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Winter 2014
MEASURMENTS OF HAPTOGLOBIN, SERUM AMYLOID A, FIBRINOGEN, AND LEUKOCYTES TO EVALUATE STRESS IN ORGANICALLY MANAGED, PASTURE-FED AND CONVENTIONAL MANAGED, TOTALLY MIXED RATION-FED JERSEY CATTLE.
Dorothy Ann Perkins University of New Hampshire, Durham
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Recommended Citation Perkins, Dorothy Ann, "MEASURMENTS OF HAPTOGLOBIN, SERUM AMYLOID A, FIBRINOGEN, AND LEUKOCYTES TO EVALUATE STRESS IN ORGANICALLY MANAGED, PASTURE-FED AND CONVENTIONAL MANAGED, TOTALLY MIXED RATION-FED JERSEY CATTLE." (2014). Master's Theses and Capstones. 994. https://scholars.unh.edu/thesis/994
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MEASURMENTS OF HAPTOGLOBIN, SERUM AMYLOID A, FIBRINOGEN, AND LEUKOCYTES TO EVALUATE STRESS IN ORGANICALLY MANAGED, PASTURE‐FED AND CONVENTIONAL MANAGED, TOTALLY MIXED RATION‐FED JERSEY CATTLE.
BY
DOROTHY ANN PERKINS Bachelor of Science, University of New Hampshire, 2001
THESIS
Submitted to the University of New Hampshire In Partial Fulfillment of The Requirements for the Degree of
Master of Science In Animal Science
December, 2014
This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Masters of Science in Animal Sciences by:
Thesis Director, Thomas L. Foxall, Ph.D., Professor, Department of Biological Sciences
Charles G. Schwab, Ph.D., Professor Emeritus, Animal and Nutritional Sciences
Andre Fonseca de Brito, DVM., Ph.D., Assistant Professor, Department of Biological Sciences
Andrew B. Conroy, Ph.D., Professor, Thompson school of Applied Science
On November 7, 2014
Original approval signatures are on file with the University of New Hampshire Graduate School.
DEDICATION
This thesis is dedicated to my beloved husband, Ralph K. Perkins Jr.
Without him, this work or my wonderful life wouldn’t be.
Thank you honey, I’ll keep my promise.
iii
ACKNOWLEDGEMENTS
What do you say to a person who never let’s go of your hand and never loses faith in you?
Thank you doesn’t seem like enough. My advisor, Dr. Thomas Foxall, took my hand when no one
else could or would. He helped me through so many personal tribulations while I worked on this thesis. He worried about me. He shared his knowledge with me. He was compassionate,
understanding, and full of insight. He urged me on even when I wanted to quit. His insistence on
excellence and sweating the details, improved my work, writing, and work ethic. His undying
encouragement helped me achieve my goals, which will open the doors to my future. Thank you
just doesn’t seem like enough.
I would like to thank my committee members, Dr. Charles Schwab, Dr. Andre Brito and
Dr. Drew Conrad for their support, experience, and input into this thesis.
Dr. Schwab you saw in me what I didn’t see in myself. Thank you for your patience,
understanding, and help when I wasn’t sure of anything on my life’s path. You are a wonderful
and compassionate human being and a talented and passionate researcher. You have made a
profound impact on my life, most of which I have yet to discover. Dr. Brito, you solidified my understanding of FA digestion in the ruminant. Your willingness to help, approachability, and
warm demeanor helped me feel supported. My work, academic writings, future publications and research projects will benefit from all your advice. Dr. Conroy, your advice about writing this thesis was more valuable than you could have imagined it would be. It helped me slow down to the
speed of life, breath through the obstacles, and take it 10 pages at a time.
iv In addition, I would like to express my sincere appreciation to Megan Seneca for collecting
all the samples and the hours she spent helping me with assays and analysis; Dan Gallant for all
the work he did with the Fibrinogen assays and WBC counts; Dr. Becky Sideman for all her help
with my statistical analysis; Amy Papineau, Shirley Clark, Mary West and Lynn Poire for their
computer and formatting help; the staff and employees of both the University of New Hampshire
Fairchild Dairy Teaching and Research Center and the University of New Hampshire Burley‐
Demeritt Organic Dairy Research Farm; and last but certainly not least, my wonderful family, colleagues and friends, for their love, support, and encouragement. I could not have prevailed
without each and every one of them. I am clearly blessed.
v
TABLE OF CONTENTS
DEDICATION ...... iii
ACKNOWLEDGEMENTS ...... iv
LIST OF FIGURES ...... ix
LIST OF TABLES ...... x
LIST OF GRAPHS ...... xi
ABSTRACT ...... xii
CHAPTER PAGE
I. INTRODUCTION ...... 1
Dietary Evolution of Humans and Cattle ...... 1
Evolution of the Human Diet ...... 2
Evolution of the Dairy Cow Diet ...... 4
Fatty Acids ...... 9
Essential Fatty Acids and Inflammation ...... 18
Fatty Acids and Disease ...... 22
Fatty Acid Digestion and Absorption in Humans and Cows ...... 24
Fatty Acid Digestion and Absorption in Humans ...... 25
Fatty Acid Digestion and Absorption in Ruminants ...... 26
The Inflammation Acute Phase Response ...... 33
Haptoglobin ...... 35
vi Serum Amyloid A ...... 36
Fibrinogen ...... 37
White Blood Cell Counts ...... 39
Stress Effects of Heat and Cold on Lactating Dairy Cattle ...... 42
II. MATERIALS AND METHODS ...... 46
Experimental Design ...... 46
Objective ...... 46
Hypothesis and Animal Management ...... 46
Sample Collection ...... 47
Blood Analysis ...... 48
Statistical Analysis and Weather Data ...... 49
III. RESULTS ...... 50
Blood Markers ...... 50
Weather Data ...... 67
IV. DISCUSSION ...... 69
Haptoglobin ...... 69
Serum Amyloid A ...... 72
Fibrinogen ...... 74
Total White Blood Cells ...... 75
V. CONCLUSIONS ...... 77
vii
LIST OF REFERENCES ...... 79
APPENDIX ...... 96
Blood Marker Data ...... 96
INSTITUTIONAL CARE AND USE COMMITTEE APPROVAL ...... 99
viii
LIST OF FIGURES
Figure 1: Seasonal Variation of Fatty Acids in Pasture ...... 5
Figure 2: Triglyceride Structure ...... 12
Figure 3: Phospholipid Structure (lecithin) ...... 12
Figure 4: Galactolipid Structure ...... 13
Figure 5: Structure of α‐Linolenic Acid ...... 15
Figure 6: Structure of Linoleic Acid ...... 15
Figure 7: Structure of Saturated Fat (stearic Acid) ...... 15
Figure 8: Structure of Conjugated Linoleic Acid ...... 16
Figure 9: Chemical Structure of cis‐and trans‐isomers of oleic acid ...... 16
Figure 10: Fatty Acid Metabolism ...... 21
Figure 11: Fatty Acid Digestion and Absorption in Humans ...... 26
Figure 12: Rumen Biohydrogenation of Linoleic Acid ...... 29
Figure 13: Rumen Biohydrogenation of α‐Linolenic Acid ...... 30
Figure 14: Fatty Acid Digestion and Absorption in Ruminants ...... 32
Figure 15: Conversion of Fibrinogen to Fibrin ...... 39
ix
LIST OF TABLES
Table 1: Omega‐6 to Omega‐3 Ratio of Seed Oils and Selected Meats ...... 3
Table 2: Saturated Fat in Common Foods in the United States ...... 10
Table 3: Unsaturated Fatty Acids Found in Common Foods in the United States ...... 11
Table 4: Fatty Acid Structure and Classification ...... 17
Table 5: Fatty Acid Composition of Typical Feedstuffs ...... 28
Table 6: Hematology: Normal Reference Values for Cattle ...... 42
Table 7: Blood Sampling Dates ...... 48
Table 8: Haptogloben Levels Within Farm by Sample Week ...... 50
Table 9: Haptogloben Levels Between Farms by Sample Week ...... 51
Table 10: Serum Amyloid A Levels Within Farm by Sample Week ...... 53
Table 11: Serum Amyloid A Levels Between Farms by Sample Week ...... 54
Table 12: Plasma Fibrinogen Levels Within Farm by Sample Week ...... 56
Table 13: Plasma Fibrinogen Between Farms by Sample Week ...... 56
Table 14: Total White Blood Cell Counts Within Farm by Sample Week ...... 58
Table 15: Total White Blood Cell Counts Between Farms by Sample Week ...... 59
Table 16: Percent Neutrophils Within Farm by Sample Week ...... 61
Table 17: Percent Neutrophils Between Farms by Sample Week ...... 62
Table 18: Percent Lymphocytes Within Farm by Sample Week ...... 64
Table 19: Percent Lymphocytes Between Farms by Sample Week ...... 64
Table 20: Neutrophil/Lymphocyte Ratios by Sample Week ...... 66
Table 21: Temperature Data ...... 68
x
LIST OF GRAPHS
Graph 1: Haptogloben Within Farm by Sample Date‐Organic ...... 51
Graph 2: Haptogloben Within Farm by Sample Date ‐ Conventional ...... 52
Graph 3: Haptogloben Between Farms by Sample Date ...... 52
Graph 4: Serum Amyloid A Within Farm by Sample Date ‐Organic ...... 54
Graph 5: Serum Amyloid A Within Farm by Sample Date ‐ Conventional ...... 55
Graph 6: Serum Amyloid A Between Farms by Sample Date ...... 55
Graph 7: Fibrinogen Within Farm by Sample Date‐Organic ...... 57
Graph 8: Fibrinogen Within Farm by Sample Date ‐Conventional ...... 57
Graph 9: Fibrinogen Between Farms by Sample Date ...... 58
Graph 10: Total White Blood Cell Counts Within Farm by Sample Date ‐Organic ...... 60
Graph 11: Total White Blood Cell Counts Within Farm by Sample Date ‐Conventional ...... 60
Graph 12: Total White Blood Cell Counts Between Farms ...... 61
Graph 13: Neutrophil Counts Within Farm by Date ‐Organic ...... 62
Graph 14: Neutrophils Counts Within Farm by Date ‐Conventional ...... 63
Graph 15: Percent Neutrophils Between Farms by Sample Date ...... 63
Graph 16: Lymphocyte Counts Within Farm by Date ‐Organic ...... 65
Graph 17: Lymphocytes Counts Within Farm by Date ‐Conventional ...... 65
Graph 18: Lymphocytes Counts Between by Date Farms ...... 66
Graph 19: Neutrophil/Lymphocyte Ratios Between Farms by Week ...... 67
xi
ABSTRACT
MEASURMENTS OF HAPTOGLOBIN, SERUM AMYLOID A, FIBRINOGEN, AND LEUKOCYTES TO
EVALUATE STRESS IN ORGANICALLY MANAGED PASTURE‐FED AND CONVENTIONAL MANANGED
TOTALLY MIXED RATION‐FED JERSEY CATTLE
By
Dorothy A. Perkins
University of New Hampshire, December, 2014
In cattle sub‐acute inflammatory disorders are difficult to assess because visual symptoms
of diseases are not easily detectable with large numbers of dairy cattle in loose housing. In addition, inflammation is not always followed by leukocytosis in cattle, which makes the use of common blood tests like total white blood cell counts less reliable as a diagnostic tool. More sensitive and objective methods are needed to evaluate health status. Research has been focused
on the use of acute phase proteins (APPs) to diagnose acute and chronic illness in cattle, because
acute phase proteins respond very quickly when an inflammation event occurs, and they can be
easily detected in blood. The most studied APPs in cattle are Haptoglobin (Hp), Serum Amyloid A
(SAA), and Fibrinogen (FB). No studies have been done comparing the physiologic response of pasture‐fed and TMR‐fed confinement cows during a pasture season using acute phase proteins
as markers of inflammation and stress.
Nine Jersey dairy cows were selected from the herd at the University of New Hampshire
Burley‐Demeritt Organic Dairy Research Farm and nine Jersey cows were matched to these cows
xii by age, parity and stage of lactation and housed at the University of New Hampshire Fairchild
Dairy Teaching and Research Center. APPs and white blood cells (WBC) were measured in the blood of these cows at 5 time points from April through September, 2012. Levels of Hp, SAA, and
Fb were determined using a commercial bovine ELISA test kit. Whole blood from EDTA tubes was
stained with Wright’s stain for differential white blood cell counts. All levels were compared with
the calculated temperature humidity index and health records.
All APP levels at both farms were within normal ranges established for healthy cattle.
However, Hp, SAA, and WBC indicated a low level inflammation response that in some instances differed significantly between the two farms. Hp, SAA, and WBC levels in the pastured herd increased significantly by sample period 3, and remained elevated through sample period 4 and
5. Hp, SAA, and WBC in the conventional herd did not increase significantly until the 4th sample period and remained elevated for the 5th sample period. The Fb levels at both farms did not follow the same pattern seen with Hp, SAA, and WBC. Health records confirmed that minor inflammatory
events may have been responsible for the rise in Fb levels, Neutrophil/Lymphocyte ratios between
the two farms showed an increased inflammation response in the pastured cows over the entire
sample period as compared to the TMR fed cows. Neutrophil lymphocyte ratios for the pastured
herd were never within the normal limit set at 0.5 for healthy cattle at any of the 5 sample time
points, where the TMR herd had normal levels at 3 sample time points.
These results indicated that the conventional herd had lower inflammation levels than
the pastured herd over the entire study period and that common environmental conditions such
as temperature and humidity may elicit a low level immune response in both management systems that can be measured in the blood using APPs and WBC.
xiii
CHAPTER I
INTRODUCTION
Dietary Evolution of Humans and Cattle
All cattle are descendants of the European Auroch (Bos primagenius). These ruminant animals have been supplying food for humans for a long time. Domesticated approximately
8000 years ago, there are representations and descriptions of Aurochs in prehistoric cave paintings (Ajmone‐Marsan et al., 2010). By looking at stable carbon and nitrogen isotope ratios, scientists have shown that these early‐domesticated cows were minimally grain fed depending on locale and climate (Bailey, 1958; Cordain, 2005). With the development of agriculture and the industrial revolution, the diets and management systems of these animals changed from a grazing system to a grain based, by‐product fed, totally mixed ration (TMR) confinement system.
This change would have altered the fatty acid (FA) profile of their feedstuffs to one higher in omega‐6 FAs and lower in omega 3 FAs. There are many who believe these animals have not had sufficient time to genetically adapt to the new dietary FA profile and management systems and evolutionary discordance is the result (Ajmone‐Marsan et al., 2010; Larson et al., 1996;
Shearer J.K., 2007; Sharif and Muhammad, 2008). Discordance manifests itself phenotypically as increased morbidity and mortality and reduced reproductive success (Eaton, 1985; Eaton, 1988;
Boaz, 2002; Cordain, 1999). Studies have shown that a FA profile that favors omega‐6 FAs over omega‐3 FAs causes health problems in humans (Cordain, 2005; Roth and Andersson, 2004;
Galhard et al., 2007). There is ample evidence that feeding high amounts of grain to cows adversely affects their health (Schroeder et al., 2003; Ellis et al., 2006; Laidlaw et al., 2006;
1
Ajmone‐Marsan et al., 2010; Larson et al., 1996; Couvreur et al., 2006; Bergamoa et al., 2003;
White et al., 2001; Evans et al.,2008; Olmos et al.,2009).
Evolution of the Human Diet
The diet of humans and their evolutionary ancestors approximately 10,000 years ago was quite different from the human diet of today that resulted from the development of agriculture and the domestication of plants and animals (Simopoulos, 1999a). The human genome developed to metabolize a very different diet with a very different essential FA profile than what humans in developed countries are currently eating. It is believed that this change has occurred much more rapidly than what our genome can adapt to in such a short time and ill health may be the result (Simopoulos,1999a; Nachman and Crowell, 2000; Roth and Andersson,
2004; Galhardo et al., 2007).
Although the diet of early humans would have varied by geographic locale, climate and specific ecological niche, studies have shown that there were universal characteristics common to all groups that have helped us understand how our current diet may predispose modern populations to chronic disease (Cordain, 2005). The diet of early hunter gatherer societies consisted of minimally processed wild plant and animal foods which are low in calories, high in protein, low in fat and carbohydrates, and high in antioxidants including vitamins A, C, and E
(Simopoulos,1999a; Nachman and Crowell, 2000; Roth and Anderson, 2004; Galhardo R.S. et al.,
2007). This type of diet would have had an essential FA profile much lower in the essential omega‐6 FAs, and much higher in the essential omega‐3 FAs, as compared to modern diets
(Cordain, 2002). Studies have shown the ratio of omega‐6 FAs to omega‐3 FAs in a typical hunter gather diet was close to 1:1 or 2:1. The current estimated ratios of omega‐6 FAs to omega‐3 FAs in a typical Western diet is 10:1, or even as high as 15 or 20:1(Eaton S.B., 1992; Brand‐Miller &
Colagiuri, 1994). This shift has been linked to grain consumption, which is high in omega‐6 FAs.
2 Hunter‐gatherers would not have included grains in their diet. Grains are hard for humans to digest unless they are processed in some way (Cordain, 2005), and as a main staple, wild cereal grains would have been small and very time consuming to harvest so would have been rarely used by hunter gather societies (Eaton, 1992; Cordain, 2000). Evidence of grain processing tools didn’t appear in the fossil records until approximately 12,000 years ago, which coincides with the development of agriculture (Wright, 1991). Agriculture enabled human populations to grow more rapidly and with this growth came the need for more food and better ways to utilize and preserve it. The industrial revolution brought more drastic change to the human diet.
Intense refining of grain by removing the germ and bran from the kernel produced flour that was finer in texture than what humans had already started to genetically adapt to. This new
flour was devoid of fat, which increased its shelf‐life and made the starches more easily digestible (Wood et al., 2008). In addition to highly refined grains, vegetable oils were also introduced into the human diet. Oils and fats have two times the energy in them as other foods.
Many of these vegetable oils are very high in omega‐6 FAs and almost devoid of omega‐3 FAs
(Table 1).
Table 1 Omega‐6 to Omega‐3 Ratio of Seed Oils and Selected Meats Source Corn Soybean Cottonseed Sunflower Canola Olive Wild Grass Grain oil oil oil oil oil oil Salmon fed fed Beef Beef Ratio 83:1 13:1 259:1 19:1 2:1 13:1 1:11 2:1 4:1 French et al., 2000; Simopoulos, 1999; National Institute of Health‐Essential FA education. (http://efaeducation.nih.gov/sig/esstable.html).
The oils were then chemically altered to create trans‐fat products that took the place of lard and butter. Another change in the human diet was due to the domestication of animals for the use of food. To promote their rapid growth humans began to feed them grains that are higher in omega‐6 FAs and reduced forages, which are high in omega‐3 FAs (Laidlaw et al.,
3 2006). Thus humans began to consume higher amounts of omega‐6 FAs not just as plant nutrients but also as animal derived nutrients, whether as meat, eggs or milk. It is estimated that only 20 to 25% of the trans fats we consume are from animal fats, the rest are from artificially hydrogenated vegetable oils (Aro et al., 1998; O’Donnell‐Megaro A.M. et al., 2011). All of the aforementioned changes in diet moved the FA profile of our foods to the ratios we see today in an extremely short time in our evolutionary time line (Eaton, 1985; Cordain, 2005).
Studies in humans have linked the consumption of higher amounts of omega‐6 FAs compared to omega‐3 FAs and increased consumption of trans fats to many chronic illnesses particularly those associated with chronic inflammation such as type II diabetes, psoriasis, heart disease, cancer, and arthritis (Miller and Colagiuri, 1994; Eaton, 1985; Eaton, 1988; Cordain et al., 2002; Cordain et al., 2005; Reyes‐Paredes et al., 2010; Dowse & Zimmet, 1993) and the result is evolutionary discordance due to drastic dietary changes (Cordain, 2005). The estimated spontaneous mutation rate for nuclear DNA is 0.5% per million years putting the mutation rate for the past 10,000 years at only 0.005%. The most drastic dietary changes in the human diet have occurred in the past 200 years (Cordain, 2005). Thus, we are biologically programmed by our genome to consume nutrients, including more balanced ratios of FAs, very differently, both qualitatively and quantitatively, from the way we currently consume them in the United States and other developed countries (Eaton and Konner, 1985; Cavalli‐Sforza et al. 1993; Simopoulos,
1999a; Nachman and Crowell, 2000; Roth and Anderson, 2004; Galhardo et al, 2007).
Evolution of the Dairy Cow Diet
Cattle (Bos taurus) evolved to utilize vegetation as their main energy source. Early farmers most likely practiced transhumance. Transhumance mimics the grazing habits of large wild ruminant herds, where animals move to new pastures following the seasons. It has been
4 shown that the FA profile of pastures, and their nutrient content, changes with species
composition, age of the plants and environmental factors (Figure 1).
Figure 1 Seasonal Variation of Fatty Acids in Pasture
α‐linolenic acid & total FAs in pasture (Wyss et al., 2010).
By following the seasonal development of plants, animals were able to harvest plants
for peak nutrient levels (Laidlaw et al., 2006; Wyss et al., 2006). When plants are at their peak
nutrient level, they are high in monounsaturated FAs and polyunsaturated FAs and high in
omega‐3 FAs (Laidlaw et al., 2006 as stated by Stypinski, 2011). Studies with dairy cattle show
that meat, milk and milk products from pastured animals have: higher ratios of omega‐3 FAs to
omega‐6 FAs, increased amounts of conjugated linoleic acid (CLA), increased amounts of
precursors for vitamin A and E, high antioxidants levels, and have a more desirable saturated FA
profile, as it relates to human health. This is especially true when pastures have large amounts
of legumes in them (Wyss et al., 2006; Ellis et al., 2006; Dhiman et al., 1999; Daley et al., 2010;
Cordain et al., 2005; Couvreur et al., 2006; Bergamoa et al., 2003; White et al., 2001; Kay et al.,
2004; Higgs, 2000). Transhumance is still practiced to some extent today in the US with our
meat animals, and pasture‐based farm animals.
5 As the developed world changed toward a more industrial urban environment where fewer people were producing their own food, the emphasis in the dairy industry focused on higher milk production to feed an ever growing population of people (Oltenacu and Algers,
2005; Lucy, 2002; Emanuelson and Oltenacu, 1998). To accomplish this goal, farms increased cow numbers. In the late 1700’s U.S. farmers started keeping cows in more confined pasture systems instead of moving them with the seasons. This new system made it possible to increase cow numbers to 10 or 15 animals per barn without increased labor. Within 100 years, technological developments in milking and cropland equipment helped reduce labor needs even more, which enabled farmers to increase animal numbers to 100 head per farm (Blaney, 2002).
Although these animals were not being moved following the growth of the pasture plants, fresh forage was still a major part of their diet. Today, further developments have made it economically viable to manage total confinement dairy operations. More specifically, a better understanding of animal nutrition has enabled the farmers to feed more precisely balanced rations, known as totally mixed rations (TMR), to maximize milk production utilizing by‐product feedstuffs of the industrial revolution (Evans et al., 2008; National Research Council (NRC) 1988;
Oetzel and Vilalba, 1993). Fifty percent of the 51,000 dairy farms in the US are in the western region of the country where access to low cost grain and large tracts of land make it more economically feasible to manage confinement herds. Many of these confinement herds milk in excess of 2,000 cows per day, usually three times a day. These farms produce 85% of the milk consumed in the U.S. (USDA, 2007, Bulletin #47). However, since the TMR rations contain high amounts of grain‐based concentrates and by‐product feeds, the FA profile of the diets of modern dairy cows is very different from what it was before the industrial revolution (Cordain et al., 2002; Jenson 1995; Evans et al., 2008; NRC 1988; Oetzel and Vilalba, 1993).
6 A recognized issue within the dairy industry as TMR‐fed herds increased in in size was an increase in the incidence of diseases that adversely affected milk production and farm profits
(Shulman, 2008). The dairy industry along with the private sector worldwide, have committed vast amounts of money to research aimed at the early detection and treatment of what we know as common management related diseases of dairy cows such as mastitis, peritonitis, retained placenta, acidosis, displaced abomasums and more. The cost to farmers across the world is in the millions if not billions of dollars a year (Ott 1999; Haribar 2010).
The average production life of a modern dairy cow in a confinement herd is 5 years or
2.5 lactation cycles. Dairy cows of the past were known to live and provide sustenance for an average of 10 to 15 years. Oltenacu and Algers (2005) reported that between 1980 and 2000, numbers of dairy cows alive beyond 48 months of age had declined from 80% to 60%. They also noted that reproductive efficiency had decreased and in that same period of time pounds of milk produced per animal went from 5,000 pounds annually to 11,000 pounds. Many disorders such as displaced abomasum or retained placenta can be linked to high‐energy grain rations
(Shearer 2007; Philipot et al., 1994; Olmos et al., 2009; Goldenburg et al., 1992). In addition, confinement cows are culled regularly for lameness due to thin soles and abscesses from walking or standing on concrete surfaces (Shearer 2007). The National Animal Health Monitoring
Service (NAHMS, 2010) reported that hairy heel wart affects huge numbers of dairy cows across the U.S., and the incidence of this disease is lower in pastured animals compared to confinement animals. The cause of this highly contagious disease is still unknown but it is suspected that many strains of environmental bacteria coupled with concrete floors are involved. Other more major illnesses directly related to overcrowding, confinement and diet include contagious mastitis, endometritis, ketosis, displaced abomasum, acidosis, ringworm and peritonitis (Schultz et al., 1978; Sharif, and Muhammad, 2008., Washburn et al., 2002; Regula et
7 al., 2004; Mee 2012). As with many diseases, the above can be related to or exacerbated by a chronic state of inflammation (Robbins, pp. 59).
Public demand for a healthier more humanely produced product and high fuel prices are making pasture based production systems more economical for the smaller dairy farms. Smaller dairy farms are more prevalent in the Northeastern part of the United States (Blaney, 2002;
Weimar and Blaney, 1994). Pasture‐based production dairy systems put more emphasis on forages as a main component of their dairy nutrition program, but also feed supplements, grains and other feedstuffs to maximize milk yield. By allowing cows to harvest their own forages during the growing season, time and tractor use can be decreased. This makes it an economical option for these smaller farms in temperate New England. By utilizing forages in a responsible well‐planned rotational grazing system, these pasture‐based operations can be profitable. (Rust
et al., 1995; Parker et al., 1992; Muller 1993; Benson 2008; Goldenburg et al., 1992; Roche et al.,
2003a; Roche et al., 2003b.). Some studies have shown pasture systems produce dairy products higher in omega‐3 FAs, lower in omega‐6 FAs and have a higher CLA content than confinement systems (Elgersma et al., 2004; Ellis et al., 2006). Because more balanced FA profiles and CLAs have been shown to enhance human health, many studies have been conducted to find feedstuffs that will increase the omega‐3 FAs and CLA content of meat and milk. It has been
shown that when animals in temperate climates are transitioned from pasture to the barn for the winter however, the FA profile of their milk and meat and blood changes within weeks of the transition (Elgersma et al., 2004; Ellis et al., 2006; Kay et al., 2004; Hutton et al., 1969; Butler et al., 2000; Stypinski 2011). Observations made by Hutton, Seeley, and Armstong (1969) showed that summer milk fat contained more long chain FAs and less short chain FAs than winter milk and abrupt changes in feeding were clearly reflected in the milk from both herds used for their study. In recent years, questions have been raised about whether these FA
8 adjustments have a negative or positive effect on animal health. Since cows do not process unsaturated fats like humans do, assumptions cannot be made without extensive and conclusive studies looking at these constituents as they relate to cattle health. (Jenkins et al., 2008; Muller
2004; Ajmone‐Marsan et al., 2010).
Organic dairies are pasture‐based systems. The difference between a conventional pasture‐based system and an organic pasture based system are the regulations on inputs (all feedstuffs, supplements, cleaning products, medicine, etc…must be certified organic), pasture management (all organic cows must have access to the outside 365 days a year and must be on pasture 120 days out of the year, regardless of the weather), and medical intervention (no antibiotics can be used or other drugs that are not approved for organic milk production) by the
National Organic Standards Board. No definitive studies have been done on whether these practices and restrictions have a positive or negative effect on dairy cow health.
Fatty Acids
Fatty Acids are the building blocks of fat/lipid molecules. They are simple chemical structures made up of chains of 4‐30 carbon atoms with hydrogen atoms attached. There are several hundred different FAs, which furnish most of the energy contained in dietary fat. Fatty acids are a major component of cell membranes and play a vital role in maintaining health of all animals. They buffer toxins and diseases, and help us absorb important vitamins. Some are considered essential because they are needed for health and must be consumed because they are not produced in the body. Fatty acids can be short chain, which range in length from 14 to
18 carbons or long chain with 20 to 24 carbons. Some are classified as saturated FAs (SFA) which have no double bonds between carbon atoms in the chain, or unsaturated meaning there is at least one double bond in the carbon chain. Saturated FA have been shown to increase the amount of cholesterol in our blood (Billet et al., 2000; Mensink and Ketan, 1992), and are nearly
9 impossible for humans to digest. Saturated FA consumption has been strongly linked to inflammatory diseases such as cardiovascular disease, arthritis, and more (Greil and Kris‐
Etherton, 2006; Gebauer, 2007). Large amounts of SFAs are consumed in the U.S. annually.
Table 2 lists the top food sources of SFAs in the U.S. diet.
Table 2 Saturated Fat in Common Foods in the United States
NHANES 2005‐2006; Applied Research Cancer Control and Population Sciences. (http://appliedresearch.cancer.gov/diet/foodsources.html).
Rank Food Item Cumulative contribution (%)
1 Regular cheese 8.5
2 Pizza 14.4
3 Grain‐based deserts 20.2
4 Dairy deserts 25.8
5 Chicken and chicken based dishes 31.2
6 Sausage, franks, bacon, and ribs 36.2
7 Burgers 40.5
8 Mexican mixed dishes 44.6
9 Beef and beef mixed dishes 48.7
10 Reduced fat milk 52.6
11 Pasta and pasta dishes 56.3
12 Whole milk 59.7
When an unsaturated FA has only one double bond it is classified as monounsaturated
(MUFA). Polyunsaturated FAs (PUFA) have two or more double bonds in their chain. The double bond in unsaturated FAs causes a “kink” in the chain of the molecule keeping them from packing tightly together thus reducing their melting point. This is the reason for increased or decreased membrane fluidity, which controls the movement of substances through and within the membranes (Yang et al., 2011). Unsaturated FAs are found mostly in foods from plants such as vegetable oils, nuts, and fish (Center for Disease Control and Prevention, 2012) (Table 3).
10 Table 3 Unsaturated FAs Found in Common Foods in the United States.
MUFA Omega‐6 PUFA Omega‐3 PUFA Sources Sources Sources Nuts Soybean oil Soybean oil
Vegetable Corn oil Canola oil oils
Canola oil Safflower oil Walnuts and others
Olive oil Flaxseed
High oleic Cold water fish safflower oil http://www.cdc.gov/nutrition/everyone/basics/fat/unsaturatedfat.html
The effects FAs have in the body depend on how they are combined with a glycerol backbone to form important lipid molecules. Phospholipids are one such important lipid molecule that is a major component of cell membranes in all plants and animals (Figure 3). They consist of a glycerol backbone with two FAs attached to a phosphate group that links an organic base. They provide fluidity and are important in lipid digestion in the small intestine.
Phospholipids are a minor component of most feedstuffs for cows. Triglycerides are made up of a glycerol backbone and three FAs (Figure 2). Their purpose is energy storage in plants and animals. Triglycerides enable the bi‐directional transference of adipose fat and blood glucose from the liver. They are the major lipid type found in grains, oilseeds, and animal fats. They are also the type of lipid that makes up milk fat. Triglycerides can be saturated or unsaturated.
Glycolipids are the major lipid type found in forages (Figure 4). Found in stems and leaves, they are similar to triglycerides except they have two or more sugars linked to one portion of the glycerol backbone instead of the third FA. The most common glycolipids found in forages are galactolipids which have galactose linked to the glycerol backbone. Galactose is a component of the milk sugar, lactose.
11 Figure 2 Triglyceride Structure
Figure 3 Phospholipid Structure (lecithin)
12 Figure 4 Galactolipid Structure
http://www.fao.org/docrep/field/003/ab470e/ab470e03.htm
Oleic acid and linoleic acid are examples of unsaturated FAs containing one or more double bonds. Oleic acid is the predominant FA in animal fats and some plant oils such as canola oil. Linoleic acid is the predominant FA in many plant oils, including cottonseed oil, soybean oil and corn oil. Linolenic acid, has three double bonds which is a polyunsaturated FA (PUFA).
Linolenic acid is the primary FA in most pasture species and in linseed oil from flax (Jenkins et al., 2011).
Omega indicates where in the chain from the methyl end the double bond resides.
Omega FAs belong to one of three families: omega‐9(n‐9), omega‐6(n‐6) or omega‐3(n‐3)(Gorlin,
1988; as reviewed by Jenkins et al., 2011). This means these FAs have double bonds between the 9th and 10th, 6th and 7th, or 3rd and 4th carbons in the chain as counted from the methyl end.
Each family has a parent FA that is converted to other biologically active acids within the same omega family. The only parent FA that can be made by body tissues is oleic acid, which is the parent compound of n‐9. Linoleic and linolenic acids are the parent compounds of n‐6 and n‐3 respectively. They cannot be synthesized by the body tissues and must be supplied in the diet.
They are required for normal tissue functions so are considered essential FAs.
13 Essential FAs (EFA) provide energy for the body but are not stored well by the body.
Essential FA have very important health roles. They regulate vascular pressures, dilate or constrict blood vessels, direct hormones to their target cells, regulate smooth muscle reflexes, regulate cell division rates, maintain the fluidity of the cell membranes which in turn controls the flow rate of products in and out of a cell, transport oxygen from red blood cells to tissues, mediate the release of inflammatory substances from cells, regulate nerve transmission and more (Jenkins et al., 2007; Palmquist, 2010; Harris, 2004; Griel and Kris‐Etherton, 2006; Gebauer et al., 2007; Kris‐Etherton and Innis, 2007).
Where the double bond exists, there is the opportunity for the molecule to be a cis or trans isomer of that molecule (Figure 10). An isomer is a geometrical configuration of a molecule and that configuration affects its properties and function. Most naturally occurring unsaturated
FAs are of the cis configuration giving the molecule a U like shape. The trans form of an unsaturated FA molecule resembles the linear shape of a saturated FA (Figure 7). Oleic acid is considered the most common cis FA (Table 3). Olive oil contains 60 to 70% oleic acid, but oleic acid is also found in a wide range of nuts. The major sources in human foods are rapeseed, soybean, butter, lard and tallow. There are two predominant sources of trans‐fats in our diet.
Those that are derived from partially hydrogenated fats and oils and those found mostly in the meat and products from ruminant animals (Kris‐Etherton et al., 2012). It is estimated that only
15 to 20% of the trans‐fats we consume are ruminally‐produced trans‐fats and studies have shown we have evolved to process the trans‐fats from animals with little or no difficulty
(Turpeinen et al., 2002). Unlike artificially produced trans‐fats, which have been strongly linked to cardiovascular disease, the ruminally‐produced trans‐fats may have beneficial health effects.
Humans and mice convert vaccenic acid directly to rumenic acid/conjugated linoleic acid (CLA).
CLAs have been found to have both anti‐inflammatory and anti‐carcinogenic effects in humans
14 and animals (Lock, 2004; Belury, 2002; Gebauer et al., 2001). Grass‐fed milk and meat contains more of these trans‐fats than grain‐fed milk and meat (Dhiman et. al., 1999; Elgersma et al.,
2004; Elgersma et al., 2003; Goldenburg et al., 1992; Couvreur et al., 2006). These ruminally produced trans FAs include vaccenic acid (VA) and the isomer of conjugated linoleic acid, cis‐9, trans‐11 (Figure 9) (Gebauer et al., 2011). Extensive studies have shown that all omega FAs are essential for normal development affecting neural growth and later infant development, and if consumed in proper ratios can help humans achieve good health throughout life (Yang et al.,
2011; Greil and Kris‐Etherton, 2006; Willis and Smith, 1989; Turpeinen et al., 2002; Kris‐Etherton et al., 2005; Gebauer et al., 2007; Mensink and Katan, 1992).
Figure 5 Alpha‐Linolenic Acid (18:3), an Omega‐3 FA
Figure 6 Linoleic (18:2), an Omega‐6 FA
Figure 7 Stearic Acid (18:0), a Saturated FA
15 Figure 8 Conjugated Linoleic Acid (18:2), cis‐9, trans‐11
Figure 9 Chemical structures of cis‐ and trans‐isomers of oleic acid.
16 Table 4 Fatty Acid Structure and Classification FA Structure Shorthand abbreviation Palmitoleic acid CH3(CH2)5CH=CH(CH2)7COOH 16 : 1n‐ 7
Oleic acid CH3(CH2)7CH=CH(CH2)7COOH 18 : 1n‐ 9
Linoleic acid CH3(CH2)4CH=CH(CH2)7COOH 18 : 2n‐ 6
Linolenic acid CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH 18 : 3n‐ 3
Arachidonic acid CH3(CH2)4CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COOH 20 : 4n‐ 6
EPA CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)3COOH 20 : 5n‐ 3
DHA CH3CH2CH=CHCH2CH=CHCH2CH=CHCH2CH=CHCH2CH 22 : 6n‐ 3 =CHCH2CH=CH(CH2)2COOH
Stearic acid CH3(CH2)16COOH 18 : 0 http://www.fao.org/docrep/field/003/ab470e/ab470e03.htm
Arachidonic acid (AA) is an omega‐6 FA that is considered conditionally essential because it can be either synthesized from the essential FA linoleic acid and incorporated into phospholipids, or if phospholipase C hydrolyzes an AA containing phospholipid, diacylglycerol forms, which can be hydrolyzed to form AA. Therefore, phospholipids can be considered a store for AA but not its source. Through the AA cascade, greater than 20 different signaling pathways occur in the body that control a large number of cellular functions. The most significant body function AA controls is inflammation (Ricciotti and FizGerald, 2011). Omega‐3 and omega‐6 FAs
interact with each other and this interaction affects the intensity of an inflammation response.
More specifically, eicosanoid and leukotriene metabolites originating from their parent omega‐3 or omega‐6 FAs are associated with beneficial and detrimental effects (Gebauer et al., 2011).
When the ratio of omega‐3 to omega‐6 FAs is in favor of omega‐3, less AA is incorporated into the cell membranes and consequently released so a less inflammatory response occurs (Garg et al.,1989). This in turn may lower the incidence of chronic diseases that are due to a chronic inflammatory state (Jenkins et al., 2007).
17 Essential Fatty Acids and Inflammation
Inflammation is a complex reaction to injurious agents such as microbes and damaged cells that consist of a vascular response, migration and activation of leukocytes, and systemic reactions. Both acute and chronic inflammations are mediated by chemical factors that are derived from plasma proteins, endothelial cells, phagocytes, lymphocytes and mast cells. They are produced in response to or activated by the inflammatory stimulus. Inflammation is a protective mechanism that can become injurious to healthy tissue when it becomes chronic due to the presence of continuous injurious stimuli (Irvine, 1982). It is initiated by the release of chemical mediators into the extra‐cellular fluid. The most important mediators are histamine, kinins, complement, and lymphokines. They are all vasodilators and as blood flow to the area increases, local blood congestion occurs, accounting for redness and heat. These mediators also increase capillary permeability so that exudates containing proteins, such as clotting factors and antibodies, can seep from the bloodstream to the interstitial fluid. These exudates cause local edema, which in turn presses on adjacent nerve endings, causing pain (Kumar et at., 2005)
Other sources of pain include the release of bacterial toxins, lack of nutrition to injured cells, insudation of white blood cells, and hypersensitivity caused by the release of prostaglandins and kinins, which are eicosanoids (Kumar et al., 2005; Golej et al., 2011; Irvine, 1982; Piomelli, 2000).
Eicosanoids and leukotrienes are metabolites of their respective parent omega FAs.
Omega‐6 and omega‐3 FAs obtained in the diet from linoleic and linolenic acid compete for incorporation into cell membranes and the products of their metabolism are involved in immune function and the inflammatory response. While inflammatory responses to acute infections and injuries mediated by these eicosanoids have certain beneficial effects, persistent inflammation has a degrading affect that can lead to debility and death (Kumar et al., 2005;
Gebauer et al., 2011). Omega‐6 FAs are converted to AA, which is the physiologically active
18 omega‐6 FA present in tissue membranes. Arachidonic acid and its omega‐3 counterpart eicosapentanoic acid (EPA) are precursors for two different series of eicosanoids, each with varying physiological activities (Kumar et al., 2005). Metabolism of AA or EPA can follow either of two enzymatic pathways; the cyclooxygenase pathway (COX), which produces thromboxanes
(TX) and prostaglandins (PG); and the lipoxygenase (LOX) pathway producing leukotrienes (LT) and hydroxyeicosatetraenoic acids (HETE). The COX pathway is a signal‐mediated pathway whereby when triggered; Protein Kinase C (PKC) is activated. PKC and cytosolic calcium then
activate Phospholipase A2 (PLA2). PLA2 then activates and releases AA from membrane phospholipids. Cyclooxygenase (COX) then converts AA into Prostaglandin G2 (PGG2) and then a further peroxidation step converts PGG2 into Prostaglandin H2 (PGH2). During this step a free radical of oxygen is released, which can cause oxidative DNA, lipid, and protein damage in tissues. PGH2 in platelets is converted by Thromboxane A2 Synthetase into Thromboxane A2
(TxA2), which is a highly thrombogenic eicosanoid. TxA2 is then released from the cell. In endothelial cells, PGH2 is converted to Prostacyclin (PGI2), which decreases platelet aggregation, causes vasodilation and potentiates edema. Prostacyclin therefore acts as a regulator of the effects of TxA2. PGI2, and TXA2 which are series II protaglandins and thromboxanes(Kumar et al.,
2005). This series eicosanoids have been linked to cardiovascular disease, asthma, arthritis, thrombosis, tumor proliferation, osteoporosis, decreased bleeding time, and more in humans
(Seamans, 2002; Simmons et al., 2004). This is why AA is considered to be pro‐inflammatory.
Leukotrienes (LT), and hydroxyeicosatetraenoic acids (HETE) are produced through the second AA pathway, the Lipoxygenase (LOX) pathway (Kumar et al., 2005). Lipoxygenases are important enzymes affecting a series of biosynthetic processes leading from AA to TX, PG, and
LT. Leukotrienes are a subgroup of eicosanoids that are similar to hormones. In contrast to hormones, which are produced in a specific organ and transported to where they are needed,
19 leukotrienes are produced in very low concentrations and are produced locally (at the site of stimulus) and have a very strong local effect. Leukotrienes are involved in the immune‐mediated inflammatory reaction of analphylaxis causing the contraction of smooth muscle. They also affect vascular permeability and strongly attract polymorphoneuclear leukocytes (neutrophils and eosinophils) (Kumar et al. 2005). HETE is an intermediate in the path of Leukotriene synthesis. It is a modulator of tubuloglomerular feedback. Tubuloglomerular feedback provides a mechanism to keep flow through the nephrons in a narrow range by detecting and rapidly correcting it on a minute‐to minute basis as well as over sustained periods of time Kumar et al.2005c). Lipoxygenases insert oxygen in the 5 carbon of AA turning it into 5‐HPETE which then goes on to become a LT. Without AA no transformation occurs thus no LT are produced. The LOX pathway is also called the 5‐LOX pathway of AA. The 5‐LOX pathway produces series II TX, PG, and series 4 LT which are also considered to be pro inflammatory (Serhan, 2002; Gebauer et al.,
2011).
Lipoxins (LX) are a class of metabolites also produced from AA, but generally inhibit neutrophil and eosinophil migration (Kumar et al., 2005). LX contain a fully conjugated tetraene derived from AA, and they have a different initial step enzyme in their pathway to producing TX,
PG, and LT. The 15‐lipoxygenase pathway has been shown to produce intermediates (series I TX,
PG, and series 5 LOX) that are antagonistic to LT. Series I Thromboxanes (TxA3) have been shown to be weaker aggregators of platelets than Series II Thromboxanes (TxA2), and series 5 lipoxygenases have lower physiological activity than series 4 lipoxygenases(Serhan et al., 2011).
It is believed that increasing the amount of lipoxins reduces the amounts of leukotrienes and vice versa. Diets rich in omega‐3 FAs, especially those derived from fish oil produce this less‐ inflammatory effect (Simmons et al., 2004; Serini et al., 2012). The most nutritionally important n‐3 FAs for humans are: α ‐linolenic acid (ALA), eicosapentaenoic acid (EPA), and
20 dihomogammalinolenic acid (DGHA) (Harris, 2004). Once consumed, ALA is converted in the body to EPA and DGHA. EPA has also been shown to inhibit chemotactic and aggregation activities of polymorphoneuclear leukocytes, and decrease cholesterol and triglycerides in plasma (Lands et al., 1971). Figure 10 shows the pathway AA, EPA, and DHGA follow to produce their respective series eicosanoids. Having a more balanced diet of n‐6 to n‐3 may produce an inflammation reaction that proceeds with mitigation of the event in a more controlled fashion, thereby inhibiting complications.
21 Fatty Acids and Disease
In humans, when omega‐3 FAs and omega‐6 FAs are balanced, the inflammatory response reacts as needed during stress or injury yet is controlled in a healthy condition. When increased amounts of omega‐6 FAs are consumed, as is typical in the Western diet, higher amounts of AA metabolites are synthesized and lower amounts of EPA or DHGA metabolites are synthesized. This creates the pro‐inflammatory physiological state that is currently seen where cardiovascular disease, diabetes, arthritis, hypertension, and cancer prevail (Seaman, 2002).
Chronic inflammatory diseases are responsible for 70% of deaths in the U.S., with heart disease being the number one cause of morbidity and mortality (Cordain et al., 2005). The World Health
Organization released a bulletin in 2013 calling for a worldwide removal of industrial trans FAs
(iTFA) from our food supply, stating the relationship of iTFA to cardiovascular disease is well established and iTFAs should be replaced with polyunsaturated fats (Gebauer et al., 2011;
Downs et al., 2013). There are different dietary sources of trans‐fats in our food supply: those formed during the industrial partial hydrogenation of vegetable oils (iTFA), those formed by biohydrogenation in ruminants (rTFA) including vaccenic acid and the naturally occurring isomer of conjugated linoleic acid cis‐9, trans‐11 (CLA). Some studies suggest it is the iTFA and not rTFA or CLAs that negatively impact heart health (Gebauer et al., 2011). Kummerow et al. (2013) showed that as iTFAs are increased in diets, prostacyclin, dose dependently, significantly decreases. In 2001, Banni et al.,2001 showed that feeding vaccenic acid to mice increased tissue levels of conjugated linoleic acids and suppressed the development of malignant lesions in mammary glands of those animals. When Higazi et al. (1994) examined the regulation of fibrinolysis by non‐esterified FAs, is was shown that a minimal chain length of 16 carbon atoms and the presence of at least one double bond, preferably in a cis configuration, was required for inhibition of the fibrinolytic activity of plasmin. Proper ratios of omega‐6 FAs to omega‐3 FAs
22 that would enhance health and reduce inflammation have not been established for cattle beyond the recognition that they are essential (Jenkins et al., 2011). As in humans, they may play an important role in animal health.
Conjugated linoleic acids (CLA) are an important class of FAs that are not considered omega‐3 or omega‐6. CLAs have been found to have both anti‐inflammatory and anti‐ carcinogenic effects in humans and animals (Lock, 2004; Belury, 2002; Parodi, 1999; Rezamand et al., 2009a; Rezamand et al., 2009b; Rezamand and McGuire, 2011; Reyes‐Parades et al.,
2010). Moloney et al. (2007) looked at the potential anti‐diabetic effect of cis‐9, trans‐11 CLA, focusing on the molecular markers of insulin sensitivity and inflammation in adipose tissue in mice. The study suggested that increased amounts of cis‐9, trans‐11 CLA in the diet may attenuate the pro‐inflammatory state in adipose tissue that predisposes to obesity‐induced insulin resistance. Bassaganya‐Riera et al. (2012) conducted a study with 13 patients with mild to moderately active Crohn’s disease. The patients were given 6 g of CLA orally for 12 weeks.
Findings were considered encouraging. The CLA was well tolerated; it suppressed the ability of
peripheral blood T cells to produce pro‐inflammatory cytokines, it decreased disease activity and increased the quality of life of the patients. CLA is also called rumenic acid because it is found predominantly in ruminant tissue and milk. Bauman and Lock (2006) and Piperova (2003) showed that through a process known as biohydrogenation, many types of CLAs are produced in the rumen of a dairy cow, with the predominate form being cis‐9,trans‐11 C18:2. In 2011,
Gebauer et al. (2011) reviewed a multitude of studies on the effects of ruminant trans fats on cardiovascular disease and cancer and found feeding studies in rodent models with either induced or spontaneous dyslipidemia did not support adverse health effects of vaccenic acid on markers of cardiovascular risk, but suggested vaccenic acid supplementation may improve dyslipidemia by lowering circulating triglycerides, attenuating artherosclerotic progression. Milk
23 fat studies demonstrated a neutral or beneficial effect of vaccenic acid and cis‐9, trans‐11‐CLA on artherosclerosis and risk factors of cardiovascular disease, and although limited, data suggests that dairy fat enriched with vaccenic acid and /or cis‐9, trans‐11 CLA may reduce tumor development (Gebauer et al., 2011; Downs et al., 2013). Although studies are presently being done with cattle, it is not known exactly how these endogenous CLAs affect the health of dairy cows and more studies are needed to ascertain this information (Jenkins et al., 2009).
Fatty Acid Digestion and Absorption in Humans and Cows
Plants are high in monounsaturated FAs and polyunsaturated FAs and high in omega‐3
FAs (Laidlaw et al., 2006 as stated by Stypinski, 2011). Studies with dairy cattle show that meat, milk and milk products from pastured animals have: higher ratios of omega‐3 FAs to omega‐6
FAs, increased amounts of CLA, increased amounts of precursors for vitamin A and E, higher antioxidants levels, and a more desirable saturated FA profile, as it relates to human health
(Wyss et al.,2006; Ellis et al., 2006; Dhiman et al., 1999; Daley et al., 2010; Cordain et al., 2005;
Couvreur S. et al., 2006; Bergamoa et al., 2003; White S.L. et al., 2001; Kay J.K. et al., 2004; Higgs
J.D., 2000). It is assumed that because a healthier EFA profile will make humans healthier, cattle on pasture will be healthier as well. Although we have learned of the adverse health effects of high amounts of omega‐6 FAs to low amounts of omega‐3 FAs in the human diet, no studies have been done to evaluate what the ratio of these essential fatty acids should be in cows for them to achieve optimal health. Whether consumed by a human or a ruminant, lipids are slightly soluble to insoluble in the aqueous environment of the digestive tract. The energy produced from fat is twice that from carbohydrates or protein sources. Because fat molecules are large and hydrophobic, they must first be broken into much smaller emulsified droplets called micelles, so they can be digested by water‐soluble enzymes in the intestines. Humans and ruminant animals break fat down very differently.
24 Fatty Acid Digestion and Absorption in Humans
In the human body there are three fat sources for obtaining energy; from diet, adipose tissue and from the liver, when excess carbohydrates are consumed. Before absorption, fat particles must be made smaller. Bile salts made from cholesterol in the liver accomplish this task. Bile salts and phospholipids are amphipathic molecules that are stored in the gall bladder
(table 5). Movement in the small intestine breaks the fat particles apart and those smaller fat molecules are then coated with bile salts and phopholipids. This emulsification step creates what we call micelles, which prevent the droplets from re‐forming. Micelles transport poorly soluble monoglycerides, FAs, fat‐soluble vitamins and cholesterol to the surface of the enterocyte where they are released for absorption. Micelles are constantly breaking down and re‐forming with the aforementioned components in this aqueous solution; they are not absorbed. Once at the surface and released, the monoglycerides and FAs diffuse across the intestinal mucosa. Once they pass through the mucosa, the monoglycerides and FAs are re‐ synthesized into triglycerides, and packaged together with cholesterol and the fat‐soluble vitamins to form a lipoprotein called a chylomicron. The chylomicron enters the lymphatic system via a lacteal. From the lymph system they enter the blood and then depending on the bodies’ requirements, reach the muscle or adipose tissue. Apolioprotein CII activates the enzyme lipoprotein lipase, which breaks down the chylomicron and hydrolyses the triglycerols to FAs and glycerol, which are utilized for energy in muscles. In the adipose tissue the FAs and glycerols are repackaged into triglycerols for storage (Figure 11).
25 Figure 11. Fatty Acid Digestion and Absorption in Humans.
http://pharmaxchange.info/press/2013/10/digestion‐of‐fats‐triglyceroles/
Fatty Acid Digestion and Absorption in Ruminants
Balancing various energy sources from carbohydrates to lipids is critical for optimizing ruminal fermentation of forages and maximizing milk production (Slyter, 1976; Mertens, 1995;
Valazadeh et al., 2010). Predicting ruminal fermentation changes due to dietary lipids is difficult
because FAs come from so many sources in cattle diets and some of those sources are in a
dynamic environment that changes constantly. It has been shown that more mature grass with
26 higher amounts of seed heads become lower in n‐3 FAs and higher in n‐6 FAs. It has also been shown that pasture species affect the FA profile of forages. Rain patterns, time of day, altitudes, summer heat or lack thereof, etc., can also play a part in that profile (Boufaied et al., 2003).
These factors and avoiding milk fat depression by over feeding fats can be a large problem on dairy farms, especially pasture based dairies (Drakley, 2007; Schneider et al., 1988). Lipids added to cattle diets have been shown to greatly disrupt fermentation in the rumen causing reduced
digestibility of non‐lipid energy sources. Rumenal digestion of structural carbohydrates can be reduced 50% or more by less than 10% added fat.(Palmquist, 1990; Jenkins 1993; Petit, 2004;
DaSilva, 2007; Beauchemin, 2007; Davis, 1990;Chalupa and Fergason, 1988; Chow et al., 1991;
Drakley, 2007). Most of the lipids in a ruminant diet come from forages, concentrates and fat supplements. Caroprese et al., (2009); Lessard et al., (2004); and Sklan et al., (1989) showed that supplementation of dairy cows with flax seed oil, which is high in omega‐3 FAs, might have a positive effect on a cow’s immune system and improve reproductive performance and conception rates, however other studies have shown adding flax seed meal may depress feed efficiency and milk production and reduces some nutrient absorption (Bauman and Lock 2005;
Brito, 2011). It has been speculated that the increased energy alone from the flax seed meal could be the reason for the benefits if animals tested are already in a negative energy state (
Jenkins et al., 2007). Forages are estimated to contain about 4 to 6% of the dry weight of the leaf tissue in the form of glycolipids (Bauman et al., 2003). The lipid content of concentrates varies depending on source but the majority of the lipids are in the form of triglycerides. Table 5 shows the FA composition of some typical feedstuffs and fat supplements used in a dairy ration.
27 Table 5 Fatty Acid Composition of Typical Feedstuffs
Cows metabolize fats very differently than humans do, turning almost all the unsaturated FAs that enter the rumen into saturated fats by way of two steps; hydrolysis and biohydrogenation (Drakley, 2007). Plants are high in monounsaturated FAs and polyunsaturated
FAs (Laidlaw et al., 2006). In forages, FAs are typically found in esterified form (triglycerides, phospholipids and glycolipids) and are typically of a cis configuration. Upon entering the rumen, bacteria hydrolyze almost all these esterified FAs into non‐esterified FAs (NEFAs), or “free FAs” and glycerol. Not all rumen microbes are capable of hydrolysis and it has been shown that cows fed high grain diets have fewer lipolytic bacteria to carry out hydrolysis than pasture fed animals
(Harfoot and Hazelwood, 1997). Complete saturation of FAs in the rumen is believed to be necessary, having evolved to protect the rumen microbes from a low pH environment that develops when carbohydrates are processed into volatile FAs (Bauman et al., 2006; Drakely,
2007). In addition, hydrolysis liberates the glycerol and sugars from the triglycerides and glycolipids, which are then readily metabolized by the rumen bacteria. The extent of hydrolysis is generally > 85% but several things can decrease this amount: dietary fat increases above 7%
28 of dry matter intake, low pH, and administration of ionophores that are used to decrease microbial populations in high grain rations to decrease the incidence of acidosis (Harfoot and
Hazelwood, 1997). After hydrolysis takes place, biohydrogenation completes the saturation of the esterified FAs. Most biohydrogentation occurs in relation to feed particles, and extracellular enzymes of microbes either associated with the feed or free in suspension. Protozoa are very active in biohydrogenation and the degree of biohydrogenation occurring is usually much less extensive when protozoa populations are suppressed or eliminated due to decreased rumen pH or the administration of ionophores which are both common in TMR fed herds (Harfoot and
Hazlewood, 1997; Jenkins et al., 2006). The major substrates for biohydrogenation are the essential FAs linoleic and linolenic acids, which eventually become stearic acid which is a saturated fat (figures 12 & 13).
Figure 12
Rumen Biohydrogenation of Linoleic Acid
cis‐9, cis‐12 C (Linoleic acid) 18:2 Isomerization (group A)
cis‐9, trans‐11 Conjugated diene (cis‐9, trans‐11 C CLA) 18:2 Hydrogenation (group A)
trans‐11‐Ocatadecenoic acid (trans‐11 C18:1) Hydrogenation (group B)
Stearic acid (C18:0)
Group A: Butyrivibrio fibrisolvens, Ruminococcus albus, and Eubacterium Group B: Fusocillus sp. and Gram‐negative rod R8/5
Harfoot & Hazlewood (1988)
29 Figure 13
Rumen Biohydrogenation of ‐Linolenic Acid
cis‐9, cis‐12, cis‐15 C (‐Linolenic acid) 18:3 Isomerization (group A, group B) cis‐9, trans‐11, cis‐15 Conjugated triene Hydrogenation (group A, group B) trans‐11, cis‐15‐Octadecadienoic acid
Hydrogenation (group B) Hydrogenation (group A)
trans‐15‐and cis‐15‐Octadecenoic acids trans‐11‐Octadecenoic acid
(trans‐11 C18:1) Not further hydrogenated even by mixed group A and group B Stearic acid (C18:0)
Harfoot & Hazlewood (1988)
The same factors that negatively affect hydrolysis, negatively affect biohydrogenation
(Jenkins, 1993). Hydrolysis and biohydrogenation produce a variety of trans‐FAs and alter chain length, position of double bonds, and produce odd chain and branch chain FAs including many different CLAs. The resulting lipid material leaving the rumen entering the abomasum has been shown to consist mostly of the saturated FAs; palmitic acid and stearic acid in a ratio of approximately 1:3 (Harfoot and Hazlewood, 1997).
FA absorption occurs predominantly in the jejunum of the small intestine of a ruminant whereas in non‐ruminants it occurs mostly in the duodenum. In ruminants as with all species of animal, micelle formation must occur for efficient FA absorption and both bile salts and pancreatic juices are required (Davis,1990). Digestion is continuous in a ruminant so their pancreatic secretions have low bicarbonate levels. This means the pH of the digesta flowing out
of the abomasum entering the small intestines is quite acidic; approximately 2.5. In non‐ ruminants pancreatic juices are produced in cycles keeping the pH around 4.5 (Noble, 1981). As the digesta passes through the proximal half of the small intestine in the ruminant, the pH remains low. The FAs that have been ingested must go from a particulate phase to a micellar phase so for this to happen and for absorption to take place, the ruminants’ bile acids are
30 essential. In ruminants, bile flow rate is 5 times greater than pancreatic secretions. This factor plus low pancreatic bicarbonate levels, limits pancreatic secretion contribution. To aide absorption of the lipids in this acid environment, the ruminant has evolved some special features. First, lysolecithin is produced. Lysolecithin is a powerful emulsifier that is only made in ruminant animals and has been shown to be the only amphiphile to significantly increase the distribution of stearic acid into the micellar phase and away from the particulate phase; increasing the solubility of stearic acid approximately twofold over other amphiphiles (Moate et al., 2004; Block et al., 2004). It is produced from lecithin (phosphatidylcholine, the main phospholipid in rumen microbial cells, pancreatic juice, and bile) by action of an enzyme called phospholipase that is secreted from the pancreas of the cow into the upper small intestine
(Harfoot and Hazelwood, 1997). Together with bile salts, lysolecithin desorbs the FAs from feed particles, bacteria and protozoa, allowing the formation of micelles (Figure14). In addition to producing lysolecithin, taurine‐conjugated bile acids predominate over glycine‐conjugated bile acids in a ratio of 3:1, which is opposite to that in young ruminants and non‐ruminants (Lock et al., 2005 and 2006). This is important because taurine‐conjugated bile acids are soluble and remain partially ionized at pHs as low as 2.5, whereas glycine conjugated bile salts are insoluble at pH 4.5 and below (Lock et al., 2006; Moore and Christie, 1984). Finally, in non‐ruminants, monoglycerides plus bile salts interact with the long and short chain esterified FAs, and NEFAs to form a bile salt/monoglyceride complex (mixed micelle), which serves as the stabilizer of the micelle (Davis, 1990). Long‐chain PUFA have non‐amphiphilic characterisitics and must be solubilized in the hydrophobic interior of a mixed micelle (Lock et al., 2006). Lipase then breaks down the fat molecules and monoacylglycerols, diacylglycerols, free FAs and glycerols are formed which move forward toward absorption.
31 Figure 14 Fatty Acid Digestion and Absorption in Ruminants.
Bauman and Lock, (2006). Proc. Tri‐State Dairy Nutrition Conference. http://tristatedairy.osu.edu/.
Lipase activity is much lower in ruminant pancreatic secretions and with low bicarbonate and low flow rate of the pancreatic juices; the current belief is that there is no significant absorption or modification of long chain FAs in ruminants. This is because the property of the lipase enzyme is similar to the lipase from non‐ruminants and optimal activity of lipase requires a pH of 7.5 to 7.8 with little activity below pH 5.0. With the small intestines of the ruminant remaining acidic until the lower portion of the jejunum it is unlikely that the limited lipase activity makes a substantial impact in lipolysis of any long chain esterified lipid reaching the small intestine of the animal (Brito, personal communication, Dec. 3, 2014). Having said this, studies do show that pasture raised animals have higher amounts of long chain FAs in their milk and meat (Caldari, 2009). How the ruminant system copes with, absorbs and utilizes all of the intermediates and long chain FAs it consumes and produces in the rumen is still being investigated and more work is needed to determine their impact on animal health (Jenkins,
32 2008; Bauman and Lock 2006). Understanding how omega‐3 PUFA affect the immune functions of dairy cattle may lead to the development of producer‐friendly feeding strategies that could decrease the incidence of management related diseases in dairy cows.
The Inflammation Acute Phase Response
The acute phase response (APR) in inflammation is non‐specific and is triggered in animals when they are subjected to external or internal challenges like injury or infection
(Eckersall et al., 2001). An APR has a multifactorial response aimed at mitigation of the problem by stopping microbial growth and repair of damaged tissue to bring the system back to homeostasis (Gronlund et al., 2005). The acute phase response consists of several clinical and pathological changes: fever; production of acute phase proteins; leukocytosis; other manifestations like: increased pulse, respiration, chills, anorexia, malaise and somnolence; and sepsis due to severe bacterial infection. One of the mechanisms used in the APR is the production and secretion of acute‐phase proteins (APPs) (Eckersall and Conner, 1988). This is brought about by pro‐inflammatory cytokines (e.g. Il‐1, Il‐6, TNF‐a), regardless of the causative factor (Alsemgeest et al., 1996; Yoshioka et al., 2002). The presence of the cytokines in blood triggers the liver to make and release APPs. It is possible to use changes in the concentrations of
APPs to monitor the course of a disease independently of its nature, monitor stress levels and aid in health diagnostics (Jawor and Stefanniak, 2006). The APP concentrations that increase due to inflammation are termed positive reacting species (e.g., haptoglobin (Hp), serum Amyloid A
(SAA), fibrinogen (Fb), and those that APPs decrease are termed negative reacting species (e.g. albumin and transferrin)(Jawor et al., 2008). Clinical diagnosis of inflammatory processes in cows is considered difficult as symptomatology is not as obvious. In addition, large groups of animals, as in dairy herds, makes accurate health observations of individual animals difficult
33 (LeBlanc et al., 2002; Murata et al., 2004; Jawor et al., 2008). Therefore the development of cow side tests utilizing APPs are being considered for utilization in the livestock industry.
Pathogenic products stimulate leukocytes to release cytokines such as IL‐1, IL‐6, and
TNF. Cytokines are short acting messenger molecules that carry information from cell to cell during an immune response to stimuli (Berne and Levy, 1990). IL‐1 and TNFα increase the enzymes (cyclooxygenases) that convert AA to inflammatory prostaglandins. Fever is produced in response to substances called pyrogens that act by stimulating prostaglandin synthesis in the hypothalamus. In the hypothalamus the prostaglandins, especially PGE2, stimulate the production of second messengers such a cyclic AMP which function to reset the temperature set‐point at a higher level, creating an inhospitable environment for the invading pathogen to exist. IL‐6 initiates the synthesis of PGE2 and because of IL‐6’s ability to cross the blood brain barrier it is considered one of the most important mediators of fever (Kumar et al. 2002b). The
PGE2 then dilates the vascular system, relaxes smooth muscle, and inhibits the release of nor‐ adrenaline from sympathetic nerve terminals in the acute phase response to injury and
infection. Il‐6, found in macrophages and adipocytes, often precedes the production of other cytokines and elevated levels in the blood are common in advanced metastatic cancer patients.
IL‐6 has also been linked to depression and weight loss (Kumar et al. 2002; Kim et al., 2003;
Dantzer et al., 2008). Kasimanickam et al., (2013) evaluated serum concentrations of TNFα, IL‐1, and IL‐6 in cows with postpartum uterine disease. The study showed cows with metritis, clinical or subclinical endometritis, low body condition scores (2‐2.5 on a scale of 1‐5) and persistent inflammatory uterine conditions had higher serum concentrations of TNFα, IL‐1 and IL‐6 than normal cows. High stress levels can lead to the overproduction of Il‐6, IL‐1 and TNFα in both humans and animals and this can stimulate the synthesis of all acute phase proteins involved in the inflammatory process: C‐reactive protein, haptogloben (Hp), serum Amyloid A (SAA),
34 fibrinogen (Fb), etc. (Leandro et al. 2007; Hiromichi et al., 1997). The usefulness of APPs for diagnosis and prognosis during the course of commonly occurring inflammatory health challenges in cows (dystocia, retained placenta, metritis, abdominal disorders, mastitis and limb disease) and calves (respiratory tract and diarrhea) and in herd health monitoring has been shown in many studies to be a valuable tool to make early determinations about health and treatment (Oda et al., 2005; Alexandrakis et al., 2000; Antonelli et al., 2009; Panek et al., 2006;
Jawor and Stefaniak, 2011). The most successfully used APPs for diagnosis of illness and disease
in cattle are considered to be Hp, SAA and Fb (Jawor and Stefanaik, 2003; Skinner et al., 1991;
Horadagoda et al., 1999; Humblet et al., 2004; Eckersall et al., 2001; Gronlund et al., 2003).
Haptoglobin
The acute phase protein haptoglobin (Hp) is produced primarily by hepatocytes but also by skin, liver, lung, and kidney. In some species, including humans, mice, and cows, it is produced in adipose tissue as well. Horadagoda et al., (1994) determined reference values in cows for Hp and Skinner et al., (1991) evaluated serum Hp in inflammations and introduced Hp as an inflammatory indicator in cattle. Hp binds free hemoglobin when red blood cells are lysed due to tissue damage. The binding inhibits hemoglobins oxidative activity and also sequesters the iron within hemoglobin, preventing iron‐utilizing bacteria from using the iron to proliferate.
Hp levels can be undetectable in healthy cattle and increase 50 to 100 times during an acute phase response. Hp has been used for clinical evaluation of inflammation, infection and stress
(Gronlund et al., 2003; Murata et al., 2004). Horadagoda et al. (1999) found that in order to differentiate between an acute and a chronic inflammatory state in cattle, APPs are much more useful than classical hematological tests (leukocyte count, neutrophil percentage, or band neutrophil percentage). In this study, SAA showed the highest sensitivity, while Hp showed a higher specificity for a particular disease state. Bertoni et al. (1997) showed a positive
35 relationship between serum Hp levels, days in milk, open days and culling rates of high producing cows. Takahashi et al., (2007) conducted a study looking at SAA and Hp levels in healthy cows and compared them to cows with Amyloidosis and chronic inflammation. Hp levels of the healthy cows were all below the limits of detection of 20ug/ml and significantly lower than the levels in cows with Amyloidosis and chronic inflammatory disease. Unhealthy cows showed a median Hp level of 455 ug/ml with a range of 180 ug/ml and 2000 ug/ml. There were no significant differences in Hp levels between animals with Amyloidosis and chronic inflammation showing that Hp cannot be used for specific diagnostics which confirms
Horadagodas (1994) results.
Serum Amyloid A
Serum Amyloid A (SAA) is expressed in the liver, but its physiological role is not well understood. Recently, a broader view of SAA expression and function has been emerging.
Expression studies show local production of SAA proteins in histologically normal, atherosclerotic, Alzheimer, inflammatory, and tumor tissues. Binding sites in the SAA protein for high‐density lipoproteins, calcium, laminin, and heparin/heparan‐sulfate have been described
(Uriel‐Shoval et al.,2000). Adhesion motifs were identified and new functions, affecting cell adhesion, migration, proliferation and aggregation have been described (as reviewed by Urieli‐
Shoval et al., 2000). Alsemgeest et al., (1994) showed ratios of Hp and SAA can be useful
parameters to distinguish healthy animals and to distinguish between animals with acute or chronic inflammatory disease, but methods in this study are not well documented. Lomborg et al., (2008) subjected otherwise healthy adult cattle as well as calves to transport, slippery floors, isolation and restraint. The results of which suggested that SAA and Hp may serve as markers of stress. SAA and Hp levels in Holstein calves increased before weaning to three days post weaning (Myung‐Hoo Kim et al., 2011), confirming Lomborgs findings that these acute‐phase
36 proteins respond to psychological as well as physical and physiological stresses. Takahashi et al.
2007 found that SAA levels in cows suffering from Amyloidosis was not a good diagnostic tool for this problem, nor was there a significant correlation for SAA and Hp levels for this disease, but the correlation between SAA and Hp levels in cows with chronic inflammation was significant. Correlations between SAA and Hp have been recorded for cows with fatty liver, experimentally induced Escherichia coli mastitis, acute and chronic peritonitis and more (Connor et al., 1986; Gruys et al., 1994; Eckersall et al., 2000; Horadagoda et al., 1994; Horadagoda et al.,
1999; Gronulund et al., 2003; Skinner et al., 1991). All reports show that levels of these acute phase proteins are undetectable in healthy animals. The concentrations of the acute phase proteins SAA and Hp were determined in the plasma of healthy cows and cows with spontaneous acute, subacute or chronic inflammatory disease. The concentration of SAA in healthy cattle was 20.0 mg/l, and Hp was undetectable. The SAA/Hp ratio differed significantly between healthy animals and animals with inflammatory disease with concentrations varying by disease and severity of disease (Alsemgeest, 1994). These findings are consistent with other studies using these acute phase proteins for diagnostic purposes where levels for healthy cattle were determined to be <0.3ug/ml to 13.5ug/ml., and the levels for unhealthy cows was
17.1ug/ml to 298.2ug/ml (Jawor et al., 2008; Horadagoda et al., 1999; Eckersall et al., 2001;
Connor et al., 1996).
Fibrinogen
Fibrinogen (Fb) is a soluble plasma glycoprotein that is synthesized in the liver by the hepatocytes in response to many inflammatory states (McSherry et al., 1970). Fb is converted from thrombin to fibrin strands during blood clot formation (thrombosis), specifically binding activated coagulation factors (Xa) and thrombin in its fibers to temporarily inhibit the enzymes which would otherwise stay active and could be released during fibrinolysis (Figure15).
37 Fibrinolysis is a process that prevents blood clotting from becoming a problem (Muszbek et al.,
2008; Ross et al., 1986; Krem and DiCera, 2002). In 1962 Barnhart showed that fibrinogen was formed in the microsomes of the liver parenchymal cells of a cow and then stored in the soluble part of the cell until required. Cattle have a great capacity to produce fibrinogen and do so in response to a variety of stimuli the foremost of which is inflammation from tissue destruction
(McSherry, 1970). Normal levels of plasma fibrinogen in cattle were established by McSherry in
1970, where Fb levels in 133 clinically normal calves, bulls, non‐pregnant and pregnant cows were compared to Fb levels in 233 cows with various inflammatory conditions. Mean plasma fibrinogen levels were determined to be 508, 505, 660, and 581mg/100ml respectively. Lower than normal levels were sometimes seen in liver disease and terminal states. These levels confirmed the findings by Stormorken, (1957) where fibrinogen was used successfully to identify cows with reticuloperitonitis. Reticuloperitonitis in cows is very difficult to detect diagnostically and many animals die as a result of misdiagnosis. The results of a study by Jafarzadae et al.,
(2004) demonstrated that the serial interpretation of total plasma protein and plasma fibrinogen with suggested cut‐off points is useful for differentiating traumatic reticuloperitonitis from vagus indigestion, liver abscesses, omasanal impaction and Johne’s disease in cattle.
Plasma concentrations of Fb and Hp decreased in bull and steer calves between the first day of arrival at a feedlot and 28 days later, indicating that these acute phase proteins could be indicative of elevated stress in the animals (Berry et al., 2004). Correlations between SAA, Hp and Fb were reported by Jawor et al.,(2008) in the treatment of limb disease of cows showing their value as a diagnostic tool for this common management related disease/disorder.
38 Figure 15 Conversion of Fibrinogen to Fibrin
J. Dunkley (2007). Wikipedia common source original file.
http://en.wikipedia.org/wiki/Thrombin#mediaviewer/File:Coagulation full.svg
White Blood Cell Counts
A critical function of inflammation is to deliver leukocytes to the site of injury and
infection and to activate the leukocytes to perform their normal functions in host defense.
Leukocytes ingest offending agents, kill bacteria and other microbes, and remove necrotic tissue and foreign substances as the healing process proceeds.
Within one or two hours of injury or infection, the endothelial cells begin to express E‐
selectin to attract effector and memory T cells to peripheral sites of inflammation, particularly in
the skin. Endothelial cell expression of E‐selectin is a hallmark of acute cytokine‐mediated
inflammation. The normal sequence of events is the induction of adhesion molecules on
39 endothelial cells. This happens by several different mechanisms: The redistribution of P‐selectin, which is mediated by histamine, thrombin, and platelet activating factor in response to injurious agents, where resident tissue macrophages, mast cells, and endothelial cells respond by secreting the cytokines (tumor necrosis factor‐1(TNF‐α), and Interleuken‐1 (IL‐1) and chemokines; or TNF‐α and IL‐1 induce the expression of ICAM‐1 (intercellular adhesion molecule) and VCAM‐1 (vascular cell adhesion molecule) on endothelial cells near areas of infection or injury. Leukocytes bind to the endothelial selectins, causing a rolling effect along the endothelial surface. While this is taking place, the chemokines enter the blood stream where they attach themselves to proteoglycans and are displayed on the endothelial cells. This in turn activates the rolling leukocytes. The leukocytes stop rolling, are reconfigured and spread out on the endothelial cells at the site of injury. The chemokines then act on the adhered leukocytes and stimulate the cells to migrate through inter‐endothelial spaces toward the site of injury or infection following a chemical concentration gradient. Once on site, the leukocytes adhere to the extracellular matrix and are retained there. In most forms of acute inflammation, neutrophils predominate in the inflammatory infiltrate during the first 6 to 24 hours; they are preplaced by lymphocytes in 24 to 48 hours. Another very important role of leukocytes is the production of cytokines that trigger the acute phase response leading to the production of acute phase proteins.
Using leukocytes to diagnose disease in cattle is not as reliable, but are still considered useful when used with other health markers like acute phase proteins because of predictable patterns in specific leukocyte numbers in response to an inflammatory stimulus (Jawor and
Stefaniak,2008; Alvarez et al., 2013; Kahn and Line, 2003). WBC concentrations are interpreted by comparison with species‐specific reference ranges (Table 6). Valid interpretation can be made only by considering the absolute numbers. Total WBC count is more variable and often
40 higher in neonates than in adults. When the total WBC is abnormal, one or more distributional abnormalities in the differential are likely. When the total WBC is normal, there still may be one or more distributional abnormalities in the differential. As a result, evaluation of the differential data is the most important component of the leukogram (Merck Veterinary Manual, 2003). The use of neutrophil: lymphocyte ratios are a common tool used to diagnose illness in humans and animals. Considered a more sensitive parameter than total leukocyte count, it has been used successfully to predict appendicitis in humans, and infections in cattle (Kahn and Line, 2003).
A leukemoid response describes a marked neutrophilia of a magnitude sufficient to indicate chronicity of an inflammatory response. Guidelines for neutrophilic leukocytosis considered to indicate a leukemoid response for ruminants is >20,000/uL (Kahn and Line 2003).
It is rare to see an adult cow with more than 18,000 to 20,000 neutrophils (neutropenia) unless exogenous corticosteroids have been administered to the animal. Corticosteroids are often used in conjunction with antibiotics to clear up a disorder such as mastitis, quickly (Wagner and
Apley, 2003; Smith et al., 2001). According to Lee and Kehrli (1998), the neutrophil expression of
CD18 increases, while the expression CD62L decreases at calving. Meglia et al.,(2001) did not find this to be true. The Meglia (2001) study showed instead a marked decline which accompanied a reduction in concentrations of vitamin A, E and the mineral Zn which all play a vital role in immunity. These results were in keeping with findings by Johnston and Chew (1984), and Spears and Weiss (2007) where a drop in the serum concentrations of these nutrients was associated with impaired immune functions and a higher incidence of disease in cattle like mastitis. Acute viral infections in cattle have been shown to cause leukopenia as a result of neutropenia, or lymphopenia, or both (Alvarez et al., 2013). Absolute lymphopenia in cattle has occurred in conjunction with stress, exogenous corticosteroid administration, some viral diseases, some acute severe infections, and endotoxemia. Frequently it is difficult to know
41 whether the lymphopenia is associated with the disease or stress from the disease. Absolute lymphocytosis that is transient is rare in dairy cattle and when present usually is associated with neutrophilia in animals recovering from an acute infection. Lymphocytosis that is persistent and repeatable usually indicates infection. These predictable patterns in neutrophils and lymphocyte numbers can therefore aide in the diagnosis of sub clinical and clinical disease when used as N:L ratios The normal N:L ratio established for healthy cattle is 1:2 or 0.5% (Rebhuns, 2007).
Table 6 Hematology: Normal Reference Values for Cattle
total White Blood Cells 4.0‐12× 103/μL
Neutrophils 15‐33%
(segmented) 0.6‐4.0× 103/μL
Neutrophils 0‐2%
(band) 0‐0.1× 103/μL
Lymphocytes 62‐63%
2.5‐7.5× 103/μL
Plasma fibrinogen 0.1‐0.6 g/dl
Adapted in part from Latimer K.S., Mahaffey E.A., and Prasse K.W., Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology, 4th ed., Wiley‐Blackwell, 2003. Reference ranges vary between laboratories. Using values supplied by the laboratory that is routinely used is recommended. http://www.merckmanuals.com/vet/appendixes/reference_guides_reference_ranges.html
Stress Effects of Heat and Cold on Lactating Dairy Cattle
Samuel Brody (1956) was concerned about the development of methods enabling selection and breeding of cattle on a global scale in different climates. It had been previously noted that the European‐evolved cattle (Holsteins, Jersey, Guernsey etc…) were very cold and heat tolerant but studies had not been formerly conducted. He therefore did a comparative analysis of three categories of cattle (European, Indian, and Santa Gertrudis) where it was demonstrated that when body temperatures of Holsteins, Zebu and Jerseys were observed at temperatures of 70F and 500F the Holstein rectal temperatures raised only 2% more at 70F
42 compared to 500F, where the Indian bred Zebu rectal temperatures raised 60% under the same conditions. At 600F the thermo regulative mechanisms of the Zebu became active with abrupt and rapid increases in respiration and vaporization rates. Beginning at 800F these mechanisms began to fail, there was a rise in body temperature, feed rejection, lower milk production and decreased body weight. These tests were conducted indoors, with humidity readings of 50‐60% with air movement of 0 .5mph. It was noted that lower night temperatures yielded higher tolerated day temperatures. These results were considered to be due in part to size, as the rectal temperature readings of the Jerseys were between that of the Holsteins and the Zebu.
Other observations associated with temperatures from 500F to 1050F where: decline in thyroid activity (which has the physiological role of balancing heat loss or regulate heat production); and a rise in blood cholesterol, and creatinine. Vaporization was considered the best method to dissipate heat and there were recommendations to erect portable shade on pasture if trees were not present. The near lethal environmental temperature for Jersey cattle was found to be
1060F and for Holsteins it was found to be 1080F. Because European‐evolved cattle were considered to be very cold tolerant and believed to be essentially an arctic species, all recommendations in the 1950’s were for minor alterations to their winter environment to keep them productive. In 1971 Tatsuo established new mathematical equations to estimate critical heat and cold temperatures to establish patterns of heat loss in dairy cattle in colder climates.
Using these new equations, Bianca (1991) and Broucek et al. (1991) showed that when environmental temperatures move out of certain high or low temperatures, dairy cattle begin to experience either heat stress or cold stress. Lower critical temperatures (LCT) were estimated to be 320F, and Upper critical temperatures were estimated to be 770F‐78.80F. The Bianca study established what is called the thermoneutral zone. Within the thermoneutral zone animals do not have to expend any extra energy to maintain body core temperature. Recommendations
43 were then made that cattle should be protected from strong winds and moisture in cold weather, especially during early stages of lactation.
An alternate approach to evaluating cooling needs in cattle is to use the Temperature
Humidity Index (THI). The THI combines both ambient temperature and relative humidity which has been shown to be more effective in evaluating environmental effects on lactating cattle than temperature alone Collier et al. (2012). Original THI values for dairy cattle were established in the early 1950s and 1960s utilizing cows at the University of Missouri with average milk yields of 15.5 kg of milk/day. THI of 700F or less were considered comfortable, 750F‐780F stressful, and values greater than 780F were considered to cause extreme stress. The experiment conducted by Collier et al. (2012) showed that today’s high yielding cows (averaging 30 to 50 kg/milk/day) start showing signs of heat stress at THI readings as low as 680F and that other environmental factors such as a lack of shade, metal roof, temperature of water etc., lowers this number even more. Linville and Pardue, (1992) showed that effects of high THI are maximal between 24 and
48 hours following a high temperature stress event, and there were higher correlations to decreased milk yield when the THI was greater than 720F to 800F over a four day interval.
Conventional dairies combat heat stress problems by employing misters, evaporative cooling pads, High pressure foggers, Portable shades, and fans (Shearer et al. 1999).
Factors that can alter the effects of cold temperatures on animals are: wind, hair depth, hair coat condition, and a combination of wet/coat. Different LCTs have been established for different hair coats. Table 8 shows the LCT for varying hair coats of Beef Cattle. Table 9 shows the effects of wind speed on air temperature. It has been determined that for every 33.80F drop below the LCT a cow compensates with an approximate 2% increase in energy use. Decreases in milk yields will persist for 2 weeks following a 48 hour heat stress event (Tarr 2013). Because of
44 the Collier study, THI threshold for lactating dairy cows producing more than 35 kg of milk per day have been reestablished at 680F.
To summarize: Ratios of n‐6 to n‐3 EFA of 2:1 have been shown to benefit humans by reducing the intensity of an inflammation response. Adding n‐3 EFAs to a cow’s diet has been shown to have possible positive effects on the immune system. Ratios of n‐3 and n‐6 FA have been shown to fluctuate in pastures over the course of the growing season. These ratios are not constant but have been shown to be close to 2:1. The question proposed in this study is whether dairy cattle managed on pasture will exhibit a different inflammatory state as validated by measures of the APPs: Hp, SAA, and Fb, than those managed in a TMR system. In the following study, clinical markers of inflammation were measured from Jersey dairy cows in two different management systems and compared to their WBC counts and compiled temperature data during the pasture season.
45
CHAPTER II
MATERIALS AND METHODS
Experimental Design
Two groups of 9 Jersey dairy cows each, matched for age and number of lactation cycles,
were established at the UNH Organic Dairy in Lee, NH and the conventional dairy at the Fairchild
Teaching and Research Dairy in Durham, NH. The organic group was managed on pasture eating
a high forage diet and the conventional group was managed in confinement consuming a total
mixed ration (TMR). The two groups were compared for measurements of inflammation before,
and during, the pasture season. Clinical markers of inflammation measured were haptoglobin
(Hp), serum Amyloid A (SAA), fibrinogen (Fb), and white blood cell counts (WBC). Measurements were compared within groups over time and between groups over time.
Objective The objective of this study was to investigate whether Jersey dairy cows on pasture, eating a diet possibly higher in omega‐3 FAs and lower in omega‐6 FAs, will differ in clinical markers of health than those of Jersey dairy cows raised indoors being fed a TMR which is possibly lower in omega‐3 FAs and higher in omega‐6 FAs.
Hypothesis Dairy cows managed on pasture will demonstrate a less inflammatory state than dairy cows managed in confinement and fed a TMR.
Animal Management
Nine Jersey dairy cows were selected from the herd at the University of New Hampshire
Burley‐Demeritt Organic Dairy Research Farm located in Lee, NH. Cows were out on pasture from
46 May 29, 2012 to October 19, 2012 where they grazed typical cool season, New England pasture
grasses such as orchard grass, reed canary grass, timothy, perennial ryegrass, white and red
clover, etc. Cows were supplemented twice a day after milking at approximately 0500 hr and
1500 hr, with baleage top‐dressed with a grain ration consisting of: corn meal, soy meal, barely, roasted soy, organic mids, vitamins and minerals (Morrison’s Custom Feeds, Inc. Barnet, VT).
Water was provided in the fields ad libitum via a livestock tub with float‐controlled water from a hose line. The total diet was approximately 75% forage and 25% concentrate. When not on pasture, late October through early May, cows were housed in a free‐stall, open barn with constant access to the outdoors. Water, and feed baleage, was provided ad libitum. In accordance to USDA organic standards, cows were on pasture a minimum of 120 days out of the year, and
had access to the outdoors all year except in severe weather. They were not treated with
antibiotics or other non‐approved drugs.
Nine Jersey cows were matched to the above organic cows by age, parity and stage of lactation, and housed at the University of New Hampshire Fairchild Dairy Teaching and Research
Center in Durham, NH. Cows were managed along with the existing Holstein herd and housed in a free‐stall pen with bedded stalls, concrete slatted floors. Water and a mineral lick was provided ad libitum. Cows were milked at each day at 0530hr and 1730hr and fed twice per day a 55:45
forage to concentrate ratio TMR consisting of corn silage, steamed flaked corn, ground corn, citrus
pulp, beet pulp, soybean hulls, , alfalfa hay, haylage, urea, vitamins and minerals. Cows were
treated for infection per standard practices of animal health with antibiotics given as needed.
Sample Collection
The following (Table 7) shows the respective dates corresponding with sample number when data is displayed by sample number and not by date.
47 Table 7 Blood Sampling Dates
Sample # Organic (Site 1) Conventional
(Site 2) 1 4/24/2012 4/28/2012
2 5/29/2012 5/29/2012 3 6/27/2012 6/28/2012 4 7/24/2012 7/25/2012 5 8/21/2012 8/22/2012
Blood was taken from the coccygeal (tail) vein using 1”, 21 gauge needles (Kendall
Monoject) and collected into 10ml Vacutainer ™ tubes with clot activator for serum and also into
10ml Vacutainer ™ tubes with K2 EDTA (BD, Franklin, NJ) for plasma. Whole blood from EDTA tubes
was used to make smears on frosted slides, which were stained with Wright’s stain for differential
white blood cell counts. Both clot and EDTA tubes were centrifuged at 3300 rpm (3652 xg) for 20
minutes. Plasma and serum were collected into 2 mL microtubes (Sarstedt, Newton, NC) in aliquots of 3 ml, and stored at ‐80⁰C until analyzed. Red blood cells from clot tubes with serum removed were also stored in the original VacutainerTM at ‐80⁰C for future analysis. All animal procedures were reviewed and approved by the UNH Institutional Animal Care and Use
Committee (IACUC approval#120506).
Blood Analyses
All serum samples were analyzed in duplicate and results averaged. Serum haptoglobin
was measured using a commercial bovine haptoglobin enzyme‐linked Immunosorbent assay
(ELISA) (Life Diagnostics, Inc., West Chester, PA) with samples diluted 2000 fold per
manufacturer’s recommendation. Serum Amyloid A and plasma fibrinogen were detected using a
commercial bovine serum Amyloid A ELISA and bovine fibrinogen ELISA (TSZ ELISA, Framingham,
MA) with samples for both tests diluted 2000 fold per manufacturer’s recommendation. Standard
curves were established by running provided standards in duplicate. Absorbance was measured
48 at 450nm using a EL 800 Plate Reader (Biotek Instruments, Inc., Winooski, VT) and collected by
Gen5 Microplate Data Collection & Analysis software (Biotek, Inc.).
Statistical Analyses
Changes in Serum Amyloid A, fibrinogen, haptoglobin, and white blood cell counts, were analyzed for significant differences within and between farms using one way ANOVA, LSMeans
Student’s t test and connecting letters report. A repeated measures split plot approach model was used to confirm or reject the statistical findings of the ANOVA and strengthen the statistical analysis. (SAS Institute. Inc. 2013. JMP®11 Discovering JMP. Cary, NC: SAS Institute Inc). Analyses and graphs were generated. Effects were considered significant at P< 0.05.
Weather Data
Weather data was compiled from weather stations located at Pease Air Force Base in
Portsmouth, NH and accessed from the National Oceanic and Atmospheric Administration (NOAA) website. The temperature data is reported for dates, 24 hours before sample dates to 48 hours after sample dates, in degrees Fahrenheit.
49
CHAPTER III
RESULTS
Blood Markers
The serum Hp levels in the organic and conventional groups per sample week were as follows (Table 8): Table 8 Haptogloben (ug/ml) Levels Within Farm by Sample Week Sample Week
Organic 1 2 3 4 5
Mean value 3.397 3.451 5.118 5.645 5.382
Range/ week 2.85‐3.94 2.90‐4 4.67‐5.56 5.17‐6.12 4.91‐5.86
Conventional 1 2 3 4 5
Mean value 3.697 3.411 3.841 5.009 4.741
Range/Week 3.19‐4.20 2.97‐3.85 3.43‐4.26 4.57‐5.45 4.18‐5.30
Comparisons within farm between sample weeks for the organic farm using Student’s t, connecting letters report, showed no significant differences (P>.05) in Hp levels between sample weeks 1 and 2. There were no significant differences (P>.05) in Hp levels between sample weeks
3, 4, and 5. There was a significant difference (P=.0001) between the first two weeks and sample
weeks 3, 4, and 5. Sample week 4 had the highest level with a mean of (5.64ug/ml).
Comparisons within farm between sample weeks for the conventional farm using
Student’s t connecting letters report, showed a significant difference (P=.0001) in levels between
50 sample week 5 and sample weeks 1, 2 and 3. There was a significant difference between sample week 4 and sample weeks 1, 2, & 3.
Student’s t, connecting letters report showed no significant differences (P>.05) in serum Hp levels between the two farms for weeks 1, 2, and 5. There was a significant difference in Hp levels
between the two farms for sample week 3, (P=.0031) and sample week 4 (P=.0351).
The serum Hp levels between farms per sample week were as follows:
Table 9 Haptogloben (ug/ml) Levels Between Farms by Sample Week
Sample Week 1 2 3 4 5 Organic 3.40 3.50 5.11 5.65 5.38 Conventional 3.70 3.41 3.84 5.01 4.74
The repeated measures spit plot approach model confirmed the statistical findings of the
ANOVA (Graphs 1, 2, &3).
Graph 1
Haptoglobin Within Farm by Sample Date‐Organic 0.007 B 0.006 B B 0.005 0.004 A A Organic
(mg/ml) 0.003
Hp 0.002 0.001 0
Sample Date Weeks not connected by letters denotes a significant difference (P=<0.05).Normal levels of Hp in healthy cattle = 0 – 350 ug/ml.
51 Graph 2 Haptoglobin Within Farm by Sample Date‐Conventional 0.006 B 0.005 B A A 0.004
/ml) A 0.003 (mg
Hp 0.002 Conventional 0.001
0
Sample Date
Weeks not connected by letters denotes a significant difference. (P=< 0.05). Normal levels of Hp in healthy cattle = 0‐350 ug/ml.
Graph 3 Haptoglobin Between Farms by Sample Date 0.007 0.006 0.005
/ml) 0.004 (mg 0.003 Organic Hp 0.002 Conventional 0.001 0
Sample Date
Hp levels in both groups were within the limits for healthy cows (0 to 350 ug/ml), for all weeks.
52 The SAA levels in the organic and conventional groups per sample week were as follows:
Table 10 Serum Amyloid A (ug/ml) Levels Within Farm by Sample Week Sample Week Organic 1 2 3 4 5 Mean value 0.101 0.107 0.126 0.132 0.129 Range/Week 0.009‐0.111 0.098‐0.117 0.118‐0.134 0.125‐0.141 0.122‐0.138 Conventional 1 2 3 4 5 Mean value 0.115 0.107 0.108 0 .132 0.132 Range/Week 0.109‐0.120 0.102‐0.112 0.103‐0.112 0.127‐0.137 0.127‐0.137
Comparisons within farm between sample weeks for the organic farm using Student’s t, connecting letters report, showed no significant differences (P>.05) in levels between sample weeks 1 and 2. There was no significant difference (P>.05) between sample weeks 3, 4, and 5.
There was a significant difference (P=<.0001) between the first two weeks and the last three weeks. Sample week 4 had the highest levels with a mean of (0.133ug/ml).
Comparisons within farm between sample weeks for the conventional farm using
Student’s t connecting letters report showed no significant differences between sample weeks 4 and 5. Sample weeks 4 and 5 were significantly different (P‐<.0001) from sample weeks 1, 2 and
3. Sample weeks 2, & 3 were not significantly different from each other, but were significantly
different from sample week 1.
Student’s t connecting letters report showed no significant differences (P=> 0.05) in
serum SAA levels between the two farms for sample weeks 2, 4 & 5. There were significant differences (P=<0.05)) between farms for sample week 1 and 3. Between farm mean values per
sample weeks were as follows:
53 Table 11 Serum Amyloid A (ug/ml) Levels Between Farms by Sample Week
Sample Week 1 2 3 4 5 Organic 0.102 0.107 0.126 0.133 0.133 Conventional 0.116 0.107 0.107 0.132 0.130 The repeated measures split plot approach confirmed the ANOVA.
Graph 4
Serum Amyloid A Within Farm by Sample Date‐Organic 0.14 B B 0.12 B A A 0.1 /ml)
(ug 0.08
SAA 0.06 Organic 0.04
0.02
0
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal levels of SAA in healthy cattle ‐ .03‐ 16 ug/ml.
54 Graph 5
Serum Amyloid A Within Farm by Week‐Conventional 0.14 C C A 0.12 B B 0.1
/ml) 0.08
0.06 Conventional SAA(ug 0.04
0.02
0
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal levels of SAA in healthy cattle = .03 – 16 ug/ml.
Graph 6 Serum Amyloid A Between Farms by Sample Date 0.14 * 0.12 * 0.1
0.08 Organic
/ml) 0.06
(ug Conventional
0.04 SAA 0.02
0
Sample Date
*Denotes a significant difference between farms (P=< 0.05)
55 The plasma Fb levels in the organic and conventional groups per sample week were as follows:
Table 12 Plasma Fibrinogen Levels (mg/dl) Within Farm by Sample Week Sample Week Organic 1 2 3 4 5 Mean value 375.8 396.7 358.4 399.4 416.6 Range/Week 335.6‐415.9 356.5‐436.9 320.9‐396.0 361.8‐437.0 369.1‐464.1 Conventional 1 2 3 4 5 Mean value 359.3 377.0 379.4 536.2 393.1 Range/Week 248.1‐470.5 321.4‐432.6 314.5‐471.8 480.6‐591.8 318.3‐443.8
Comparisons between sample weeks within the organic farm using Student’s t connecting letters report, showed no significant differences in levels for all sample weeks (P>.05).
Comparisons between weeks within the conventional farm using Student’s t, connecting letters report showed no significant differences (P>.05) between sample weeks 1,2,3, and 5, but did show a significant difference in Fb levels (P=.0010) between sample week 4 and sample weeks
1,2,3 and 5.
Student’s t connecting letters report showed no significant differences (P>.05) in plasma
Fb levels between the two farms for sample weeks 1, 2, 3 and 5. There was a significant difference
between farms for sample week 4 (P=.0263).
Table 13 Plasma Fibrinogen (mg/dl) Between Farms by Sample Week Sample Week 1 2 3 4 5 Organic 390.7 395.0 358.4 522.9 402.7 Conventional 371.5 377.0 379.4 407.4 381.1
The repeated measures spit plot approach model confirmed the statistical findings of the
ANOVA (Graph 7, 8, & 9).
56 Graph 7
Fibrinogen Within Farm by Sample Date‐Organic 600
500
400 (mg/dl) 300 Organic Fb
200
100
0
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Yellow line denotes lower threshold of 200 mg/dl of Fb in healthy cattle.
Graph 8 Fibrinogen Within Farm by Sample Date‐Conventional 600
500
400
300 (mg/dl)
Fb 200 Conventional 100
0
Sample Date Weeks not connected by letters denotes a significant difference (P=< 0.05). Yellow line denotes lower threshold of 200 mg/dl of Fb in healthy cattle.
57 Graph 9 Fibrinogen Between Farms by Sample Date 600
500 *
400 Organic
(mg/dl) 300
Fb 200
100
0
Sample Date
Fb levels at both farms were within limits set for healthy cattle (200‐1200mg/dl), for all sample weeks. * denotes significant difference between farms.
The total WBC counts in the organic and conventional groups were as follows:
Table 14 Total White Blood Cell Counts within Farm by Sample Week
Sample Week
Organic 1 2 3 4 5 Mean value 9.9 7.5 8.6 12.4 16.2 Range/Week 7‐12.7 4.7‐10.4 6.2‐11.0 9.9‐14.8 12.7‐19.6 Conventional 1 2 3 4 5 Mean values 11.0 6.9 9.6 15.2 11.3 Range/Week 8‐14.1 3.9‐10 6.9‐12.2 12.6‐17.8 7.5‐15.0 * All values expressed as (X103 cells/ul).
Comparisons between sample weeks within the organic farm using Student’s t connecting letters report, showed no significant differences (P>.05) in levels between sample weeks 1,2 and 3. There were no significant differences (P>.05) between sample weeks 1 and 4.
There were no significant differences (P>.05) between sample weeks 4 and 5. There were significant differences (p=.0014) between the first three sample weeks and sample week 5.
58 There was also a significant difference between sample weeks 2 & 3 with sample week 4.
Sample week 5 had the highest mean of (16.2 X103 cells/ul).
Comparisons between sample weeks within the conventional farm using Student’s t connecting letters report, showed no significant differences (P>.05) in counts between sample weeks 1, 2, 3, and 5. There were no significant differences between sample weeks 4 and 5.
There was however a significant difference (P=.0014) between sample week 4, and sample weeks 1,2 and 3. Sample week 4 had the highest mean value of (15.6 X103 cells/ul).
Student’s t, connecting letters report showed no significant differences (P>.05) in WBC counts between the two farms for sample weeks 1, 2, and 3. There were no significant differences (P=.0119) between farms for sample weeks 4 and 5.
Table 15 Total White Blood Cell Counts Between Farms by Sample Week
Sample Week 1 2 3 4 5 Organic 9.9 x103 7.5 x103 8.6 x103 13.1 x103 5.1 x103 Conventional 11.0 x103 6.9 x103 9.6 x103 15.6 x103 3.8 x103 * All values expressed as (X103 cells/ul).
The repeated measures spit plot approach model confirmed the statistical findings of the ANOVA (Graph 10, 11, 12).
59 Graph 10
Total White Blood Cells within Farm by Sample Date‐ Organic
) 18 C 16
cells/ul 14 C
3
^
12 BC B B 10 (X10 8 Organic
WBC 6 4 2 0
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal tWBC in healthy cattle = 4500 – 10000cells/ml. (denoted by Yellow lines).
Graph 11 Total White Blood Cells within Farm by Date ‐ Conventional 16 B
) 14 A AB 12 A cells/ul
3 10
^ 8 A (X10 6 4 WBC Conventional 2 0
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal tWBC in healthy cattle = 4500 – 10000cells/ml. (denoted by Yellow lines).
60 Graph 12 Total White Blood Cell Counts Between Farms
18 ) 16 * 14 * cells/ul 12 3
^ 10 Organic 8 (X10
Conventional 6
WBC 4 2 0
Sample Date
Total WBC counts exceeded normal limit (>10,000cellls/ul) at both farms in sample week 4 and 5. Limits were exceeded at the conventional farm on sample week 1. (denoted by yellow line). * denotes a significant difference (P=< 0.05) between farms.
Neutrophil counts in the organic and conventional groups were as follows:
Table 16 Percent Neutrophils within Farm by Sample Week Sample Week Organic 1 2 3 4 5 Mean value 37.3 49.2 34.9 30.8 30.0 Range/Week 29.2‐45.5 41.0‐57.3 27.8‐41.9 23.7‐37.8 20.0‐40.0 Conventional 1 2 3 4 5 Mean value 29.0 35.8 28.9 29.1 26.0 Range/Week 23.2‐34.8 30.0‐41.6 23.9‐33.9 24.1‐34.1 18.9‐33.1 *Neutrophil counts are reported as a percentage of total WBC.
Comparisons between sample weeks within the organic farm using Student’s t, showed a significant difference (P<.0148) in mean values between sample week 2 and sample weeks 1,
3, 4, and 5.
Comparisons between sample week within the conventional farm using Student’s t, showed there was a significant difference (P>.05) between sample week 2 and sample week 5.
61 Student’s t, connecting letters report showed there was a significant difference
(P=<0.05) between farms for sample week 2 with mean values of (49.2% for the organic herd, and 35.8% for the conventional herd).
Table 17 Percent Neutrophils between Farms by Sample Week Sample Week 1 2 3 4 5 Organic 37.3 49.2 34.9 30.8 30.0 Conventional 29.0 35.8 28.9 29.1 26.0 *Neutrophil counts are reported as a percentage of total WBC.
The repeated measures spit plot approach model confirmed the statistical findings of the ANOVA(Graph 13, 14, & 15).
Graph 13 Neutrophil Counts within Farm by Date ‐Organic
60 B 50 A 40 A A A 30 Organic 20 Neutrophils
(%) 10 0
Sample Date
Neutrophils exceeded normal limits (15.6‐39%) at organic farm (denoted by yellow line), on sample week 2. Weeks not connected by letters denotes a significant difference (P=< 0.05).
62 Graph 14
Neutrophil Counts within Farm by Date ‐ Conventional
60
50 AB
40 A 30 A A AC
20 Neutrophil
Conventional
(%) 10
0
Sample Date
% Neutrophils were within normal limits (15.6 ‐39%) on all sample weeks at conventional farm. Weeks not connected by letters denotes a significant difference (P=< 0.05).
Graph 15
% Neutrophils ‐ between Farms by Sample Date
60% 50% * 40%
30% Organic Neutrophils 20%
10%
0%
Sample Date
Neutrophil levels exceeded normal range for healthy cattle of (15.6‐39% denoted by yellow line) within the organic group on sample week 2. * denotes significant difference between farms.
63 Lymphocyte counts in the organic and conventional groups were as follows:
Table 18 Percent Lymphocytes Within Farm by Sample Week Sample Week Organic 1 2 3 4 5 Mean value 52.3 38.5 50.3 49.4 47.8 Range/Week 44.9‐59.8 31.0‐46.0 43.8‐56.7 42.9‐55.8 38.6‐56.9 Conventional 1 2 3 4 5 Mean value 62.3 52.3 57.0 58.0 58.5 Range/Week 55.8‐68.9 45.8‐58.9 51.3‐62.7 52.3‐63.7 50.5‐66.5 *Lymphocyte counts are reported as a percentage of total WBC.
Comparisons between sample weeks within the organic farm using Student’s t, showed there was a significant difference (P=<0.05) between sample week 2 and sample weeks 1, 3,
and 4.
Comparisons between sample weeks within the conventional farm using Student’s t, showed there was a significant difference (P=< 0.05) between sample week 2, and sample week 1.
Student’s t, connecting letters report showed there was a significant difference (P=0.0038) between farms for sample weeks 1 and 2.
Table 19 Percent Lymphocytes Between Farms by Sample Week Sample Week 1 2 3 4 5 Organic 52.3 38.5 50.3 49.4 47.8 Conventional 62.3 52.3 57.0 58.0 58.5 *Lymphocyte counts are reported as a percentage of total WBC.
The repeated measures spit plot approach model confirmed the statistical findings of the ANOVA (Graph 16, 17, & 18).
64 Graph 16 Lymphocyte Counts Within Farm by Date‐Organic 64 60 56 A 52 A A AC 48 Organic 44 BC 40 Lymphocytes 36 (%) 32 28
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal range for healthy cattle = 60‐63% (denoted by yellow line).
Graph 17 Lymphocyte Counts Within Farm by Date ‐ Conventional
64 A 62 60 AC AC 58 AC 56 54
Lymphocytes BC 52
(%) Conventional 50 48 46
Sample Date
Weeks not connected by letters denotes a significant difference (P=< 0.05). Normal range for healthy cattle = 60‐63% (denoted by yellow line)
65 Graph 18 Lymphocyte Counts Between Farms by Date 70 60 * 50 * 40 Organic 30 Conventional
Lymphocytes 20
(%) 10 0
Sample Date There was a significant difference between farms for sample weeks 1 & 2, denoted by *
Neutrophil /lymphocyte ratios between farms are as follows:
Table 20 Neutrophil/Lymphocyte Ratios by Sample Week Sample Week Organic 1 2 3 4 5 ratio .7 1.2 .7 .6 .7 Conventional 1 2 3 4 5 ratio .5 .7 .5 .5 .4 *Normal N:L ratio in cattle is 2:1 or (.5%).
The percentages of Neutrophils /Lymphocytes in the organic herd were much closer together than the accepted normal ratio of 2:1 (.5), at all sample times. The percentages of
Neutrophils to Lymphocytes in the conventional herd were closer together in sample weeks 2, showing a normal ratio of .5 on sample week 2 and a below the normal ratio on sample week 5.
The conventional herd had accepted normal ratios of .5 in sample weeks 1,3, and 4 were normal. N:L ratios between farms by sample weeks showed significant differences (P=<.05) in sample week 1,2, and 3. N:L ratios between farms were not significantly different on sample weeks 4 and 5.
66
Graph 19
Neutrophil/Lymphocyte Ratio Between Farms by Sample Week 1.2
1 1
0.8 0.7 0.7 0.7 0.7
0.6 0.6 (%) 0.6 Pasture 0.5 0.5 0.5 N:L Confinement 0.4
0.2
0 12345
Ratios were significantly higher in the organic herd on sample weeks 1, 2, and 3. N:L ratios in the organic herd were above accepted ratios (.5) established for cattle on all sample weeks.
Weather Data
Ambient air temperature, and temperature humidity index varied by sample week.
Ambient air temperatures, and/or THI temperatures were at or above the critical heat temperature for Jersey cattle of 680Fwere recorded 11 times in sample weeks 3, 4, and 5.
67 Table 21 Temperature Data Date Ambient Air Temp 0F Relative Humidity THI % 0F
4/23/2012 54 98.9 54 *indicates 4/24/2012 52.5 98.9 52 4/25/2012 52.5 98.9 52 days when 4/26/2012 51 98.9 50 5/28/2012 62 73.6 61 5/29/2012 62 96.8 62 *5/30/2012 76 89 *77 6/26/2012 62 88.5 62 *6/28/2012 70 65.8 *70 6/29/2012 67 62.1 66 *6/30/2012 75 76.7 *76 *7/23/2012 86 71.6 *96 *7/24/2012 82 67.9 86 *7/25/2012 80 57.8 82 *7/26/2012 70 77.1 70 *8/20/2012 76 75.9 77 *8/21/2012 78 77.7 80 *8/22/2012 79 68.8 81 *8/23/2012 86 72 96 temperatures were at or above critical heat levels for Jerseys.
68
CHAPTER IV
DISCUSSION
Haptoglobin
In our study all Hp levels in both herds showed detectable levels of Hp, but those levels remained within the reported physiological range (0‐50ug/ml) for cattle with mean values of
(4.74ug/mL for the organic herd and 5.84ug/mL for the conventional herd). Although considered healthy, there were trends in these low levels and they did differ by farm.
Hp levels in the organic herd increased by the third sample time point from 3ug/ml for sample time point 1 & 2, to 5ug/ml for sample time point 3; 6ug/ml for sample time point 4; and
5ug/ml for sample time point 5. They remained elevated but stable for sample time points 3, 4,
5 and 6, indicating a possible immune system response (Graph 1). The pattern seen in Hp levels would have coincided with the early spring pre‐pasture season, with animals being put out on pasture on May 21. In pasture based systems it is customary to put animals out on pasture when the grass has reached sufficient height to be able to withstand grazing pressure. Dependent on weather; this usually occurs from May 15th to June 1 in New Hampshire. In 2012, grasses in New
Hampshire were not ready until the end of May (as reported by growers in Merrimack County to
UNH Cooperative Extension, 2012). By sample time point 3 the weather started to get hot with temperatures reaching critical highs for the remainder of the sample time points (Table 27).
Animals were brought back into the barn for the winter on September 24, 2012. Stressors inherent to pasture based systems are: increased physical activity, change in environment, changes in feedstuffs (hay to fresh feed), fluctuating temperatures, lack of shade, warm drinking water, exposure to flies, parasite infections, etc. Alsemegeest et al. (1995) showed Hp levels in
69 cows do not increase in response to physical stress whereas SAA concentrations do. The levels of Hp used to determine stress in this study were above the established physiological range for of 0‐50ug/ml. Unaccustomed exercise has been shown to result in temporary, repairable skeletal muscle damage in humans (Ebbling and Clarkson, 1989). Changes in environment have been shown to increase signs of stress in cows using parameters other than blood markers to make these determinations (Rushen et al. 2001). Munksgaard et al. (2001) showed that cows have a clear observable avoidance response to rough handling of other cows. This demonstrated ability to observe, distinguish, and react to perceived danger. This stress response could show up metabolically. The organic cows in our study were switched from a barn/paddock situation to a pasture system. Cows are considered prey animals. Strong prey instincts like hyper vigilance would be a predictable reaction to possible predation and would elicit a stress response that could show up metabolically (Boissy 1998; Boissy et al. 1998; Boissy et al. 2005; Wohlt et al.
1994; Waiblinger et al. 2006; Murphey et al 2007; Forkman et al. 2007).
West et al. (2003) conducted a study where lactating cows were exposed to moderate and hot/ humid weather to determine the effects of increasing ambient temperature, relative humidity, or temperature‐humidity index (THI) on intake, milk yield, and milk temperature.
Environmental conditions had minor effects on intake and milk yield during the cool period, but dry matter intake and milk yield declined linearly with respective increases in air temperature.
Our study did not measure milk yields. The organic cows in our study are kept on pasture per the rules from the National Organic Standards set forth by the National Organic Standards
Board. The cows at the organic farm have no shade provided to them and the organic standards
rules do not allow managers to keep them in/off pastures when weather will affect them adversely. It should be noted that it is not against the rules to erect shade structures in the fields or allow free access to barns. At the UNH organic farm, the use of a management intensive
70 grazing system where electric fence is moved every 12 hours makes putting up temporary, moveable shade structures that can withstand wear and tear of large animals would be costly, time consuming, and difficult.
In comparison, the Hp levels at the conventional dairy stayed fairly steady through the first 3 sample time periods with mean values of (3.6ug/ml; 3.4ug/ml;3.8ug/ml) but significantly increased at sample time point 4 with a mean of (5.0ug/ml) and remained high for sample time point 5 with a mean of (4.7ug/ml) (Graph 2). The conventional cows could be considered to be housed in a fairly constant environment throughout the year, where they are provided shade, fans for cooling, fly management, etc. Factors that may have elicited a stress response in this group of cows could be: changes in personnel; heat stress, or subclinical illness. Both groups of cows would have undergone a personnel change due to work study students graduating or leaving school in May. As reported by farm managers, the feed for this group is never changed with the exception of increasing the energy ration for cows 14 days after freshening. Herd health records kept for the conventional herd recorded four cows from our study that could have brought the overall mean value up (cow #74; cow #2209; and cow #2210). Cow # 74 freshened on 9/7/2012 and cow # 2209 freshened on 6/20/2012. It has been shown there is an
APP reaction in cows 4 weeks prior to calving and up to 2 weeks after (Jawor et al. 2011). A third cow (#2210) was diagnosed with mastitis on 8/4/2012. Further investigation did not reveal any malfunctions in ventilation or fans used to cool the cows. The farm manager (personal correspondence 2014) reported the fans are turned on as needed starting in June and stay on through September unless cool, breezy conditions occur in which case they would have been shut off. No other cooling systems are used. It was also suggested that the fan system may not be large enough, as it does not reduce heat to below outside air temperatures when heat and humidity persist for more than one or two days. Actual temperatures between the outside air
71 and the inside air in the barn were not formally recorded. In 1956 Samuel Brody showed the responses of conventional cattle from different areas of the world to climate change. Increasing temperatures from 50 degrees F. to 105 degrees caused thyroid activity to decline, and there was a rise in blood cholesterol, creatinine, and in respiratory alkalosis, which he believed partly explained the decline of milk yield and change in milk composition of highly productive cattle in tropical regions. Brody tested various methods to cool cattle in this study. Water temperature was shown to have a positive or negative impact on body temperature. The best water temperatures to provide to cattle in hot weather were found to be approximately 50‐550F. The
water source for the conventional cows is from a well and is piped into the barn from beneath the ground and delivered via automatic on demand waterers. Although water temperatures were not recorded, it could be concluded that the temperature of the water supplied to the conventional cows is near or at geothermal range of 50 degrees, and the water supplied to the organic cows is close to ambient air temperature or hotter. Sample time points 3, 4 and 5 had recorded ambient air temperatures, THI, or both, above the upper critical temperature of 680F for Jersey cattle (Table 27). All of the aforementioned averse conditions could have elicited an
Hp response.
Serum Amyloid A
In our study both farms had overall mean SAA values well below the limits set for healthy animals (0.121ug/mL for the organic dairy and 0 .118ug/mL for the conventional dairy).
Even though, levels were not in the ranges described that would indicate illness, SAA levels in our cows did seem to fluctuate with seasonal stressors that were identified as possible APP triggers of Hp. Speaking to physical activity as a source of elevated APPs, Kujala et al. (2010) showed cows with lameness have elevated levels of SAA from day 0 to 7‐8. Increased physical activity does create a lame situation in animals that are accustomed to limited activity (Davidson
72 et al. 2003). This would have been the case with the organic cows when they were turned out on pasture on May 21, 2012. Prior to turn out the cows are kept in a paddock area that is attached to the barn so they have year round access to the outdoors. This area does not have a lot of room to move around so physical activity would have been limited. Once turned out for the pasture season, the organic cows are then brought across the field twice a day to be milked.
Following the pattern associated with the grazing season, the SAA levels in the organic group were lower the first two sample time points which was before turn out, (0.101 ug/ml for sample time point 1 and 0.107 ug/ml for sample time point 2), and then rose in weeks 3,4, 5
(0.126ug/ml for sample time point 3; 0.133ug/ml for sample time point 4; and 0 .130ug/ml for sample time point 5), which was after turn out. Mallonee et al. (1985) showed heat stressed cows lose more potassium in sweat than cows that are not heat stressed. Schneider et al. (1984) showed that decreased feed intake occurs when cows are heat stressed. A deficiency of dietary potassium may result from reduced feed intake (Pradhan and Hemken, 1968). When cows are experiencing heat stress, they often pant (Brady 1956). Panting can result in respiratory alkalosis resulting from hyperventilation (Benjamin, 1978). In addition from June through the end of the
August fly season occurs, and effective treatment of flies for organic cows is limited. Cows bothered by flies stop feeding, and huddle close together. The huddling can increase body temperature and result in reduced feed intake and increased body temperatures (Benjamin,
1978). In addition, it has been shown that flies transmit summer mastitis (Sol, 1983). Additional stressors for the organic group not mentioned earlier could also be parasite loads. The organic cows are not treated with anthelmintics for parasites and studies show SAA increases in response to gastrointestinal parasite infections (Glass et al., 2003; Cray et al., 2009). The cows were not tested for parasite loads.
73 The conventional group does not experience internal parasite infections, which are contracted during grazing, and flies are controlled with pesticides. They do however experience external parasite infestations, summer mastitis, and heat stress as previously discussed. All these stressors can cause reduced feed intake and an immune system response. SAA levels for the conventional group showed a significant difference in levels between sample time point one
(0.116ug/ml) and sample time point 2 (0.107ug/ml) and sample time point 3 (0.108ug/ml). The slightly higher level seen in sample time point 1 may have been due to 2 cows that freshened
(cow #73 and cow #74). The first 3 sample time points were significantly lower than sample time point 4 (0.131ug/ml) and sample time point 5 (0.132ug/ml) (Graph 2). These findings would coincide with the hot humid weather described earlier and may also be explained by health incidents recorded by dairy manager and explained previously for Hp levels.
Fibrinogen
The plasma Fb concentrations in this study for both groups were well within the normal limits for healthy cows, having mean values of (386.7mg/dl, for the organic herd and 416.3mg/dl for the conventional herd). There were no significant differences in Fb concentrations for the organic cows for all sample time points, but graph 6 does show an insignificant increase at sample time points 4 and 5. Health records for the organic farm showed cow #17, and #47 freshened on 6/6/2012 and cow # 1059 was being treated for udder trauma on 6/7/2012. In addition, cow # 23 freshened on 8/2/2012. These health events, although minor, would have elicited an inflammation response that would have slightly elevated Fb concentrations. It should be noted that the health records for both farms lack standardization, and appeared to be incomplete as they did not include information about all animals in the study. They are therefore not printed in this work.
74 Fb levels were similar for the conventional group until sample time point 4 where the levels significantly increased. Then at sample time point 5 the levels returned to what was seen the first 3 sample time points (Graph 7). As with the organic group heat was suspected as a contributing factor in the increase seen on sample time point 4, but weather data showed the temperatures did not come down significantly in week 5 (Table 27). This indicated that Fb was probably responding to something other than heat stress. Fly pressure would not have been a factor as cows in the conventional group are treated for flies. Health records showed cow #2209 freshened on 6/20/2012 and cow #2210 was experiencing severe stress, categorized as a downer cow from an undescribed illness on 8/4/2012. On 8/11/2012 cow #2210 was diagnosed with mastitis and then removed from the study. This could explain the decrease in levels and indicates Fb does not respond to increased body temperatures. More studies should be done to understand the relationship between Fb levels and common stress.
Total White Blood Cell Counts
A critical function of inflammation is to deliver leukocytes to the site of injury and infection and to activate the leukocytes to perform their normal functions in host defense.
Inconsistent numbers of blood samples made using our WBC data difficult. WBC at the organic farm showed counts to be above normal on sample time points 4 and 5 (Table 17). These increase counts could have been due to all conditions mentioned earlier for Hp and SAA or Fb as
WBC are first responders in all inflammation events, producing cytokines which trigger an APP response. WBC at the conventional farm were above normal limits at sample time points 1, 4 &
5 indicating an inflammation event before the pasture season and hot weather. As stated earlier health events such as calving and mastitis were recorded in health records for this farm. WBC differential data showed that the organic cows had a consistently higher N:L ratio than the conventional cows over time (Table 26). These cows had N:L ratios that were consistently 2%
75 above the normal range of .5. The conventional group only reached above normal N:L ratios on sample time point 2 and sample time point 4, indicating that the organic herd was under more stress than the conventional herd during the entire study whether they were in the barn or outside. As stated earlier, inconsistent numbers of samples due to various reasons and the lack of standardized health records made data evaluation difficult. A more tightly controlled study could yield different results.
76
CHAPTER V
CONCLUSIONS
Hp, SAA, Fb and WBC have been used successfully in the past to determine the health status in cattle and other animals including humans. Studies have also shown that APPs respond to stress as well as acute, sub‐acute or chronic inflammatory conditions. In this study, although
average concentrations for all health markers tested were within normal reference ranges for healthy cows at all sample time points, the low levels did indicate an immune system response.
For the purpose of identifying potential health differences between farms, the health markers tested showed that the organic and conventional cows differed significantly in sample week 3 with the organic farm showing higher levels at the start of the pasture season. The increases in levels that were seen especially in July and August were not statistically significant between farms but did show that even low stress situations elicits an APP response in both management systems that can help identify inadequate management related problems like inadequate ventilation or a need for shade. These results reinforce previous findings about the effect of stress factors on cows and measures should be taken by managers to identify and reduce these stressors, in both management systems as they can result in lower milk yields, illness or even death. This study also indicated that heat does not elicit an increase in Fb levels which confirms previous studies that this APP does not rise in response to increased body temperatures
While this study was designed to observe the effects of the environment, diet and management on cattle health, and the results reinforced past studies about the usefulness of
APPs as a tool to determine herd health, there are inherently many uncontrolled variables within farm systems that made it difficult if not impossible to isolate differences exclusively.
77 More tightly controlled studies with better standardized health record keeping, more samples over a longer period of time possibly focusing on one farm with more animals, or doing within farm crossover studies could result in more meaningful conclusions. In addition, new studies to reestablish or confirm baselines using newer more sensitive assays would strengthen the validity of findings in all future work where measuring these health markers to determine health in cattle is a goal.
78
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APPENDIX
Blood Marker Data Date Cow Site Week Fb Hp SAA WBC Neut Lymph 4/24/2012 18 1 1 209.673 0.106 0.105 13.5 46 47 4/24/2012 23 1 1 166.397 0.105 0.102 14.25 31 54 4/24/2012 24 1 1 168.293 0.104 0.1 4.6 23 62 4/24/2012 35 1 1 170.82 0.845 0.094 0 0 0 4/24/2012 36 1 1 167.977 0.114 0.096 8 45 50 4/24/2012 43 1 1 190.404 0.101 0.116 0 0 0 4/24/2012 46 1 1 192.931 0.107 0.09 10.25 28 66 4/24/2012 1059 1 1 219.465 0 0 8.5 51 35 4/24/2012 73 2 1 117.414 0.111 0.113 10.75 14 78 4/24/2012 74 2 1 189.282 1.478 0.101 5.75 16 76 4/24/2012 1001 2 1 137.762 0.105 0.132 17.25 24 67 4/24/2012 2086 2 1 118.28 0.114 0.116 9.25 29 63 4/24/2012 2209 2 1 173.263 0.112 0.118 0 0 0 4/24/2012 2210 2 1 188.416 0.111 0.107 10 35 56 4/24/2012 2356 2 1 241.893 0.101 0.112 10.25 43 49 4/24/2012 2378 2 1 232.732 0.352 0.105 14 46 45 5/29/2012 18 1 2 185.982 0 0 6 49 38 5/29/2012 23 1 2 217.886 0.102 0.107 6.25 52 34 5/29/2012 24 1 2 199.881 0.109 0.098 6.25 54 43 5/29/2012 35 1 2 202.408 0.112 0.112 6 45 38 5/29/2012 36 1 2 189.457 0.104 0.109 12 30 49 5/29/2012 43 1 2 191.352 0.893 0.116 8 30 62 5/29/2012 46 1 2 198.617 0.108 0.113 0 0 0 5/29/2012 1059 1 2 194.195 0.107 0.109 8.75 65 29 5/29/2012 73 2 2 228.31 0.108 0.093 8.25 27 58 5/29/2012 74 2 2 180.296 0.112 0.103 0 0 0 5/29/2012 1001 2 2 180.928 0.104 0.103 6.5 40 49 5/29/2012 2086 2 2 172.083 0.101 0.118 4 31 61 5/29/2012 2209 2 2 171.451 0.101 0.107 6.5 41 51 5/29/2012 2210 2 2 178.401 0.113 0.114 8.75 34 47 5/29/2012 2356 2 2 188.825 0.099 0.108 7.5 42 48 5/29/2012 2378 2 2 207.778 0.133 0.098 0 0 0 6/27/2012 17 1 3 176.506 0.111 0.425 9.75 29 52 6/27/2012 18 1 3 193.563 0.115 0.389 5.75 38 47 6/27/2012 24 1 3 185.034 0.132 0.469 5.75 47 43
96 6/27/2012 35 1 3 180.928 0.134 0.427 9 34 52 6/27/2012 36 1 3 187.246 0.126 0.421 12.75 24 47 6/27/2012 43 1 3 163.87 0.123 0.406 14.25 30 58 6/27/2012 46 1 3 160.711 0.126 0.416 0 0 0 6/27/2012 47 1 3 185.926 0.142 0.407 7.75 32 57 6/27/2012 1059 1 3 265.155 0.126 0.451 3.75 45 46 6/27/2012 73 2 3 206.514 0 0.099 4.75 28 58 6/27/2012 82 2 3 212.832 0.11 0.11 7.75 26 62 6/27/2012 84 2 3 194.195 0.104 0.108 16.5 19 61 6/27/2012 1001 2 3 186.298 0.111 0.116 10.75 22 59 6/27/2012 2086 2 3 173.979 0.102 0.117 5.75 30 59 6/27/2012 2209 2 3 173.979 0.106 0.106 0 0 0 6/27/2012 2210 2 3 179.979 0.11 0.101 11.25 40 48 6/27/2012 2356 2 3 186.298 0.107 0.104 15.5 29 51 6/27/2012 2378 2 3 194.195 0.117 0.107 4.25 37 58 7/24/2012 17 1 4 235.499 0.272 0.469 19 35 49 7/24/2012 18 1 4 161.139 0.134 0.441 9.5 35 41 7/24/2012 24 1 4 182.827 0.126 0.451 11 26 59 7/24/2012 35 1 4 184.598 0.147 0.445 9.25 34 36 7/24/2012 36 1 4 193.45 0.128 0.42 19.5 24 54 7/24/2012 43 1 4 169.106 0.127 0.434 18 27 54 7/24/2012 46 1 4 171.319 0.132 0.434 12 21 69 7/24/2012 47 1 4 259.843 0.134 0.416 9.5 40 44 7/24/2012 1059 1 4 275.335 0.134 0.466 10.25 39 38 7/24/2012 73 2 4 259.843 0.122 0.426 19 28 62 7/24/2012 82 2 4 208.499 2.76 0.466 19 47 40 7/24/2012 84 2 4 237.27 0.126 0.446 18.75 31 53 7/24/2012 1001 2 4 263.384 0.125 0.469 12.25 20 68 7/24/2012 2086 2 4 239.925 0.127 0.428 10.75 29 62 7/24/2012 2209 2 4 180.172 0.124 0.436 12 32 61 7/24/2012 2210 2 4 283.302 0.131 0.423 11.75 32 46 7/24/2012 2356 2 4 309.859 0.121 0.42 18 34 54 7/24/2012 2378 2 4 375.367 0.134 0.439 19 27 58 8/21/2012 17 1 5 166.644 1.776 0.492 24.25 27 57 8/21/2012 18 1 5 212.402 0.136 0.45 16.5 43 43 8/21/2012 35 1 5 251.215 0.125 0.439 17.5 22 51 8/21/2012 36 1 5 209.951 0.129 0.439 16.25 18 56 8/21/2012 47 1 5 189.115 0.129 0.338 14.5 37 41 8/21/2012 1059 1 5 183.395 0.146 0 22.25 33 34 8/21/2012 73 2 5 171.955 0.126 0.442 11.25 16 74 8/21/2012 82 2 5 166.236 0.129 0.45 11 27 61 8/21/2012 84 2 5 163.784 0.258 0.432 5.5 18 62 8/21/2012 2086 2 5 193.2 0.371 0.434 4.75 32 54
97 8/21/2012 2209 2 5 236.916 0.121 0.432 0 0 0 8/21/2012 2356 2 5 211.177 0.115 0.44 15.5 29 43 8/21/2012 2378 2 5 0 0.126 0.239 7.25 32 56
98 Institutional Care and Use committee protocol (IACUC # 120506).
99