), which suggested that fat-rich

diets may facilitate better gluco-regulation. Treiber et al. (2005)(table 9) suggested that horses

fed a starch-rich diet (SS) had reduced insulin sensitivity in untrained resting horses com-

pared to horses fed a fat-rich diet (FF). The starch- rich diet altered insulin signalling (Treiber

Discussion

36

et al., 2005). Measurement of insulin levels would have been necessary to relate the decrease

in glucose to insulin sensitivity.

It has been suggested that the lack in significant difference in blood glucose levels, might be

because horses were able to compensate for low insulin sensitivity. In foals and obese horses,

noninsulin-dependent GLUT1 glucose transporters on β-cells were found and these may be

able to compensate for low insulin sensitivity (Hoffman et al., 2003; Treiber et al., 2005),

thereby difference in blood glucose levels were not detectable. Whether this could be a factor

to consider in this study remains unknown, due to the function of β-cells has not been exam-

ined in four-year old horses.

Wilson et al. (2007) confirmed that high blood glucose levels were associated with intake of

starch over a longer period of time. In addition to the lack of significant differences between

treatments (LS_HF and HS_LF) in this study, could be explained by a relatively short treat-

ment period. Studies concerning how diet affected horses’ performances have varied in the

length of treatment periods (from 30 days - 390 days, table 9). Zeyner et al. (2002) had a long

treatment period (390 days, table 9) and found higher plasma glucose levels in horses fed a

starch-rich diet compared to horses fed a low starch diet. Furthermore, Vervuert et al. (2009)

had a short treatment period (30 days, table 9) and found increased plasma blood glucose

levels when adding more starch to the horses´ diet (table 9). Therefore, the effect of the length

of the treatment period is not clear.

Though no significant difference in whole blood glucose levels between treatments (LS_HF

and HS_LF) was found in this study, other studies have found an effect of feed. Wilson et al.

(2007) found that foals fed starch-rich diets had a steeper raise in plasma blood glucose levels

post-feeding compared to foals fed a fat-rich diet. Studies which had added fat to the diet

showed conflicting blood glucose levels. Meyers et al. (1989) conducted a study where horses

were allocated to different amounts of added fat (table 9) and found that horses fed 10% added

fat had lower plasma glucose levels compared to horses fed 5% added fat and horses where

no fat was added. In contrary to Meyers et al. (1989), Hambleton et al. (1980) had found that

high levels of fat (16%, table 9) increased plasma glucose levels. Corresponding the findings

of Zeyner et al. (2002) where higher plasma glucose levels were found for horses given a

higher amount of fat compared to horses fed a diet with lower amounts of fat (table 9). Both

studies had considerable high amounts of starch in the diet with high levels of added fat (table

9). The amount of starch, Zeyner et al. (2002) used in the fat-rich diet was higher than the

amount of starch used in the HS_LF diet in this study. Vincze et al. (2016) found lower glucose

levels when 15% of the daily intake was provided as oil and in this study the amount of starch

was lowered considerable compared to the starch-rich diets (table 9). Therefore, the increased

levels of blood glucose in Zeyner et al. (2002) and Hambleton et al. (1980) might be explained

by the high amounts of starch together with high fat levels, though in addition to that high fat

levels can result in glucose sparring. In glucose sparring fat inhibits key enzymes in for exam-

ple glycolysis. This inhibits glucose breakdown and therefore blood glucose levels increase.

Discussion

37

A study made by Jansson and Lindberg (2012) considered how horses were affected when

only provided haylage compared to when horses were provided haylage and a starch-rich

concentrate (table 9). Horses only fed haylage had lower plasma glucose and insulin levels

compared to horses fed haylage and a starch-rich concentrate (table 9). Finally, Vervuert et al.

(2009) examined the effects of increased amounts of starch on serum levels and found that

increased amounts of starch, increased serum glucose levels. In this study, the effect of fat is

not considered and the amount of fat in the feed was not available. Also Vervuert et al. (2009)

lacked information whether if the horses were exercised during treatment and information of

previous exercise. Exercise and the type of exercise influences glucose dynamics and thereby

glucose uptake (Treiber et al., 2006). Previous exercise and exercise type are therefore relevant

factors to consider. O'Connor et al. (2004) found that exercised horses allocated fish oil, tended

to have lower insulin levels compared to horses fed corn oil and that plasma glucose levels

returned to baseline levels quicker when horses were fed fish oil. Fish oil contains a higher

amount of omega-3 fatty acids compared to corn-oil. Omega-3 fatty acids may be beneficial to

reduce oxidative stress. Oxidative stress is free radicals, which are produced as a by-product

of oxidation in the electron transport chain (summary, appendix C1). Free radicals are not a

problem, as long as they don’t exceed the capacity of the anti-oxidant system (summary, ap-

pendix C1). When the antioxidant system´s capacity is exceeded, free radicals are not removed

quickly enough and this results in muscle tissue damage. Omega-3 fatty acids are suggested

to be anti-inflammatory, whereas omega-6 fatty acids are pro-inflammatory. No studies have

been made on horses regarding oxidative stress, but rats showed that if they were given more

complex carbohydrate, they had less oxidative stress compared to rats given starch (Robert et

al., 2008). Robert et al. (2008) suggested that fat metabolism is upregulated when the rats are

given complex carbohydrate instead of using starch as energy fuel. This indicated that starch

increased oxidative stress, because glycolytic metabolism was upregulated, contrary to when

fat metabolism was upregulated, oxidative stress was decreased. Fat is more energy dense,

therefore less molecules are oxidised in the electron transport chain, hence lowering the

amount of electron exchange across the membrane.

In general, the available studies varied in the amounts used of fat and starch, whereas some

studies only looked at the effect on blood glucose of either fat or starch (table 9). Another

considerable difference between this current study and previous studies, was that when

horses in this study were trained at their max. achievable speed this was the lowest level in

other studies. The studies also varied in age, whereas the horses in this current study were

exercised and were four years old, thus the available studies are conducted on either older

horses or horses that have previously experienced exercise. Age and previous exercise are

factors known to affect exercise capacity (Lawan et al., 2013; Seeherman and Morris, 1991).

Limited knowledge available and lack of standardisation between methods used in the differ-

ent studies made comparison between current study and previous studies difficult.

Studies also varied in races used, and it has been shoed that different races had different

oxidative capacities, for example are Arabians more adapted to endurance compared to

Discussion

38

Thoroughbreds (Bergero et al., 2005). Though no knowledge about the oxidative capacity of

Danish Warmblood was available, though they may be more similar to Thoroughbreds, due

to some degree of thoroughbred is often found in their pedigree.

4.2. Muscle fatigue Muscle fatigue is the limiting factor for muscles during exercise. Different parameters have

been associated with muscle fatigue, one of them is the accumulation of lactate. As described

earlier pyruvate is fermented to lactate during anaerobic condition in the muscle, which then

accumulates. The lactate threshold, where ATP generation shifts from aerobic metabolism to

anaerobic metabolism has been examined on horses in several studies (Davie and Evans, 2000;

Lindner et al., 1992; Roneus et al., 1994). Though without considering how factors such as

feed, race, genetics, age and previous training affected the capacity to postpone muscle fatigue

during exercise. The purpose of this study, was not to pressure the horses to a degree where

they only used starch as an energy source in anaerobic metabolism, but instead to reach the

place of maximal fat oxidation during aerobic metabolism and to see how the differences in

the starch/fat ratio affected the muscles.

The lactate levels measured, indicated that the energy requirement of the treadmill training

was ensured by aerobic metabolism, since the values in general were below two mmol/L.

Two-four mmol/L is where the imbalance between lactate production and removal of lactate

starts (Campbell, 2011). Vincze et al. (2016) suggested that when lactate values are below two

mmol/L, then the energy requirement is ensured by aerobic energy supply. During aerobic

exercise, both starch and fat can be utilized, whereas under anaerobic conditions, fat is not a

possible energy source (Lindner et al., 2009). Thus, it was important that the horses remained

on aerobic metabolism.

In ET-1, no difference between the treatments in whole blood lactate levels, horses fed HS_LF

tended to increase in whole blood lactate levels during exercise whereas horses fed LS_HF

did not increase in whole blood lactate levels. In ET-2, horses fed LS_HF decreased in lactate

as a consequence of exercise, whilst horses fed HS_LF increased. This indicated that horses

fed HS_LF, would reach the lactate threshold slightly quicker compared to the horses fed

LS_HF. Jansson and Lindberg (2012) found that horses fed haylage and a starch-rich concen-

trate had higher plasma lactate levels, compared to horses only fed haylage. Furthermore,

Meyers et al. (1989) found a tendency that horses fed fat had lower plasma lactate concentra-

tions, compared to horses fed a starch-rich diet, though the p-value was higher than the p-

value defined for tendencies in this study (P>0.16). Finally, O'Connor et al. (2004) found no

effect on plasma lactate levels between horses fed fish oil and horses fed corn oil during exer-

cise.

The difference found between treatments in whole blood lactate levels found in ET-2, could

possibly be explained by the exercise form. ET-2 simulated a more endurance-based exercise,

which favoured aerobic metabolism and therefore able to generate ATP from both fat and

starch. Horses fed LS_HF had more fat available in the feed and therefore fat metabolism

Discussion

39

might have been upregulated, which reduced the breakdown glucose, which potentially low-

ered lactate levels and would possibly postpone the accumulation of lactate. Measurement of

LDH is needed to support the findings of whole blood lactate levels. The measured heart rates

in ET-2, showed that horses fed HS_LF had numerical higher heart rates (both Max HR and

Avg HR) when exercised. This could indicate that horses fed HS_LF might have been shifting

between aerobic metabolism and anaerobic metabolism during ET-2.

Studies which examine the effect of feed on blood lactate levels lack the consideration, if age

might have had an effect of the lactate accumulation. Smarsh and Williams (2016) suggested

that young horses rely more on aerobic metabolism during exercise, therefore a young age

could have had a reducing effect on blood lactate levels. Vincze et al. (2016) states that it seems

that horses around the age of seven years, with a good fitness expressed a higher activity of

lactate clearance which prevented formation of significant peaks of blood lactate. This should

be examined closer, due to the fact that Vincze et al. (2016) based this statement on a study

conducted on four horses from 6-11 years which were “normally” trained. Other studies con-

ducted, except for Hambleton et al. (1980), have used horses older than six years, when exer-

cise was included and only few considered lactate levels.

Calcium has also been associated with muscle fatigue. Not many studies have been con-

ducted, which contribute with knowledge as to how calcium affects horses during exercise.

Calcium has showed to have an important role regarding fatigue during exercise. Calcium is

responsible for initiating the power stroke of the muscle. Hence a reduced calcium release

from the sarcoplasmic reticulum, would result in muscle fatigue, due to the fact that no power

stroke would be initiated.

In ET-1, horses fed LS_HF showed stable whole blood calcium levels, whereas horses fed

HS_LF decreased in whole blood calcium levels. Pre-exercise whole blood calcium levels were

significantly lower compared to the horses fed HS_LF, though post-exercise whole blood cal-

cium levels in both treatments were similar in ET-1. Due to calcium release is initiated when

the motor neuron is stimulated by movement, it may be possible that horses fed LS_HF had

less movement in the pen and therefore the motor neuron had not signalled to generate an

action of potential, because the physiological state of the horses was “resting”. Whereas,

horses fed HS_LF might have been more active in the pen before being exercise tested. No

observations were made regarding the activity in the pen, which would have been necessary

to relate the significantly higher whole blood calcium levels pre-exercise to enhanced activity

for horses fed HS_LF in the pen. In ET-2 horses fed LS_HF had significantly higher blood

calcium levels compared to horses fed HS_LF. In contrary to ET-1, where blood samples were

taken immediately after the horses left the pen, blood samples were taken after the horses had

walked for approximately two minutes in ET-2. Therefore, an explanation could be that horses

fed LS_HF had a greater release of whole blood calcium levels as a consequence of the two

minutes of movement. Whole blood calcium levels decreased for both treatments, though the

horses fed LS_HF were significantly higher in blood calcium levels post-exercise compared to

the horses fed HS_LF in ET-2.

Discussion

40

Besides calcium and lactate being associated with muscle fatigue, it has been shown that dur-

ing treadmill exercise time to fatigue is directly related to the intensity of exercise and that

higher heart rates are associated with a faster time to fatigue (Hodgson et al., 1990). In this

study a positive correlation was found between lactate and heart rate (Max HR and Avg HR)

in horses fed HS_LF in ET-1, which showed high lactate levels were related to both high Avg

HR and Max HR, supporting the findings of Hodgson et al. (1990). In THRM-2 horses fed

LS_HF had significantly higher heart rates compared to horses fed HS_LF, which stands in

contrast to the other heart rate measurements. It is possible that this is due to 7 of 10 horses

fed LS_HF were startled by external factors (Tractor, people, cat etc.) in the environment.

Waller and Lindinger (2010) found that horses given a fat-rich diet (25 % of digestible energy)

had a lower respiration exchange ratio and decreased glucose utilization during prolonged

exercise. In this study, all horses increased in O2 levels as a consequence of exercise, as ex-

pected. HS_LF increased significantly, whereas LS_HF only tended to increase.

Discussion

41

Table 9. Comparison between the different studies used in the discussion. NA = Not available

Discussion

42

4.3. Behaviour It was hypothesised that horses fed HS_LF would show stronger fear reactions, compared to

horses fed LS_FH, when a novel object was presented. Stronger fear reactions would be re-

sembled by increased heart rates, latency to come and eat from the feed container and object

focus. However, no significant differences were found in neither behavioural nor physiologi-

cal responses in the fear tests. Horses fed LS_HF tended to show a longer latency to come and

eat from the feed container and a longer duration of sniffing towards the object in T2. In-

creased sniffing may be indicative of curiosity towards the objects, there by investigating the

object, which could lead to an increased feeding latency. In this study, the physiological meas-

urements on exercising horses, showed no significant differences between the treatments in

whole blood glucose levels. However, Treiber et al. (2005) found that even though no differ-

ence was found between starch-rich and fat-rich diets in blood glucose levels, horses fed fat-

rich diets had lower insulin levels in the blood. When horses were fed starch-rich diets they

tended to have lower insulin activity (Treiber et al., 2005) and therefore a greater production

of adrenalin, which initiates the breakdown of glycogen stores as a result of the cells not re-

ceiving the necessary glucose as a consequence of lower insulin sensitivity. The increased pro-

duction of adrenalin could potentially increase stress levels. Insulin was not measured in the

whole blood analysis conducted in the present study, and thus it is not possible to relate the

tendency towards increased sniffing to insulin levels.

Other studies have found that the level of investigative behaviour was higher for horses fed a

fat-rich diet. Nicol et al. (2005) examined foals pre-weaning, during weaning and post-wean-

ing, given diets containing similar amounts of fat as in the diets used in this study. Though

the amount of starch given was slightly different compared to our study. Additionally, two

different weaning methods were used. “Paddock-weaning” where the mares were removed

from the field, leaving the foals in a familiar field and “barn-weaning” were foals were

brought simultaneously into a large unfamiliar barn without the mares. Foals fed a fat-rich

diet cantered less and showed more explorative behaviour, within the first three hours after

weaning, regardless of weaning method. Furthermore Nicol et al. (2005) conducted three tests

(novel object test, novel human test and handling test, table 10) two months post weaning. In

the novel object test, foals fed a fat-rich diet investigated more and approached the novel ob-

ject quicker compared to foals fed a starch-rich diet. No description of investigative behaviour

was included in the article. In the handling test, foals fed a fat-rich diet approached the obsta-

cle (table 10) quicker than foals fed a starch-rich diet (Nicol et al., 2005). Finally, in the novel

human test, foals fed a fat-rich diet showed less “moving away” from the unfamiliar human

(Nicol et al., 2005). Physiological parameters to support the behavioural findings were not

used (Nicol et al., 2005). Bulmer et al. (2015) used both behavioural measurements and phys-

iological measurements in a handling test and a novel stimulus test (sound, table 10). Though

the effect of fat was not considered, only the effect of starch and fibre. Horses fed a fibre-rich

diet had lower maximum heart rates in the novel stimulus test. Similar responses were found

in the handling test where horses fed a fibre-rich diet had lower maximum and average heart

Discussion

43

rate compared to horses fed a starch-rich diet. Furthermore, horses fed a starch-rich diet had

more interrupted eating bouts compared to horses fed a fibre-rich diet (Bulmer et al., 2015).

Redondo et al. (2009) found that horses fed a fat-rich diet showed reduced startle reactions

and moved less when startled by a pop up box compared to horses fed a starch-rich diet.

Furthermore, horses fed a fat-rich diet had a reduced increased heart rate when startled. Pro-

nounced higher levels of starch and fat was used compared to this study. Finally, Holland et

al. (1996) found that horses fed diets containing fat tended to have lower subjective evalua-

tions in a reactivity test compared to horses fed a diet without fat (table 10). The horses fed a

diet without fat had a longer latency to complete a walk, where an umbrella was opened half-

way, compared to horses fed a lecithin- corn oil diet (table 10). The handlers giving the sub-

jective evaluations were blinded towards treatment. (Jansson, 2010) researched the effect of

adding starch as a supplement to haylage (table 10). The riders scored the horses while riding,

but information about the scoring characteristics and whether the riders were blinded towards

treatment was missing. No effect was found on temperament during exercise was found.

Though, horses which were only fed haylage, eating was more common post-exercise com-

pared to horses fed haylage and concentrate (Jansson, 2010). A novel object test was also con-

ducted where the horses were kept individually on a paddock with a folded tarpaulin. No

difference was found between the diets. Information about the formation of the tarpaulin,

habituation to paddock or testing procedure was not available. In the present study, only a

tendency towards more sniffing was found in T2 and the effect of the treatments were less

pronounced compared to earlier studies.

Treatments used in previous studies varied a lot. In some studies the effect of fat, was not

considered (Bulmer et al., 2015; Freire et al., 2009; Jansson, 2010), whilst others used consider-

able higher amounts of starch compared to this study (Freire et al., 2009; Redondo et al., 2009).

Furthermore, Holland et al. (1996) only examined the effect of added fat and the type of fat

added. Therefore, the current knowledge regarding how the fat/starch ratio influences be-

haviour is relatively small.

Nicol et al. (2005) and Bulmer et al. (2015) used a novel object/stimulus repeatedly and a de-

gree of habituation was suggested in both studies. Even though habituation accorded for all

horses, horses fed fat-rich/fibre-rich diets showed most investigative behaviour (Nicol et al.,

2005) and lowest HR (Bulmer et al., 2015). All horses in this study had previously experienced

the ground sheet used in T2, and further research is needed to clarify whether diet may affect

the rate of habituation. Although the effect of the diets on behaviour was weak in the current

set-up it is unknown whether these diets would have resulted in different reactions in other

types of tests, e.g. a handling test.

The previous studies vary on many factors for example the age and experience of the horses,

treatment, tests and stimulus. Bulmer et al. (2015) only included horses that were calm and

easy to handle (stabled at a college). Bulmer et al. (2015) suggested that this could be the rea-

son for the lack of significant differences in the behaviour.

Discussion

44

In order to investigate whether feed type could affect feed motivation and thus potentially the

latency to feed in the tests, a feed motivation test was conducted. The time to consume one kg

of feed was measured. This was done under the assumption, that more feed motivated horses

would consume their feed faster. Feed consumption tended to differ between the treatments,

where horses fed LS_HF consumed their feed slower compared to horses fed HS_LF. How-

ever, no correlations were found between feed consumption and latency or any of the other

parameter examined. Though because there was a tendency that time used to consume the

feed was different between the treatments, it would have been favourable to have made an

object test without feed involved.

Discussion

45

Table 10. A comparison of the important articles used in the discussion.

Conclusion

46

5. Conclusion The aim of this study was to examine the effect of the fat/starch ratio in young horses’ diet on

muscle endurance and temperament. In contrast to what was hypothesised, no significant dif-

ference was found in whole blood glucose levels between horses fed a Low Starch/High Fat

diet (LS_HF) and horses fed a High Starch/Low Fat diet (HS_LF). During aerobic exercising,

results indicated that horses fed HS_LF tended to reach muscle fatigue quicker than horses

fed LS_HF, indicated by a tendency were horses fed HS_LF increased in whole blood lactate

levels, whereas horses fed LS_HF decreased in whole blood lactate levels. Additionally, horses

fed HS_LF had lower whole blood calcium levels in a single step exercise test compared to

horses fed LS-HF, which further supports a quicker muscles fatigue in HS_LF fed horses than

in LS_HF fed horses. This was in agreement with the hypothesis that horses fed LS_HF would

show better muscle endurance than horses fed HS_LF. Finally, no difference in fear reactions

between the diets was found, though horses fed LS_HF tended to show more investigative

behaviour than horses fed HS_LF.

Perspectives

47

6. Perspectives In this study, results showed that horses fed LS_HF decreased in whole blood glucose levels

in the single step exercise test (ET-2) and tended to decrease in the incremental exercise test

(ET-1). Horses fed HS_LF did not decrease significantly in whole blood glucose levels. This

could indicate a higher insulin sensitivity in horses fed LS_HF than in horses fed HS_LF. In-

sulin measurements were not included in this study, though it would be necessary, in the

future, to include insulin measurements to be able to relate blood glucose levels to insulin and

to support the statement of horses fed LS_HF may have higher insulin sensitivity. Further-

more, one of the factors which influence was unclear, was the time factor. How long exposure

to higher blood glucose levels is needed, before a difference is seen in blood glucose levels.

This is necessary to examine in the future. In addition, it is still uncertain how much starch

and fat should be fed to support the horses´ muscles during exercise.

The results of this study, suggested that horses fed LS_HF might be able to postpone muscle

fatigue longer than horses fed HS_LF. Due to horses fed LS_HF tended to have lower whole

blood lactate levels and higher whole blood calcium levels in the single step exercise test (ET-

2). The intensities (speed and duration of exercise) used in this study were relatively low com-

pared to other studies. Therefore, if there is already an effect on whole blood lactate levels and

whole blood calcium levels at very low intensity aerobic exercise, a bigger effect may be found

by increasing the intensity. In the future, the exercising intensity should maybe be higher and

pressure the horses to where they shift from aerobic to anaerobic metabolism, to see if horses

fed LS_HF actually are able to postpone muscle fatigue. Finally, the effect of the horses having

experienced exercise should be exaimed, due to exercise is thorught to affect fat oxidation.

Endurance training increases the oxidation of fat through increased density of the

mitochondria in the skeletal muscle, which increases the capacity for fat oxidation. It also

results in a proliferation of capillaries within the skeletal muscle, which enhances the delivery

of fatty acid to the muscle (Horowitz and Klein, 2000).

In this study, there was no significant effect between treatment on fearfulness. Though it may

be related to the general temperament of this particular group of horses. In the future, it

would be interesting to examine how a starch-rich diet affects horses with different tempera-

ments.

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Appendix A

A1

Appendix A

Stages Description

Stage 1 The horse is able to be taken out of the pen and accepts fitting of a halter.

Positive reinforcement (feed and scratching on the neck) is used to moti-

vate the horse to leave the pen and to accept the halter.

Stage 2 The horse is able to walk forward, stop and turn in response to rope signals

(negative reinforcement).

Stage 3 The horse is able to enter the test arena area with the handler and eat from

the feed container.

Stage 4 The horse accepts fitting of the heart rate equipment, incl. wetting of the

skin and application of electrode gel. The horse is able to eat from the feed

container in the test arena while wearing the equipment.

Stage 5

The horse can be released by the entrance of the test arena and walks

straight to the feed container and stays there eating for 120 s.

Table 11. Protocol used to habituate the horses to handling and for the object tests. When a horse for filled the criteria for stage 5, the horse was ready for testing

Appendix A

A2

Table 12. Habituation protocol (11 steps) was used to habituate the horses to the treadmill. When a horse for filled the crite-ria for stage 11, the horse was ready for testing.

Stages Description

Stage 1

The horse eats from a feed container placed two meters away from the

treadmill for two min. The treadmill is not active.

Stage 2 The horse eats from a feed container placed 0.5 meters away from the

treadmill for two minutes. The treadmill is not active.

Stage 3 The horse places a front leg on to the ramp of the treadmill and keeps it

there for one minute. Positive reinforcement (Feed and scratching on

the neck) is used to motivate the horse too take a step onto the ramp.

Feed is offered in a feed container, held by the handler standing on the

treadmill in front of the horse.

Stage 4 The horse places both front legs on to the ramp of the treadmill and

keeps them there for two minutes. As in stage 3, positive reinforcement

is used. The horse should also be able to be move backwards down the

treadmill ramp, in response to rope signal.

Stage 5 The horse stands with all four legs, in the middle of treadmill for two

minutes, while eating from a feed container held by the handler stand-

ing in front of the horse.

Stage 6 The horse eats from the feed container placed two meters away from

the treadmill for two minutes, while the treadmill is active.

Stage 7 The horse eats from the feed container placed 0,5 meters away from the

treadmill for two minutes, while the treadmill is active.

Stage 8 The horse stands in the middle of the treadmill, the treadmill is turned

on and the horse walks on the treadmill for two minutes. Positive

(feed) and negative (pressure from the halter and the hind bar of the

treadmill) reinforcement is used for motivating the horse to walk on

the treadmill.

Stage 9 The horse walks on the treadmill for two minutes. The horse is NOT of-

fered feed from the feed container.

Stage 10 The horse walks on the treadmill for five minutes. The horse is NOT of-

fered feed from the feed container.

Stage 11 The horse walks on the treadmill for ten minutes, positive reinforce-

ment is used. The horse is ready for testing.

Appendix A

A3

Table 13. The training programs used during the 6 weeks of training.

Program Training intensity

1 • 2.5 minutes warm up on the treadmill at speed 4.8 km/h (level 0)

• 5 minutes on the treadmill at speed 5.2 km/h (level 1)

• 5 minutes on the treadmill at speed 6.2 km/h (level 2)

• 2.5 minutes of cooling down on the treadmill 4.8 km/h (level 0)

Total time: 15 min

2 • 4 minutes of warm-up on the treadmill at speed 5.2 km/h (level 1)

• 5 minutes on the treadmill at speed 6.2 km/h (level 2)

• 5 minutes on the treadmill at speed 7.8 km/h (level 3)

• 4 minutes of cooling down on the treadmill 5.2 (level 1)

Total time: 18 min

3 • 4 minutes of warm-up on the treadmill at speed 5.2 km/h (level 1)

• 4 minutes on the treadmill at speed 6.2 km/h (level 2)

• 3 minutes on the treadmill at speed 7.8 km/h (level 3)

• 3 minutes on the treadmill at speed 8.6 km/h (level 4)

• 4 minutes of cooling down on the treadmill 5.2 km/h (level 1)

Total time: 18 min

4 • 4 minutes of warm-up on the treadmill at speed 6.2 km/h (level 2)

• 4 minutes on the treadmill at speed 7.8 km/h (level 3)

• 4 minutes on the treadmill at speed 8.6 km/h (level 4)

• 3 minutes on the treadmill at speed 9.5 km/h (level 5)

• 4 minutes of cooling down on the treadmill 5.2 km/h (level 1)

Total time: 19 min

5 • 4 minutes of warm-up on the treadmill at speed 6.2 km/h (level 2)

• 4 minutes on the treadmill at speed 7.8 km/h (level 3)

• 3 minutes on the treadmill at speed 8.6 km/h (level 4)

• 3 minutes on the treadmill at speed 9.5 km/h (level 5)

• 2 minutes on the treadmill at speed 10.4 km/h (level 6)

• 4 minutes of cooling down on the treadmill 5.2 km/h (level 1)

Total time: 20 min

5a • 4 minutes of warm-up on the treadmill at speed 6.2 km/h (level 2)

• 4 minutes on the treadmill at speed 7.8 km/h (level 3)

• 3 minutes on the treadmill at speed 8.6 km/h (level 4)

• 3 minutes on the treadmill at speed 9.5 km/h (level 5)

• 1 minutes on the treadmill at speed 10.4 km/h (level 6)

• 1 minutes on the treadmill at speed 11.2 km/h (level 7)

• 4 minutes of cooling down on the treadmill 5.2 km/h (level 1)

• Total time: 20 min

Appendix A

A4

Table 14. An overview of the events during the nine weeks of training and data collection period. The horses had an adaptation

period of 14 days, which is week 1 and 2 in the table (F). The horses were trained four times/week for five weeks (week 3-7) and

two time in the sixth week. Training days varied between the weeks. The horses were not trained the remaining days (DO).

The horses were tested in an incremental exercise test (ET-1) in week 8 and an single step exercise test (ET-2) in week 9. AC

= Acclimation to Treadmill, F = Feed Adaption period, T = Training day, BB = Baseline blood sample, DO = Day off, the

equines were not conditioned, P1= Program 1, P2 = Program 2, P3 = Program 3, P4 = Program 4, P5 = Program 5.

Week Monday Tuesday Wednesday Thursday Friday Saturday Sunday

1 F F F F F F F

2 F F F F F F F

3 DO T – P1 T- P1 T – P1 T- P1 + HR DO DO

4 T - P1 + HR T – P2 DO DO T– P2 T – P2 DO

5 DO T - P2 T– P2 T – P3 T – P3 DO DO

6 DO T – P3 + HR DO DO T– P3+HR T – P4 T– P4

7 T – P4 DO T – P4 DO T – P5 T – P5 DO

8 T – P5 T – P5a DO ET-1 ET-1 DO DO

9 DO DO DO ET-2 ET-2 DO DO

Appendix B

B1

Appendix B Table 15. Correlations for both treatments: Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) between car-

bon dioxide (CO2), oxygen (O2), calcium, glucose, lactate, maximum heart rate (MAX HR) and average heart rate (AVG

HR) both pre- and post-exercise the horses performing an incremental exercise test (ET-1). Part 1.

Table 16. Correlations for ET-1 for both treatments Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) be-tween carbon dioxide (CO2), oxygen (O2), calcium, glucose, lactate, maximum heart rate (MAX HR) and average heart rate (AVG HR) both pre- and post-exercise the horses performing an incremental exercise test (ET-1). Part 2.

ET-1

LS_HF HS_LF

Pre Post Pre Post

CO2 (%) and O2 (%)

rs = - 0.10 P = 0.8 (n=6)

rs = - 0.41 P = 0.2 (n=9)

rs = - 0.39

P = 0.3 (n=7)

rs= - 0.26 P = 0.7 (n=6)

CO2 (%) and ca (%)

rs = 0.03 P =0.9 (n=8)

rs = 0.16 P = 0.7 (n=9)

rs = 0.48

P = 0.4 (n=6) rs= 0.25

P = 0.5 (n=8)

CO2 (%) and glucose (mmol/L)

rs = - 0.01 P = 0.98 (n=8)

rs = 0.54 P = 0,11 (n=9)

rs = 0.40

P = 0.3 (n=7)

rs= - 0.44 P = 0.3 (n=8)

CO2 (%) and lactate (mmol/L)

rs = - 0.76* P = 0.04 (n=7)

rs = - 0.37 P = 0.3 (n=9)

rs = 0.54

P = 0.2 (n=7) rs= 0.43

P = 0.4 (n=6)

CO2 (%) and MAX HR (bpm)

rs = 0.12 P = 0.8 (n=8)

rs = 0.58 P = 0.09 (n=9)

rs = 0.05

P = 0.9 (n=8) rs= 0.67

P = 0.06 (n=8)

CO2 (%) and AVG HR (bpm)

rs = 0.22 P = 0.6 (n=8)

rs = 0.61

P = 0.08 (n=9)

rs= - 0.05 P = 0.2 (n=8)

rs= 0.74 P = 0.03 (n=8)

ET-1

LS_HF HS_LF

Pre Post Pre Post

O2 (%) and ca (mmol/L)

rs = 0.00 P = 1.0 (n=6)

rs = 0.18 P = 0.6 (n=9)

rs = 0.00

P = 1.0 (n=6) rs= - 0.38

P = 0.3 (n=7)

O2 (%) and glucose (mmol/L)

rs = - 0.74

P = 0.10 (n=6) rs = - 0.35

P = 0.3 (n=9)

rs = - 0.20 P = 0.7 (n=6)

rs= - 0.34 P = 0.4 (n=7)

O2 (%) and lactate (mmol/L)

rs = 0.16 P = 0.7 (n=7)

rs = 0.14 P = 0.7 (n=9)

rs = 0.11

P = 0.8 (n=7) rs= - 0.09

P = 0.9 (n=6)

O2 (%) and MAX HR (bpm)

rs = - 0.69

P = 0.05 (n=8) rs = - 0.11

P = 0.8 (n=9)

rs = - 0.71 P = 0.04 (n=8)

rs= - 0.29 P = 0.5 (n=8)

O2 (%) and AVG HR (bpm)

rs = - 0.15 P = 0.7 (n=7)

rs = 0.54 P = 0.11 (n=9)

rs = - 0.48

P = 0.2 (n=8) rs= - 0.61

P = 0.09 (n=8)

Appendix B

B2

Table 17. Correlations for ET-1 for both treatments Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) be-tween carbon dioxide (CO2), oxygen (O2), calcium, glucose, lactate, maximum heart rate (MAX HR) and average heart rate (AVG HR) both pre- and post-exercise the horses performing an incremental exercise test (ET-1). Part 3.

ET-1

LS_HF HS_LF

Pre Post Pre Post

Ca (mmol/L) and glu-cose (mmol/L)

rs = 0.06 P = 0.9 (n=8)

rs = 0.38 P = 0.3 (n=9)

rs = 0.38

P = 0.3 (n=7) r s = - 0.81

P = 0.01 (n=8)

Ca (mmol/L) and lactate (mmol/L)

rs = - 0.51 P = 0.2 (n=7)

rs = - 0.17 P = 0.6 (n=9)

rs = 0.30

P = 0.5 (n=7) rs = 0.55

P = 0.2 (n=7)

Ca (mmol/L) and maxi-mum heart rate (bpm)

rs = 0.44 P = 0.2 (n=8)

rs = 0.46 P = 0.2 (n=9)

rs = 0.29

P = 0.5 (n=8) rs =0.19

P = 0.6 (n=9)

Ca (mmol/L) and aver-age heart rate (bpm)

rs = 0.45 P = 0.2 (n=8)

rs = 0.33 P = 0.4 (n=9)

rs = 0.53

P = 0.2 (n=8) rs = 0.15

P = 0.7 (n=9)

Glucose (mmol/L) and lactate (mmol/L)

rs = 0.29 P = 0.49

(n=7)

rs = - 0.13 P = 0.7 (n=9)

rs = 0.94

P < 0.001 (n=7) rs = - 0.74

P = 0.04 (n=7)

Glucose (mmol/L) and maximum heart rate (bpm)

rs = 0.40 P = 0.3 (n=8)

rs = 0.89 P <0.001 (n=9)

rs = - 0.23

P = 0.5 (n=8) rs = - 0.20

P = 0.6 (n=9)

Glucose (mmol/L) and average heart rate (bpm)

rs = 0.37 P = 0.3 (n=8)

rs = 0.93 P <0.001 (n=9)

rs = - 0.10

P = 0.8 (n=8) rs = - 0.08

P = 0.8 (n=9)

Lactate (mmol/L) and maximum heart rate (bpm)

rs = - 0.30 P = 0.4 (n=8)

rs = - 0.18 P = 0.6 (n=9)

rs = - 0.19

P = 0.6 (n=8) rs = 0.74

P = 0.03 (n=8)

Lactate (mmol/L) and average heart rate (bpm)

rs = - 0.43 P = 0.3 (n=8)

rs = 0.13 P = 0.7 (n=9)

rs = - 0.22

P = 0.6 (n=8) rs =0.68

P <0.001 (n=10)

Appendix B

B3

Table 18. Correlations for ET-2 for both treatments Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) be-tween carbon dioxide (CO2), oxygen (O2), calcium, glucose, lactate, maximum heart rate (MAX HR) and average heart rate (AVG HR) both pre- and post-exercise the horses performing a single exercise test (ET-2). Part 1.

ET-2

LS_HF HS_LF

Pre Post Pre Post

O2 (%) and ca (mmol/L)

rs = 0.62

P = 0.09 (n=8) rs = - 0.64

P = 0.2 (n=6)

rs = 0.28 P = 0.5 (n=7)

rs = 0.11 P = 0.8 (n=9)

O2 (%) and glu-cose (mmol/L)

rs = 0.14 P = 0.7 (n=8)

rs = 0.06 P = 0.9 (n=6)

rs = - 0.35

P = 0.5 (n=6)

rs = - 0.44 P = 0.2 (n=8)

O2 (%) and lac-tate (mmol/L)

rs = - 0.02 P = 0.5 (n=9)

rs = 0.18 P = 0.7 (n=7)

rs = - 0.29

P = 0.5 (n=7) rs = - 0.12

P = 0.8 (n=8)

O2 (%) and MAX HR (bpm)

rs = - 0.28 P = 0.4 (n=9)

rs = 0.635 P = 0.07 (n=8)

rs = - 0.60

P = 0.1 (n=8) rs = - 0.29

P = 0.4 (n=9)

O2 (%) and AVG HR (bpm)

rs = - 0.02 P = 0.5 (n=9)

rs = 0.24 P = 0.5 (n=8)

rs = 0.06

P = 0.8 (n=8) rs = 0.07

P = 0.8 (n=9)

Appendix B

B4

Table 19. Correlations for ET-2 for both treatments: Low Starch/High Fat (LS_HF) and High Starch/Low Fat (HS_LF) be-tween carbon dioxide (CO2), oxygen (O2), calcium, glucose, lactate, maximum heart rate (MAX HR) and average heart rate (AVG HR) both pre- and post-exercise the horses performing an incremental exercise test (ET-2). Part 2.

ET-2

LS_HF HS_LF

Pre Post Pre Post

Ca (mmol/L)

and glucose (mmol/L)

rs = - 0.29

P = 0.5 (n=7) rs = 0.67

P = 0.1 (n=6)

rs = 0.09 P = 0.8 (n=6)

rs = 0.22 P = 0.6 (n=8)

Ca (mmol/L)

and lactate (mmol/L)

rs = 0.04

P = 0.9 (n=7) rs = - 0.49

P = 0.2 (n=8)

rs = - 0.49 P = 0.2 (n=7)

rs = 0.36 P = 0.4 (n=8)

Ca (mmol/L)

and maximum heart rate (bpm)

rs = - 0.44

P = 0.2 (n=8) rs = - 0.55

P = 0.1 (n=8)

rs = 0.04 P = 0.9 (n=8)

rs = 0.04 P = 0.9 (n=9)

Ca (mmol/L)

and average heart rate (bpm)

rs = - 0.43

P = 0.3 (n=8) rs = - 0.54

P = 0.1 (n=8)

rs = 0.40 P = 0.3 (n=8)

rs = - 0.38 P = 0.3 (n=9)

Glucose (mmol/L) and lactate (mmol/L)

rs = - 0.18

P = 0.7 (n=7) rs = - 0.07

P = 0.8 (n=7)

rs = 0.15 P = 0.7 (n=6)

rs = 0.11 P = 0.8 (n=8)

Glucose (mmol/L) and maximum heart rate (bpm)

rs = - 0.58

P = 0.09 (n=9) rs = - 0.28

P = 0.5 (n=8)

rs = -0.52 P = 0.2 (n=8)

rs = 0.45 P = 0.2 (n=9)

Glucose (mmol/L) and average heart rate (bpm)

rs = - 0.58

P = 0.09 (n=9) rs = - 0.27

P = 0.5 (n=8)

rs = - 0.40 P = 0.3 (n=8)

rs = 0.10 P = 0.8 (n=9)

Lactate (mmol/L) and maximum heart rate (bpm)

rs = 0.20

P = 0.6 (n=8) rs = - 0.02

P = 0.95 (n=8)

rs = 0.17 P = 0.7 (n=8)

rs = 0.00 P = 0.98 (n=8)

Lactate (mmol/L) and average heart rate (bpm)

rs = 0.22

P = 0.6 (n=8) rs = 0.33

P = 0.4 (n=9)

rs = - 0.19 P = 0.6 (n=8)

rs = - 0.46 P = 0.2 (n=8)

Appendix B

B5

Table 20. Correlation between latency, average heart rate (Avg HR), maximum heart rate (Max HR), object focus, touch, sniff and feed motivation in object test 1.

Table 21. Correlation between latency, average heart rate (Avg HR), maximum heart rate (Max HR), object focus, touch, sniff and feed motivation in object test 2.

Object test 1 Latency Avg HR Max HR Object focus

Touch Sniff

Feed motiva-tion (sec)

rs = 0.26 P = 0.20

rs = 0.05 P = 0.83

rs = - 0.08 P = 0.72

rs = 0.19 P = 0.42

rs = 0.06 P = 0.81

rs = 0.25 P = 0.29

Latency (sec)

- rs = 0.62 P = 0.004

rs = 0.47 P = 0.037

rs = 0.92 P < 0.001

rs= - 0.26 P = 0.26

rs = - 0.22 P = 0.36

AVG HR (bpm)

- - rs = 0.87 P < 0.001

rs = 0.76 P < 0.001

rs = - 0.12 P = 0.62

rs = - 0.36 P = 0.11

MAX HR (bpm)

- - - rs = 0.62 P < 0.004

rs = - 0.26 P = 0.26

rs = - 0.49 P = 0.03

Object focus (sec)

- - - - rs = - 0.22 P = 0.35

rs = 0.30 P = 0.20

Touch (sec)

- - - - - rs = 0.47 P = 0.04

Object test 2 Latency Avg HR Max HR Object focus

Touch Sniff

Feed motiva-tion (sec)

rs = 0.36 P = 0.12

rs = 0.09 P = 0.70

rs = 0.06 P = 0.79

rs = 0.27 P = 0.25

rs= - 0.32 P = 0.17

rs = 0.36 P = 0.12

Latency (sec)

- rs = 0.62 P = 0.004

rs = 0.61 P = 0.004

rs = 0.65 P = 0.002

rs = - 0.03 P = 0.91

rs = 0.19 P = 0.41

AVG HR (bpm)

- - rs = 0.79 P < 0.001

rs = 0.46 P = 0.04

rs = 0.02 P = 0.93

rs = - 0.23 P = 0.32

MAX HR (bpm)

- - - rs = 0.52 P = 0.02

rs = 0.10 P = 0.66

rs = - 0.11 P = 0.64

Object focus (sec)

- - - - rs = - 0.42 P = 0.05

rs = 0.19 P = 0.43

Touch (sec)

- - - - - rs = 0.21 P = 0.36

Appendix C

C1

Appendix C

Summary

When horses exercise, muscles are activated and the need for energy and oxygen increases.

The breakdown of oxygen by the oxidative metabolism results in formation of free radicals

(oxidative stress). Free radicals are scavengers, which damage tissues and therefore an over-

production of free radicals in the can damage muscles tissue and thereby weakening the func-

tion of the muscle. Muscles and other tissues are dependent on an antioxidant system to re-

move free radicals. The antioxidant system is divided into two sub-systems; the primary and

secondary antioxidation system. The primary antioxidant system consists of enzymes for ex-

ample superoxide dismutase and glutathione peroxidase, which are dependent on minerals.

The secondary antioxidation system uses vitamins as its protection mechanism. A antioxida-

tion system has a specific capacity and when the amount of free radicals is within the capacity

of the body, the free radicals are removed. Is the amount of free radicals exceeds the capacity

of the antioxidation system, the removal of free radicals slows down and tissues are damaged.

There are primary to ways of reducing tissue damage and it is either by enhancing the anti-

oxidant capacity by ensuring minerals and vitamins or by enhancing the amount of omega-3-

fatty acids in the diet. Omega 3 fatty acids are oxidated to Eicosapentenoic acid and/or Do-

cosahexanoic acid, which are prostaglandins which instead of being proinflammatory they

are anti-inflammatory. Therefore, promoting the breakdown of omega 3 fatty acids instead of

omega 6 fatty acids, which are oxidized to arachidonic acid, this may reduce the inflammation

associated with free radicals.

The conclusion of this literary study is that the different studies are ambiguous, but the ten-

dency is that increasing the levels of omega-3-fatty acids will decrease the occurrence of in-

flammation associated with exercise. Additionally, ensuring the accurate amounts of minerals

and vitamins will increase the antioxidant system capacity to remove free radicals.

Dietary effect on exercise induced inflammation in riding horses By Carina Beblein 2016