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LSU Historical Dissertations and Theses Graduate School
1995 Dietary Chromium Supplementation for Pigs and Chickens. Terry Lynn Ward Louisiana State University and Agricultural & Mechanical College
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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 DIETARY CHROMIUM SUPPLEMENTATION FOR PIGS AND CHICKENS
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Animal Science
by Terry Lyim Ward B.S., Oklahoma State University, 1987 M.S., Louisiana State University, 1989 December, 1995 UMI Number: 9618333
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UMI 300 North Zeeb Road Ann Arbor, Ml 48103 DEDICATION
It is with great love and with warmest memories that I dedicate this volume o f work to the memory of Kaye Donna Newell (May 17, 1945 - March 19, 1995).
Kaye Newell was my mother-in-law and a fellow nutritionist. She tried with all sincerity to improve the nutritional health of her fellow human beings. Along the way I believe she was just as successful in improving the spiritual well-being of many individuals. May we all strive, in our lives, to be as helpful to others as Kaye
Newell was in her life. ACKNOWLEDGEMENTS
I would like to express my gratitude to my graduate committee (Dr. L.
Dwain Bunting, Dr. J. Marcos Fernandez, Dr. Michael Keenan, Dr. Donald L.
Thompson, Jr., and Dr. Gary E.Wise) for their assistance in my graduate program and their willingness to share the knowledge they possess. Special thanks are extented to my committee chair, boss, and mentor, Dr. L. Lee Southern. The knowledge of nutrition and appreciation for research that Dr. Southern has been able to instill in me is priceless. In addition, Dr. Southern has been a good friend; I will value his friendship always. There have been many other students, staff, and faculty that have been helpful in my graduate program. I will not take the pages it would require to list all of them. However, I will mention a few special individuals. For the first taste of excitement about research, I would like to thank Kevin Watkins, a true friend and colleague. Others that I owe special thanks for going beyond the call of duty include Angelica Chapa, Neal Matthews, Frederick "Chip” LeMieux, Dan
Howard, Laura Roland Gentry, Leeann Sticker, and Scott Boleman. Thanks to my family and other friends for their support. Thanks to my dad for the appreciation of hard work and my mom for the appreciation of education. Special thanks to
Tommy, my brother, who sacrificed so that I might have the opportunity to pursue the career I love. To Donnita, my wife and soul-mate, go the greatest thanks and love. Words cannot express the joy you have brought in to my life. Thanks to my kids, Cody and Leah, for making the long days and all-nighters worth it with
"Daddy’s Home!". Finally, thanks to God, for it is by His grace that I am what I am.
iv PREFACE
The following document is in partial fulfillment of the Doctor of Philosophy
degree. I believe this is true; no single document could contain all of the
components which measure an individuals worthiness to receive such a degree, if
such can be measured. I believe an individuals’ philosophy is made up of
experiences as well as higher-learning. Certainly, just as knowledge of a particular
field is learned, a person’s philosophy is also learned to a certain extent. The
components of my philosophy begin with God, my creator. Secondly, my mom and
dad, who taught me to work hard physically and mentally, have influenced my
philosophy of life. The words from Ecclesiastes 9:10,“Whatsoever thy hand findeth
to do, do it with thy might; for there is no work, nor device, nor knowledge, nor
wisdom, in the grave, whither thou goest”, have special meaning to me. On a lighter side, other occurrences and experiences have also influenced my philosophy.
One of the things I try to remember occasionally is the world according to Freddy from Jimmy Buffett’s Tales Fmm Margaritaville (pg 185). Lesson One: Never forget—they are always the enemy. Lesson Two: Just remember, [some people] are bom that way, and they usually don’t change. Lesson Three: You do not want to go to jail. Lesson Four: When you start to take this job seriously, you’re in trouble.
Lesson Five: It takes more time to see the good side of life than it takes to see the bad. Lesson Six: If you decide to run with the ball, just count on fumbling and
v getting the [stuffing] knocked out of you a lot, but never forget how much fun it is just to be able to run with the ball. Although I consider myself to be more refined than Freddy, the crude words do not discount their truthfulness in life. May the following document be just another component of the lifelong development of my philosophy and perhaps someone else’s as well. TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
PREFACE ...... v
LIST OF TABLES...... ix
LIST OF FIGURES...... xi
LIST OF ABBREVIATIONS ...... xii
ABSTRACT...... xv
CHAPTER
I. INTRODUCTION ...... 1
H. REVIEW OF IJTERATURE ...... 3 Introduction ...... 3 Genera] ...... 3 Growth Performance ...... 4 Body Composition ...... 5 Metabolism ...... 6
HI. EFFECT OF DIETARY CHROMIUM SOURCE ON GROWTH, CARCASS CHARACTERISTICS, TISSUE CHROMIUM CONCENTRATIONS, AND PLASMA METABOLITE AND HORMONE CONCENTRATIONS IN GROWING-FINISHING PIGS ...... 16 Introduction ...... 16 Materials and Methods ...... 17 Results ...... 23 Discussion ...... 35 Implications ...... 42 IV. EFFECT OF DIETARY CHROMIUM SOURCE ON GLUCOSE TOLERANCE AND INSULIN SENSITIVITY IN GROWING PIGS . . . 43 Introduction ...... 43 Materials and Methods ...... 44 Results and Discussion...... 49 Implications ...... 60
V. INTERACTIVE EFFECTS OF DIETARY CHROMIUM TRIPICOUNATE AND CRUDE PROTEIN LEVEL IN GROWING- FINISHING PIGS PROVIDED INADEQUATE AND ADEQUATE PEN SPACE: EFFECTS ON GROWTH PERFORMANCE, CARCASS CHARACTERISTICS, AND PLASMA METABOLITES AND HORMONES ...... 61 Introduction ...... 61 Materials and Methods ...... 62 Results...... 67 Discussion ...... 74 Implications ...... , ...... 79
VI. EFFECT OF DIETARY CHROMIUM TRIPICOUNATE ON GROWTH PERFORMANCE, BODY COMPOSITION, PLASMA METABOLITES AND HORMONES, AND GLUCOSE TOLERANCE AND INSULIN SENSITIVITY IN BROILERS...... 80 Introduction ...... 80 Materials and Methods ...... 81 Results...... 95 Discussion ...... 107 Implications ...... 117
VH. OVERALL SUMMARY AND CONCLUSIONS...... 118
REFERENCES...... 120
VITA 155 LIST OF TABLES
3.1 Composition of the basal diets, as-fed b a sis ...... 18
3.2 Effect of dietary chromium source on growth performance and carcass characteristics of growing-finishing p i g s ...... 25
3.3 Effect of dietary chromium source on serum glucose, NEFA, insulin, and growth hormone concentrations in growing-finishing p ig s ...... 27
3.4 Effect of dietary chromium source on serum urea nitrogen, cholesterol, and triacylgycerol concentrations of growing-finishing p ig s ...... 29
3.5 Effect of dietary chromium source on tissue chromium, copper, zinc, and iron concentrations of p ig s ...... 32
4.1 Composition of the basal diet, as-fed basis ...... 45
4.2 Effect of dietary chromium source on daily gain, feed intake, and gain/feed in growing barrows (Trial 1 and Trial 2 com bined) ...... 50
4.3 Effect of dietary chromium source on plasma metabolites and hormones in growing barrow s ...... 51
4.4 Effect of dietary chromium source on glucose kinetics in growing barrow s ...... 53
4.5 Effect of dietary chromium source on insulin kinetics in growing barrow s ...... 54
5.1 Composition of the basal diets, as-fed b a sis ...... 63
5.2 Effect of dietary chromium, crude protein level, and pen space on growth performance and carcass characteristics of growing-finishing p ig s ...... 68
ix 5.3 Effect of dietary chromium, crude protein level, and pen space on plasma glucose, NEFA, insulin, and growth hormone concentrations of growing-finishing p i g s ...... 72
6.1 Composition of the basal diet, as-fed basis (Exp. 1 ) ...... 82
6.2 Composition of the basal diet, as-fed basis (Exp. 2 ) ...... 85
6.3 Composition of the basal diets, as-fed basis (Exp. 3 ) ...... 87
6.4 Composition of the basal diets, as-fed basis (Exp. 4 ) ...... 90
6.5 Effect of dietary chromium on growth performance, N balance, and breast and leg muscle and whole-body composition of 5 to 19-day-old broilers (Exp. 1 ) ...... 96.
6.6 Effect of dietary chromium on growth performance, organ weights, body composition, and plasma metabolites and hormones of 5 to 33-day-old broilers (Exp. 2) . . . 97
6.7 Effect of dietary chromium on growth performance of 5 to 54-day-old broilers (Exp. 3 ) ...... 100
6.8 Effect of dietary chromium on organ and tissue weights of 5 to 54-day-old broilers (Exp. 3 ) ...... 102
6.9 Effect of dietary chromium on plasma metabolites and hormones of 5 to 54-day-old broilers (Exp. 3) ...... 103
6.10 Effect of dietary chromium on body composition of 5 to 54-day-old broilers (Exp. 3 ) ...... 105
6.11 Effect of dietary chromium on growth performance, and glucose and insulin clearance in broilers (Exp. 4) ...... 108
x LIST OF FIGURES
4.1 Effect of dietary chromium on glucose tolerance in pigs during an intravenous glucose tolerance t e s t ...... 55
4.2 Effect of dietary chromium on insulin clearance in pigs during an intravenous glucose tolerance te st ...... 56
4.3 Effect of dietary chromium on glucose clearance in pigs during an intravenous insulin challenge t e s t ...... 57
4.4 Effect of dietary chromium on insulin clearance in pigs during an intravenous insulin challenge t e s t ...... 58
6.1 Effect of dietary chromium on glucose clearance in broilers during an intravenous glucose tolerance te s t ...... 109
6.2 Effect of dietary chromium on insulin clearance in broilers during an intravenous glucose tolerance te s t ...... 110
6.3 Effect of dietary chromium on glucose clearance in broilers during an intravenous insulin challenge t e s t ...... I l l
6.4 Effect of dietary chromium on plasma glucose concentration in broilers during a least-sampling minimal model (LSMM) glucose tolerance test ...... 112
6.5 Effect of dietary chromium on plasma glucagon concentration in broilers during a least-sampling minimal model (LSMM) glucose tolerance te s t ...... 113
xi LIST OF ABBREVIATIONS
ADFI average daily feed intake
ADG ...... average daily gain
B W body weight
Ca ...... calcium
C P crude protein
Cr ...... chromium
CrAc chromium acetate
CrCl chromium chloride hexahydrate
C rN G C G ...... chromium-nicotinate-glutamate-cysteine-glycine complex
C rN ic ...... chromium nicotinate
CrOx chromium oxalate
CrPic ...... chromium tripicolinate
Cu ...... copper
CV ...... coefficient of variation
D a rk ...... leg plus thigh meat
DM ...... dry matter
DP ...... dressing percentage
EDTA ...... ethylenediaminetetraacetic acid
Fe ...... iron
FSH ...... follicle stimulating hormone
xii GH ...... growth hormone
G L M general linear model
GTF ...... glucose tolerance factor
G u t ...... digestive tract plus organs
h ...... hour
HDL ...... high density lipoprotein
I ...... iodine
IVGTT ...... intravenous glucose tolerance test
IVICT ...... intravenous insulin challenge test
1 c ...... clearance rate
LBM ...... lean body mass
LDL ...... low density lipoprotein
LH ...... luteinizing hormone
LMA ...... longissimus muscle area
LSD ...... least significant difference
LSMM ...... least sampling minimum model m in ...... minute
M n ...... manganese
N ...... nitrogen
NEFA ...... nonesterified fatty acid
PBS ...... phosphate buffered saline p G H ...... porcine growth hormone
PM ...... percentage of muscling p p b ...... parts per billion ppm ...... parts per million ppS T ...... porcine pituitary somatotropin
RIA ...... radioimmunoassay
RNA ...... ribonucleic acid
S e ...... selenium
TRF ...... tenth rib fat
TPN ...... total parenteral nutrition
T t n...... half-life w k ...... week
W h ite...... breast meat
Zn ...... zinc ABSTRACT
Three experiments were conducted with pigs and four experiments were
conducted with chickens to determine the effects of supplemental dietary Cr on
growth performance, carcass characteristics, tissue minerals and plasma metabolite
and hormone concentrations. In pigs (Exp. 1) 200 ppb Cr from seven different
sources tended to decrease growth performance during the grower phase and
increase growth performance during the finisher phase. Dietary Cr had minimal
effects on plasma metabolite and hormone concentrations and Cr did not affect
carcass characteristics. In general, dietary Cr increased tissue Cr concentration,
except for longissimus muscle (LM). In Exp. 2, pigs were fed 400 ppb Cr as Cr
chloride, Cr nicotmate, or Cr picolinate (CrPic) to determine the effect of these Cr
sources on i.v. glucose tolerance and insulin sensitivity. None of the Cr sources
affected glucose clearance rate or half-life, or insulin clearance rate or half-life. In
Exp. 3, the interactive effects of two levels of dietary lysine, two levels of dietary
Cr, and two levels of pen space were evaluated. During the finisher phase, Cr increased average daily gain and gain/feed of pigs fed 80% of the lysine requirement but not in pigs fed 120% of the lysine requirement. In addition, Cr decreased tenth rib fat thickness (TRF) in pigs given adequate pen space but increased TRF of pigs given inadequate pen space. Dietary Cr supplementation did not affect LM area.
Three experiments were conducted to determine the effect of CrPic on growth performance, plasma metabolites and hormones, and carcass composition of
xv broilers. In broilers fed CrPic for two weeks, Cr decreased percentage of dry
matter of the carcass and tended to decrease percentage carcass fat. In broilers fed
CrPic for four weeks, Cr supplementation increased ventriculus weight and
increased carcass ash percentage. In broilers fed CrPic for seven weeks, Cr
decreased abdominal fat pad weight. Dietary Cr had minimal effects on plasma
metabolites and hormones in broilers. In a fourth experiment with broilers, CrPic did not affect glucose tolerance or insulin sensitivity. Under the conditions of these experiments dietary Cr did not affect body composition and metabolism of pigs and broilers. CHAPTER I
INTRODUCTION
Dietary chromium (Cr) has been reported to have beneficial effects in many aspects of the metabolism of man and animals. Chromium is believed to function by facilitating the action of insulin. Some of the historic findings in Cr nutrition include improvements in glucose tolerance and insulin sensitivity, as well as plasma lipid status, in man and animals. Recently some researchers have reported dramatic improvements in body composition of growing-finishing pigs that were fed Cr tripicolinate (CrPic), an organic source of Cr, but not pigs that were fed Cr chloride
(CrCl), an inorganic source of Cr. Specifically, these researchers have reported increased longissimus muscle area and decreased tenth rib fat thickness in pigs fed
CrPic. Other researchers have reported increased clearance rate of glucose in pigs fed CrPic.
Additional research, primarily in ruminants, suggests that Cr improves the immune status of livestock. This research indicated that it was necessary for an animal to be stressed to elicit a response to Cr supplementation. If Cr is to become a feed additive for the feed industry in the United States (Cr is approved for use in some foreign countries), then the specific conditions (environment, etc.) in which Cr is beneficial to the animal need to be characterized. In addition, forms of Cr
(organic and inorganic) other than CrPic and CrCl need to be evaluated. Therefore, the following experiments were conducted to evaluate the effects of form of Cr and environmental stress (decreased pen size) on growth performance, carcass characteristics, and metabolic status of growing-finishing pigs. In addition, experiments also were conducted to evaluate the effect of CrPic in broiler diets growth performance, body composition, and glucose tolerance and insulin sensitivity. CHAPTER n
REVIEW OF LITERATURE
Introduction
There have been many excellent reviews (Anderson, 1981, 1986, 1989;
Ducros, 1992; Hambidge, 1974; Mertz 1969, 1993; M ertzetal., 1974; Offenbacher and Pi-Sunyer, 1988; Saner, 1986; Wallach, 1985) and book chapters (Anderson,
1987; Anderson, 1988a,b; Canfield, 1979; Doisy etal., 1976; Linder, 1985; Mertz,
1975, 1979, 1982; Saner, 1979) written about Cr nutrition. Certainly the most comprehensive review of the Cr literature was by Mertz (1969). Many of the aspects that were covered in that review will not be covered in the current review.
Some of these topics include the chemistry of Cr, Cr in plants, and Cr analysis methodology. These topics are important but not the primary focus of the current body of research. Many of the reviews that have been written segregate human from animal literature. The present review will include the current pertinent literature regarding Cr in man and animals. Specifically, this review will concentrate on the effects of Cr on growth performance, body composition, metabolism, and immune function.
General
The essentiality of Cr in humans has been well documented. The most convincing evidence was the dramatic recovery of TPN patients that displayed signs of diabetes (Jeejeebhoy et al., 1977). In fact, the suggested safe and adequate daily
intake for Cr is SO to 200 pig, as established by a panel of the United States National
Academy of Sciences (NAS, 1980). However, the typical diet does not meet the
suggested daily intake (Anderson and Kozlovsky, 1985). Foods that contain higher
concentrations of Cr typically are not eaten in large quantities (e.g., mushrooms,
brewer's yeast, black pepper, prunes, raisins, nuts; Anderson, 1988). Refined foods
have lower amounts of Cr than unrefined foods (Anderson, 1988). Recent evidence
suggests that animal foodstuffs also may not contain adequate Cr to meet the
animals’ requirement (Page et al., 1993).
Growth Performance
The literature regarding the effects of dietary Cr on growth performance of
animals is equivocal. Early work by Schroeder et al. (1963a,b) showed that dietary
Cr improved the growth rate of mice and rats. However, more recent data with
livestock species has yielded variable results. In a publication which contained
multiple experiments, Page et al. (1993) reported increased, decreased, and no change in growth rate of pigs due to dietary Cr supplementation as chromium
tripicolinate (CrPic). Lindemann et al. (1993) reported that pigs fed marginal lysine
(or crude protein) diets had improved growth performance and feed efficiency in response to CrPic supplementation; however, pigs fed diets adequate in lysine did not have improved growth performance when fed CrPic. Other investigators have reported no effect of dietary Cr supplementation on growth performance of pigs (Page et al., 1993a; Renteria and Cuardn, 1993), lambs (Kitchalong et al., 1995), calves (Bunting et al., 1994), or steers (Claeys et al., 1994).
Body Composition
The work that initiated the most recent thrust of Cr research was the finding by Page et al. (1993b) that dietary Cr supplementation as CrPic (an organic source of Cr) dramatically improved body composition of pigs. Specifically, supplementation of typical com-soybean meal diets with 200 to 400 ppb Cr as CrPic increased longissimus muscle area and decreased tenth rib fat thickness. These two carcass traits are of significant economic value to an industry that is trying to maintain (or gain) market share in an increasingly competitive environment.
Furthermore, the necessity of the organic form of Cr for improved body composition of pigs was discovered (Page et al., 1993a,b).
After the initial data of Page et al. (1993b), other investigators have reported similar improvements in body composition due to dietary Cr supplementation from organic sources. Lindemann et al. (1995) reported decreased tenth rib backfat thickness and increased longissimus muscle area in pigs fed CrPic. Other authors have reported more subtle differences in body composition. Boleman et al. (1995) and Mooney and Cromwell (1995) both reported little to no effect of dietary Cr as
CrPic on the linear carcass measurements of pigs. However, carcass dissection and whole-body composition analysis revealed that dietary Cr increased the rate of lean deposition and decreased the rate of fat deposition in pigs. Smith et al. (1994) also reported decreased backfat thickness of pigs fed Cr nicotinate (CrNic).
Kitchalong et al. (1995) reported that dietary Cr as CrPic supplementation decreased fat thickness over the tenth rib of lambs. Conversely, Claeys et al. (1994) reported higher quality grades for steers fed CrNic (thus increased intermuscular fat) and decreased ribeye area of steers fed 400 ppb Cr as CrCl or 800 ppb Cr as CrNic.
Hasten et al. (1992) reported that female students that were beginning weight training had increased body weight (BW), primarily due to increased lean body mass
(LBM). However, male students showed no change in BW or body composition due to Cr supplementation. Evans (1989) reported that male football players had increased BW, LBM, and calf circumference when they combined dietary Cr supplementation with weight training. However, Alniada et al. (1995) reported no beneficial effect of a nutritional supplement that contained Cr as CrPic on males during resistance training. Other authors have reported increased LBM (Hasten et al., 1993) and decreased body fat (Hasten et al., 1994, 1995) in rats.
Metabolism
Introduction. The reason for the increased lean and decreased fat in the bodies of humans and carcasses of animals given supplemental dietary Cr is not known.
However, Cr is known to be involved in a myriad of steps of metabolism, including protein, carbohydrate, and lipid metabolism. Chromium is thought to exert its effects through the potentiation of insulin action. However, the anabolic nature, and more specifically, the lipogenic nature of insulin makes the proposed mechanism of action of Cr more puzzling. The mystery probably is confounded by the fact, that after decades of research, the cellular mechanism of action of insulin is not well established.
Erotein. Apart from the multitude of reports from around the world of increased muscling of animals and humans given supplemental dietary Cr, little research has been conducted to determine the cellular or molecular mechanisms behind this occurrence. Britton et al. (1968) reported that lambs fed molasses or Cr as CrCl had increased N utilization. This would suggest that changes in lean composition that have since been reported by other authors also may have occurred; body composition was not determined in that experiment. Other investigators have attempted to study the cellular mechanism of action of Cr. Ono et al. (1994) reported that supplemental dietary Cr for pigs injected with porcine somatotropin resulted in increased muscle fiber area compared to pigs injected with somatotropin only. Dietary Cr alone did not affect muscle fiber area. Morris et al. (1994) reported no effect of dietary Cr supplementation on cardiac growth, protein, or myofibrillar protein content. Hasten et al. (1995) reported that rat skeletal myoblasts had increased 3H-leucine uptake, suggesting perhaps an enhancement of protein synthesis. Rats treated with an intraperitoneal dose of Cr as CrCl had increased nucleolar and ribosomal RNA synthesis (Okada et al., 1984). However, the true implications of Cr for protein synthesis are not known. Lipid. Low serum Cr has been correlated with coronary artery disease in
humans (Newman et al., 1978; Schroeder et al., 1970; Simonoff et al., 1984).
Riales (1979) reported that brewer's yeast (containing Cr) increased HDL- cholesterol and decreased LDL-cholesterol without affecting total serum cholesterol.
These findings are consistent with others that have postulated that Cr is responsible
for such effects (Riales and Albrink, 1981; Elwood et al., 1982; Roeback et al.,
1991; Abraham et al., 1992). In fact, Abraham et al. (1980; 1991) reported that Cr supplementation of rabbits reduced the surface area of aortic plaques that had been induced by feeding cholesterol.
Press et al. (1990) reported that supplemental CrPic decreased total cholesterol, LDL-cholesterol, and apolipoprotein B (the principal protein of the LDL fraction). Supplemental Cr as CrPic also increased apolipoprotein A-l, the principal protein of the HDL fraction, but did not significantly affect HDL-choIesterol concentration. However, Hermann et al. (1995) reported that Cr as CrCl was effective in lowering serum total cholesterol but increased apolipoprotein B in adults over age fifty. Lefavi et al. (1991) reported that a nicotinic acid-chromium complex was effective in reducing serum total cholesterol and increasing the total cholesterol:HDL-cholesterol ratio in male athletes. In addition, Schroeder (1968) and Schroeder et al. (1971) reported that supplemental Cr reduced serum total cholesterol in rats and Kitchalong et al. (1995) reported that supplemental Cr reduced serum cholesterol and NEFA in lambs. In addition, CrPic supplementation has been reported to decrease serum total cholesterol concentrations in pigs (Page et al., 1993b), lambs (Kitchalong et al., 1995), and calves (Bunting et al., 1994).
However, Rabinowitz et al. (1983) reported that CrCl, brewer's yeast containing glucose tolerance factor (GTF), and brewer’s yeast without GTF did not affect plasma total cholesterol or triglycerides in diabetic men and Amoikon et al. (1995) reported that CrPic increased serum total cholesterol in pigs. However, it is possible that changes in the lipoprotein fractions did occur in that study but were not detected (i.e., they were not measured). Other investigators also have reported a lack of effect of dietary Cr on lipid measurements in humans (Gondy and Wilson,
1994; Thomas and Gropper, 1995), chickens (Maurice and Jensen, 1978), calves
(Bunting et a)., 1994), and lambs (Kitchalong et al., 1995).
Glucose and Insulin. It is difficult to separate the effect of Cr on glucose and insulin metabolism. However, the glucose and insulin responses to dietary Cr will be separated. One of the first defined roles of Cr in mammals was the alleviation of hyperglycemia in rats (Mertz et al., 1961, 1965, 1969; Schroeder, 1966). The most striking evidence for the efficacy of Cr in alleviating hyperglycemia is in total parenteral nutrition (TPN) patients (Jeejeebhoy et al., 1977; Freund et al., 1979).
These patients required insulin injections to lower their blood glucose levels.
However, following the addition of Cr to the TPN solution, the requirement for insulin was dramatically decreased, if not completely negated. The symptoms of diabetes also were alleviated. Further evidence of a role for Cr in glucose 10 homeostasis was found for Turner’s syndrome patients. Supplemental Cr was
effective in decreasing hyperglycemia in these patients (Saner et al., 1983).
Other investigators have reported equivocal responses from supplementing the
diets of the elderly with Cr. Offenbacher and Pi-Sunyer (1980) reported that
chromium-rich brewer’s yeast improved glucose tolerance of elderly subjects.
Similarly, Potter et al. (1985) reported that Cr as CrCl was effective in lowering the
plasma glucose response to an oral glucose tolerance test, but plasma insulin levels
were not affected. Bourn et al. (1985) reported that Cr supplementation was
effective in lowering plasma glucose and insulin concentrations in post-menopausal
women. However, Martinez et al. (1985) and Offenbacher et al. (1985) reported
that supplemental Cr did not change glucose tolerance or insulin status in elderly
subjects. Mossop (1983) and Vinson and Bose (1984) reported that diabetic patients
had lower fasting plasma glucose concentrations when they received supplemental
dietary Cr. However, other investigators have found no response to supplemental
Cr in diabetic patients (Sherman et al., 1968; Uusitupa et al.t 1983, 1992). The
reasons for these different results are not clear. Some investigators have suggested
that only those patients with abnormal glucose tolerance will respond to Cr
supplementation. However, previously in the text it was noted that diabetic patients did not always respond to Cr supplementation. The unknown factors of diabetes and its associated complications makes it a challenging subject. Glinsmann and Mertz
(1966) reported that diabetic patients responded to Cr supplementation, but patients with normal glucose tolerance did not. Freiberg et al. (1975) reported that diabetic
or “doubtful11 (marginal diabetics) patients had lowered plasma glucose after
supplementation with brewer’s yeast, presumably due to its Cr content. Anderson et
al. (1991), Uu et al. (1977, 1978), and Polansky et al. (1991) reported similar
findings comparing patients with normal and abnormal glucose tolerance. Anderson
et al. (1985, 1987) reported that both hypoglycemic and hyperglycemic subjects had
improved glucose status when they received supplemental Cr. However, Evans
(1989) and Levine et al. (1968) reported that some patients with abnormal glucose
tolerance responded to Cr supplementation, but other patients with abnormal glucose tolerance did not respond to Cr supplementation. The unknown cause(s) of
"responders” and "nonresponders" further complicate the study of Cr nutrition in man and animals.
The animal data with glucose response to Cr supplementation are just as variable as the human data. Some investigators have found that supplemental Cr decreased plasma glucose concentrations, whereas others have reported no effect of supplemental Cr on glucose status of animals. Stoecker and Oladut (1985) reported that Cr supplementation lowered the plasma glucose response 60 min after a glucose tolerance test in guinea pigs. This may indicate that glucose was cleared from the plasma at a foster rate; however, clearance rate was not determined. Similarly,
Amoikon et al. (1995) reported that supplemental Cr as CrPic increased the clearance rate and decreased the half-life of glucose from the plasma of pigs during an intravenous glucose tolerance test (IVGTT). In a like fashion, Cr as CrPic has
been reported to increase glucose clearance rate in calves (Bunting et al., 1994) and
lower glucose response to IVGTT in lambs (Kitchalong et al., 1995). Evock-Clover
et al. (1993) also reported that supplemental dietary Cr as CrPic was effective in
“normalizing” the plasma glucose levels of pigs injected with porcine somatotropin.
However, other investigators have found no effect of supplemental Cr on the glucose
tolerance of steers (Claeys et al., 1994), lambs (Fomea et al., 1994; Sam sell and
Spears, 1989), or hens (Jensen et al., 1978).
Some investigators have suggested that Cr directly affects the endocrine
pancreas. However, there is a lack of agreement about the direction of these effects.
Ghafghazi et al. (1980) reported that Cr decreased insulin secretion in isolated
pancreatic islets. However, Strifrier et al. (1993) reported that dietary Cr enhanced
insulin secretion in perfused rat pancreas. Hubner et al. (1989) reported that dietary
Cr decreased insulin response to an i.p. glucose tolerance test in gestating rats.
Furthermore, the insulin content of the entire pancreas of these dams was decreased,
but the insulin content of the entire pancreas from the neonatal rats (from the Cr
supplemented dams) was increased. It is interesting that these rats supplemented with Cr did not have a change in their glucose tolerance, yet insulin secretion was obviously affected by Cr.
Some investigators have reported that supplemental dietary Cr differentially affects insulin binding in tissues. Ward et al. (1994) reported that dietary Cr as 13
CrPic decreased insulin binding to plasma membranes of pig liver cells. Berrio et
al. (1995), from the same laboratory, reported that dietary Cr as CrPic increased
insulin binding in isolated pig fat cells and erythrocyte membranes. These data may
indicate that Cr decreases glucose utilization by the liver, and therefore more
glucose is available for uptake by peripheral tissues. However, Steele et al. (1982)
reported that dietary Cr as CrCl did not affect insulin binding in hepatic tissues.
The discrepancy between these findings and those of Ward et al. (1994) might be
explained by the difference in forms of dietary Cr. Ward et al. (1994) used an
organic source of Cr (CrPic).
Other investigators have indicated that Cr may act on the enzymes of
gluconeogenesis and glycolysis. Shiau and Chen (1993) reported that supplemental
dietary Cr lowered glucose-6-phosphatase activity in tilapia, but phosphofructokinase
activity was higher in tilapia supplemented with Cr. These same investigators found
that Cr supplementation increased weight gain, energy deposition, and liver glycogen content in fish fed glucose as a carbohydrate source. Campbell et al.
(1989) reported that dietary Cr enhanced liver glycogen synthase activity in sedentary rats but not in exercise-trained rats.
Stress and Immune Function. Some investigators have postulated that some type of "stress” must be imposed before a subject will respond to Cr supplementation (Anderson, 1994; Waddington, 1994). Indeed, Mertz and Roginski
(1967) found that rats fed a low-Cr, low-protein diet had depressed growth and this growth depression was alleviated by supplementing the diet with Cr. Subjecting the rats to exercise or excessive bleeding also improved growth and survivability in response to supplemental Cr. Exercise, physical trauma, and high sugar intake have been reported to increase Cr losses in man (Anderson, 1982, 1988, 1989). Other investigators also have shown a negative correlation between plasma Cr and plasma glucose levels (Morris et al., 1988a,b).
The relationship between Cr status and glucose utilization may be further strained by physiological stress. Pekarek et al. (1975) reported that acute infection with sandfly fever dramatically decreased serum Cr levels and glucose disappearance rates in humans. Investigators in the animal sciences also have indicated that supplemental Cr may be beneficial in the presence of stressors. Chang and Mowat
(1992) reported that transport-stressed feeder calves had reduced serum cortisol when they were provided supplemental dietary Cr. In addition, supplemental dietary
Cr increased serum immunoglobulin M and total immunoglobulins in calves fed diets with soybean meal but had no effect in calves fed urea-com supplementation.
Mowat et al. (1993) and Wright et al. (1993) reported that dietary Cr decreased morbidity in transport-stressed feeder calves. The improvements in gain and morbidity have been linked to immune status of these animals. Moonsie-Shageer and Mowat (1993) reported that supplemental dietary Cr decreased morbidity and increased antibody titers and immunoglobulin Gj in stressed feeder calves.
Supplemental Cr also increased indicators of cell-mediated immunity in periparturient dairy cows and in dairy cows during early lactation (Burton et al.,
1993). Other investigators have noted positive effects of Cr supplementation on immune status of calves (Chang et al., 1994; Lindell et al., 1994; Wright ct al. ,
1994) and pigs (van Heugten and Spears, 1994). CHAPTER n i
EFFECT OF DIETARY CHROMIUM SOURCE ON GROWTH, CARCASS CHARACTERISTICS, TISSUE CHROMIUM CONCENTRATIONS, AND PLASMA METABOLITE AND HORMONE CONCENTRATIONS IN GROWING-FINISHING PIGS
Introduction
Page et al. (1993a) reported no effect of Cr chloride hcxahydrate (CrCl) on body composition or metabolites in pigs; however, Cr tripicolinate (CrPic) was reported to decrease tenth rib backfat thickness and increase longissimus muscle area
(Page et al., 1993a,b). Other authors have also reported improvements in body composition of swine from supplemental dietary CrPic (Renteria and CuanSn, 1993;
Mooney and Cromwell, 1994; Boleman et al., 1995; Lindemann et al., 1995;
Mooney and Cromwell, 1995). At least one possible reason for Ihe difference in response to supplemental dietary Cr may be the form of Cr. The chemical composition of Cr compounds has been reported to affect the biological activity of these compounds (Evans and Pouchnik, 1993). Page et al. (1993b) reported no effect of CrCl, picolinic add, or the combination of CrCl and picolinic add on body composition of pigs. However, the chemical complex of Cr and picolinic acid in
CrPic increased the proportion of muscle and decreased the proportion of fat in pigs
(Page et al., 1993a,b). Thus it appeals that the complex of Cr and picolinic acid (or perhaps some other organic molecule) is necessary for Cr to elicit beneficial effects on carcass composition of pigs. Therefore, the objective of this experiment was to
16 17 determine the effects of different Cr sources (inorganic and organic complexes) on growth, carcass characteristics, metabolite and hormone concentrations, and tissue mineral concentrations in growing-finishing pigs.
Materials and Methods
Animals and Dietary Treatments
Crossbred (Yorkshire x Hampshire x Duroc) pigs were used [avg. initial body weight (BVV)=18.1 kg]. The pigs were allotted to treatment on the basis of BW in a randomized complete block design; ancestry was equalized across treatments.
Each treatment was replicated four times with four pigs per replicate pen. There were four pens of four pigs each which were fed a control diet (no Cr supplementation). Sex (two gilts and two barrows) was equalized within pen.
The pigs were fed a com-soybean meal basal diet (Table 3.1) formulated to provide 120% of the (NRC, 1988) lysine requirement during both the grower and finisher phases of production. The dietary treatments consisted of an unsupplemented control diet (Basal) and the Basal diet supplemented with 200 n z
Cr/kg diet as either chromium chloride hexahydrate (CrCl; Sigma Chemical Co.,
St.Louis, MO), Cr acetate (CrAc), Cr oxalate (CrOx), Cr nicotinate (CrNic), Cr tripicolinate (CrPic-1; USDA source), Cr nicotinate-glycine-cysteine-glutainate complex (CrNGCG), or Cr tripicolinate (CrPic-2; commercial source, Nutrition 18
Table 3.1. Composition of the basal diets, as-fed basis
Item Grower Finisher
%
Com 73.28 80.38
Soybean meal, 44% CP 24.72 17.90
Defluorinated rock phosphate .75 .33
Oyster shell flour .70 .84
Salt .25 .25
Vitamin premix* .25 .25
Trace mineral premix” .05 .05
‘Provided the following per kilogram of diet: riboflavin, 4.4 mg; d- pantothenic acid, 22 mg; niacin, 22 mg; vitamin B12, 22 /ig; d-biolin, 220 /ig; choline chloride, 440 mg; vitamin A, 4400IU; vitamin D3, 4401U; vitamin £,11
1U; menadione dimethylpyrimidinol bisulfite, .25 mg; S e ,. 1 mg.
’’Provided the following per kilogram of diet: Zn, 75 mg; Fe, 87.5 ntg; Mil,
30 mg; Cu, 8.75 mg; I, 1 mg; Ca, 9 mg. 21, San Diego, CA). Chromium acetate, CrOx, CrNic, CrPic-1, and CrNGCG were
supplied by Dr. Richard Anderson, Nutrient Requirements and Function Laboratory,
USDA, Beltsville, MD. These particular compounds were chosen based on the
ability to obtain them, the use of some of them in previous research (CrPic, CrNic,
and CrCl), and the need to evaluate additional organic compounds. The pigs were
allowed to consume the treatment diets on an ad libitum basis. During the grower
phase of production the pigs were penned in 1.8-m x 2.4-m pens in an enclosed
facility with totally slatted floors. During the finisher phase of production the pigs
were penned in l.S-m x 2.9-m pens in a curtain-sided facility with totally slatted
floors. Pigs were switched to finisher diets when the average BW of a block was
approximately S3 kg. This experiment was conducted from January through April
o f 1988.
Blood Sampling
All pigs were bled via the anterior vena cava following 20 h without access to
feed and then again at 2 h postfeeding on days 21 and 77 of the experiment. Blood
for serum glucose and nonesterified fatty acid (NEFA) analysis was placed into
tubes containing IS mg sodium fluoride, whereas blood for other serum metabolite
and hormone analyses was placed into tubes without the glycolytic inhibitor. The
blood samples were centrifuged for 20 min at 1,600 X g at 4°C. Serum was collected and frozen (-20 °C) for later analyses. 20
Carcass Traits
Pigs were removed from the experiment when their BW was approximately
100 kg (actual final average BW = 98.1 kg). Pigs were killed via stunning
(electrical or captive bolt) followed by exsanguination at the Louisiana State
University Agricultural Center Animal Science Department Meats Laboratory or a
commercial facility. Hot carcass weights were taken with heads on the carcass and
leaf fat in the carcass. Longissimus muscle area (LMA) at the tenth .rib interface
and tenth rib fat (TRF) thickness three-fourths the distance from the midline were
measured after approximately 20 h at 2°C. Longissimus muscle area and TRF were adjusted to 104.3 kg (National Swine Improvement Federation, 1987), and percentage of muscling (PM) was calculated by methods described by the National
Pork Producers Council (1991).
Tissue samples were collected from common sections of the liver, kidney, pancreas, and heart of two pigs from two replicate pens (four replicate pens for CrCl and CrPic-2 treatments) at the time of slaughter for tissue Cr, Zn, Cu, and Fe analyses. In addition, the tenth rib portion of the longissimus muscle was removed from the same pigs for Cr, Zn, Cu, and Fe analyses.
Chemical^Analyses
Serum was analyzed for glucose (Sigma Chemical Co, 1990) and NEFA
(NEFA-C Kit, ACS-ACOD Method; Wako Chemicals USA, Inc., Richmond, VA) concentrations using spectrophotometric procedures outlined in commercially- 2 1
available kits. Serum was analyzed for total cholesterol, urea N, and triacylglycerol
concentrations by automated “centrifichem" procedures (conducted by USDA
personnel, Nutrient Requirements and Function Laboratory, Beltsville, MD). Tissue
mineral analysis was conducted according to the procedure of Hill et al. (1986)
(conducted by USDA personnel, Nutrient Requirements and Function Laboratory,
Beltsville, MD).
Serum insulin was assayed by the RIA method of Amoikon et al. (1995).
Guinea pig anti-bovine insulin antiserum (Code No. 65-101, Lot No. GP616; ICN
ImmunoBiologicals, Lisle, IL) was used at a 1:60,000 final dilution. Purified porcine insulin (26.1 /tU/ng; Sigma Chemical Co.) and bovine ,2SI-insulin (ICN
Biomedicals, Inc., Costa Mesa, CA) were used as the standard and radioligand, respectively. Sheep anti-guinea pig antiserum produced in our laboratory was used at a 1:4 dilution as the precipitating antibody. The intraassay CV for the insulin
RIA as determined by a pooled bovine plasma sample was 7.7%.
Serum GH was measured by a double-antibody RIA technique based on antiserum (AFP-10318545; A.F. Parlow, Pituitary Hormone and Antisera Center,
Harbor-UCLA Medical Center, Torrance, CA) against porcine GH (pGH). Highly purified pGH was radioiodinated via the chloramine-T method (Greenwood et al.,
1963; 2.5 ng of chloramine-T//ig of GH, 0 °C for 30 sec). The antiserum was diluted 1:150,000 in PBS containing .033 M EDTA, .112% normal rhesus monkey serum (Calbiochem, San Diego, CA), and 33% noninhibitory horse serum; 300 piL 2 2
of this solution was used per tube. The horse serum was added to minimize and to
stabilize nonspecific binding of radioiodinated pGH to the assay tubes. Anti serum
(Calbiochem) against rhesus monkey immunoglobulin-G was diluted 1:21 and was
used at 200 pL per tube to precipitate the primary antibody. Cross-reactivities of
other porcine pituitary hormones in the assay were as follows (percentage relative to
pGH): prolactin, .08; FSH, .12; LH, .001; and thyroid-stimulating hormone, .004
(A.F. Parlow, personal communication). Inhibition curves produced by serial
dilutions of porcine sera and pituitary extracts were parallel to those produced by the
reference standard (USDA-pGH-Bl; National Hormone and Pituitary Agency,
Baltimore, MD). Sensitivity of the assay averaged . 1 ng; the sample size was 200
(iL in a typical assay. Intra- and interassay CV in the GH RIA were 9.6 and 9.8%,
respectively.
Statistical Analyses
Data from these experiments were analyzed by ANOVA (Steel and Torrie,
1980) using the general linear model (GLM) procedure of SAS (198S). The data were analyzed as a randomized complete block design with initial BW as the blocking factor. The pen of pigs was the experimental unit for all data. Statistical analysis of dressing percentage included final BW in the model as a covariate. Data are presented as means with LSD means separation procedures to indicate treatment differences at P < .05, unless otherwise indicated. In addition, a single degree-of- 23
freedom contrast was done to compare the main effect of Cr supplementation (i.e.,
the basal diet vs all Cr diets).
Results
Growth Performance
During the grower phase of production, average daily gain (ADG) was
reduced (P < .01) in pigs fed Cr (basal diet vs all Cr diets, Table 3.2). Average
daily gain was reduced (P < .05) in pigs fed CrCl, CrAc, or CrOx compared with the pigs fed the basal diet (Table 3.2). Chromium supplementation as CrNic, CrPic-
1, CrNGCG, or CrPic-2 resulted in intermediate ADG. The decreased ADG did not result (P > .05) from differences in feed intake, although there was a tendency
(P < . 12) for Cr to decrease feed intake; therefore, differences in feed efficiency were observed. Pigs fed CrCl were less efficient (P < .05) than pigs ted CrAc,
CrOx, CrNic, CrPic-1, or CrPic-2. However, pigs fed the basal diet or CrNGCG had intermediate gain:feed.
During the finisher phase of production the main effect of Cr was opposite of that observed during the grower phase. Pigs fed diets containing Cr tended to eat more feed (P < . 17) and gain more weight (P < . 10). Average daily gain was lower (P < .05) for pigs fed CrOx than for pigs fed CrPic-2; pigs fed the other dietary treatments had intermediate (P > .05) ADG. The response to supplemental
Cr was more variable during the finisher phase compared with the grower phase.
Pigs fed CrCl had greater (P < .05) average daily feed intake (ADFI) than pigs fed 24
the basal diet or CrAc, CrOx, or CrPic-1. Pigs fed CrPic-1, CrPic-2, and CrNGCG
had greater (P < .05) ADFI than pigs fed CrAc or CrOx.
During the overall combined grower and finisher phases there was no main
effect of Cr (P > .20). Average daily gain of pigs fed CrOx was lower than ADG
of pigs fed CrNic, CrNGCG, or CrPic-2. Pigs fed the basal diet or CrCl, CrAc, or
CrPic-1 had intermediate ADG. Pigs fed CrCl or CrNGCG had greater ( P < .05)
ADFI than pigs fed CrAc, CrOx, or CrPic-1. Pigs fed the basal diet, CrNic, or
CrPic-2 had intermediate ADFI. Feed efficiency was less (P < .05) in pigs fed
CrCl compared with pigs fed CrAc, CjO x, CrNic, CrPic-1, or CrPic-2. Pigs fed
the basal diet or CrNGCG had intermediate feed efficiency.
famiM Traits
Longissimus muscle area, TRF, and PM were not affected (P > .05) by
dietary treatment (Table 3.2). Dressing percentage was less (P < .05) in pigs fed
CrAc or CrOx compared with pigs fed CrCl. Pigs fed the basal diet, CrNic, CrPic,
or CrNGCG had intermediate DP. There was no (P > .20) main effect of Cr
supplementation on carcass traits.
Semm Mfttahnlitea and Hormones
Day 21. In pigs that had not had access to feed for 20 h, serum glucose was lower (P < .05) in pigs fed Cr (main effect). Serum glucose was lower (P < .05) in pigs fed CrNic, CrNGCG, or CrPic-2 than in pigs fed the basal diet; pigs fed other Cr sources had intermediate serum glucose concentrations (Table 3.3). Serum Table 3.2. Effect of dietary chromium source on growth performance and carcass characteristics of growing-finishing pigs* Chromium source ______Pooled Criterion Basal Chloride Acetate Oxalate Nicotinate CrPic-1 Complex CrPic-2 SEM Grower phase ADG, kg .81b .70* .74°* .71* .76** .75** .77* .77* .02 ADFI, kg 2.34 2.31 2.03 2.02 2.16 2.06 2.28 2.11 .12 Gain/feed .348* .307 .368b .354b .354b .366" .340* .365" .015 Finisher phase
ADG, kg .82* .89* * 00 °sr .81c .89* .88* .90* .91" .03 ADFI, kg 3.10* 3.49" 3.09* 2.89* 3.29** 3.18** 3.40* 3.39* .09 Gain/feed .263 .256 .284 .280 .272 .277 .265 .270 .011 Overall ADG, kg .81* .79* .81* .76* .82b .81* .84" .84" .02 ADFI, kg 2.69** 2.87b 2.53* 2.44* 2.69** .261** 2.84" 2.73* .07 Gain/feed .301* .276* .319* .311b .3Q5b .312” .295* .307" .009 Carcass characteristics LMA, cm2 29.6 30.4 30.1 31.2 29.3 29.9 29.4 29.9 .8 TRF, cm 3.0 3.0 2.9 2.9 3.2 2.9 3.1 2.9 .1 PM, % 45.2 45.9 46.0 46.8 44.6 45.9 44.7 45.5 .7 DP, % 81.1* 81.3b 80.2* 80.2* 80.7* 80.3* 80.5* 80.9* .4 'Data are means of four replicates of four pigs per replicate; average initial BW = 18.1 kg. The pigs were fed a com-soybean meal diet without (Basal) or with 200 ftg Cr/kg diet as Cr chloride, Cr acetate, Cr oxalate, Cr nicotinate, Cr tripicolinate (CrPic-1 ;USDA synthesized), Cr nicotmate-glycine-cysteine-glutamate complex, or Cr tripicolinate (CrPic-2; Commercial source); LMA=longissimus muscle area, TRF=tenth rib fat thickness, PM=percentage of muscling, DP=dressing percentage. **Means in the same row with uncommon superscripts differ. P < .05. 26
glucose was not different (P > .05) between treatments in samples collected 2 h
postfeeding. Serum NEFA levels were not different (P > .05) between treatments
for pigs that had not had access to feed for 20 h. However, 2-h postfeeding serum
NEFA levels were lower (P < .05) in pigs fed CrPic-1 than pigs fed CrCl. Other
treatment diets resulted in intermediate serum NEFA levels. Serum cholesterol was
not affected (P > .05) by dietary treatment in pigs that had not had access to feed
for 20 h (Table 3.4). However, 2 h postfeeding serum cholesterol was less
(P < .05) in pigs fed CrNGCG than in pigs fed CrCl, CrAc, or CrPic-1; other
treatments resulted in intermediate cholesterol concentrations. Seium biacylglycerol
concentrations for pigs that had not had access to feed for 20 h were lower
(P < .05) in pigs fed CrNic, CrNGCG, or CrPic-2 than pigs fed CrOx; pigs fed
other dietary treatments had intermediate serum triacylglycerol concentrations.
Serum urea N, insulin, and GH concentrations were not different (P > .05) between
dietary treatments for both the 20 h without access to feed and 2 h postfeeding
samples. In addition, there was no main effect of Cr (P > . 10) on any serum
metabolite (except glucose) or hormone on Day 21.
Day 77. In pigs that had not had access to feed for 20 h, serum NEFA
concentrations were lower (P < .09) in pigs fed Cr (main effect). Serum NEFA
concentrations were lower (P < .05) in pigs fed CrPic-2 than pigs fed the basal diet
(Table 3.3). All other dietary treatments resulted in intermediate NEFA concentrations. For the 2 h postfeeding blood samples, serum NEFA concentrations I I
Table 3.3. Effect of dietary chromium source on serum glucose, NEFA, insulin, and growth hormone concentrations in growing- finishing pigs* ______Chromium source Pooled Criterion ______Basal Chloride Acetate Oxalate Nicotinate CrPic-1 Complex CrPic-2 SEM
Glucose, mmol/L Day 21 Unfed 4.4b 4.2** 4.3* 4.3* 4.0® 4.1* 4.0® 4.0® .1 Fed 9.7 9.5 10.5 10.8 11.3 11.2 10.7 9.8 .8 Day 77 Unfed 4.1 4.2 4.2 4.3 4.3 4.1 4.1 4.3 .2 Fed 6.0 5.6 5.6 5.6 5.7 5.9 5.7 5.7 .4
NEFA, mEq/L Day 21 Unfed 1.050 .944 .883 .986 1.060 1.036 1.034 .984 .065 Fed .173* .170* .194* .199* .183* .160b .165* .167* .013 Day 77 Unfed .669b .521* .441* .625* .459* .581* .539* .433® .080 Fed .159* .246” .163* .172* .144® .220* .146® .245b .032 table con’d)
S3 I
Insulin, pU/mL Day 21 Unfed .49 .56 .28 .60 .10 .28 .31 .16 .20 Fed 64.0 53.1 43.6 52.6 56.0 61.7 58.7 52.4 9.7 Day 77 Unfed 1.93* 1.84e 2.34* 2.05* 2.10* 2.66* 3.01* 4.20* .77 Fed 32.7 33.4 25.9 27.7 44.1 44.8 39.2 27.6 9.2
Growth hormone, ng/tnL Day 21 Unfed 5.2 3.5 6.6 4.3 5.0 4.5 4.4 4.6 1.1 Fed 4.7 4.5 4.7 4.4 3.8 4.4 3.8 3.8 .8 Day 77 Unfed 1.5 1.2 1.0 2.4 .7 1.9 1.1 .6 .6 Fed 1.2 2.3 2.3 2.2 1.5 2.5 2.6 1.6 .7 ’Data are means of four replicates with four pigs per replicate; average initial BW = 18.11$. The pigs were fed a com-soybean meal diet without (Basal) or with 200 pg Cr/kg diet as Cr chloride, Cr acetate, Cr oxalate, Cr nicotinate, Cr tripicolinate (CrPic-l;USDA synthesized), Cr nicotinate-glycine-cysteine-glutamate complex, or Cr tripicolinate (CrPic-2; Commercial source). b,cMeans in the same row with uncommon superscripts differ, P < .05.
to 00 Table 3.4. Effect of dietary chromium source on serum urea nitrogen, cholesterol, and triacylglycerol concentrations of growing- finishing pigs* ______Chromium source Pooled Criterion Basal Chloride Acetate Oxalate Nicotinate CrPic-1 Complex CrPic-2 SEM Urea nitrogen, mmol/L Day 21 Unfed 10.6 10.4 9.8 10.0 9.7 9.1 9.5 10.0 .5 Fed 9.8 9.8 9.9 9.9 9.8 9.9 9.4 9.8 .5 Day 77 Unfed 8.6* * 11.6* 10.0* 9.6* 8.8* 8.9* 8.9* 7.6s 1.0 Fed 10.3 12.4 10.8 10.1 10.0 10.1 10.6 8.9 1.2
Cholesterol, mmol/L Day 21 Unfed 2.8 2.9 2.9 2.9 2.7 2.9 2.7 2.8 .1 Fed 2.9*“ 3.1* 2.9* 2.8* 3.0* 2.7* 2.9* .1 Day 77 Unfed 2.5* 2.5* 2.7* 2.4* 2.5* 2.6* 2.5* 2.6* * * Fed 2.7 2.7 2.6 2.6 2.5 2.7 2.6 2.7 .1
Triacylglycerol, mg/dL Day 21 Unfed 98.6 99.8* 102.5* 139.5* 82.8* 103.2* 90.0* 91.8* 15.1 Fed 59.1 53.6 71.4 59.3 66.2 72.1 53.5 63.7 8.0 Day 77 Unfed 29.3 36.6 38.3 39.5 32.8 37.8 38.7 27.9 4.7 Fed 28.6 32.4 27.8 31.6 24.6 30.9 31.7 24.3 2.9 (table con’d) Data are means of four replicates with four pigs per replicate; average initial BW = 18.1 kg. The pigs were fed a com-soybean meal diet without (Basal) or with 200 pg Cr/kg diet as Cr chloride, Cr acetate, Cr oxalate, Cr nicotinate, Cr tripicolinate (CrPic-1;USDA synthesized), Cr nicotinate-glycine-cysteine-glutamate complex, or Cr tripicolinate (CrPic-2; Commercial source). *,cMeans in the same row with uncommon superscripts differ, P < .05. 31
were lower (P < .05) in pigs fed CrNic or CrNGCG than in pigs fed CiCJ or
CrPic-2 forms; other dietary treatments resulted in intermediate NEFA
concentrations. In pigs that had not had access to feed for 20 h, serum insulin
concentrations were greater (P < .05) in pigs fed CrPic-2 than in pigs fed CrCl;
other dietary treatments resulted in intermediate serum insulin concentrations. In
pigs that had not had access to feed for 20 h, pigs fed CrPic-2 had lower serum urea
N concentrations than in pigs fed CrCl. All other dietary treatments resulted in
intermediate serum urea N concentrations for the 20 h without access to feed and no
differences (P > .05) were observed between treatments for the 2 h postfeeding
samples. In pigs that had not had access to feed for 20 h, plasma cholesterol was
lower (P < .05) in pigs fed CrOx than in pigs fed CrAc. In the 2 h postfeeding
samples, no differences (P > .05) between treatments were observed. Serum
glucose, GH, and triacylglycerol concentrations were not affected (P > .05) by
dietary treatments in either the 20 h without access to feed or 2 h postfeeding
samples. There was no main effect of Cr (P > . 10) on any serum metabolite
(except NEFA) or hormone on Day 77.
Tissue Minerals
Chromium. Chromium supplementation (basal diet vs all Cr diets) increased
heart (P < .10), kidney (P < .11), liver (P < .03), and pancreas (P < .09) Cr concentrations but not (P > . 10) longissimus muscle Cr concentration (Table 3.5).
Heart Cr concentration was greater (P < . 10) in pigs fed CrOx compared with pigs Table 3.5. Effect of dietary chromium source on tissue chromium, copper, zinc, and iron concentrations of pigs* Chromium source ______Pooled Criterion Basal Chloride Acetate Oxalate Nicotinate CrPic-1 Complex CrPic-2 SEB Chromium, ng/g dry tissue Heart 8.4® 18.7** 21.9" 9.2* 16.3** 19.8* 11.8*® 3.7 Kidney 9.7* 14.9* 77.7b 50.4* 35.3* 37.8* 10.4* 42.2** 14.4 Liver 8.6* 17.1* 35.0> '34/3* 13.2* 20.4® 9.3* 19.2* 4.1 Pancreas 2.6* 29.6b 1.8® 4.1®* 12.2* 8.3®* 3.7®* 17.6® 3.7 Longissimus muscle 2.2 6.4 4.8 4.1 2.6 5.0 6.8 2.5 2.1
Copper, A^g/g dry tissue Heart 20.1* 19.6^ 20.2* 19.8* 20.4b 19.7* 19.8* 19.1® .5 Kidney 19.9 19.7 20.6 20.6 19.9 23.6 19.5 18.3 2.0 Liver 25.6 21.2 22.4 21.0 24.6 21.6 22.0 20.5 2.0 Pancreas 4.5* 4.8* 4.6* 4.9b 3.9® 4.4* 4.3* 4.4* .4 Longissimus muscle 1.8 1.8 1.7 2.0 1.8 1.9 1.7 2.1 .2
Zinc, /^g/g dry tissue Heart 86.0* 89.2* 85.2® 87.2* 92.4b 90.0* 84.5® 83.9® 2.6 Kidney 102.5 100.8 103,9 102.2 99.7 109.2 102.6 93.9 8.2 Liver 271.4b 225.3* 266.8* 241.8* 210.7* 236.6* 205.0* 190.8® 29.8 Pancreas 133.3b 129.9b 146.3* 130.2* 82.7® 138.6® 105.3* 110.8* 17.8 Longissimus muscle 44.0* 44.4* 49.4b 46.3* 48.1* 47.1* 47.3* 42.6® 2.4 (table con’d) Iron, £tg/g dry tissue Heart 199.0"* 205.0“ 206.8"* 199.4"* 208.2"* 193.5* 212.3"* 215.8" 7.2 Kidney 171.3d 200.31* 172.8" 180.7®" 210.8" 181.8*" 171.9" 206.8"* 9.9 Liver 654.9bed 601.8W 781.0* 556.8®" 775.3" 668.1"®" 516.2" 721.7*“ 73.8 Pancreas 39.1“ 47.6“ 42.4"* 38.9"® 51.5" 47.7"* 35.6* 48.1" 5.6 Longissimus muscle 14.8 14.7 14.4 16.0 14.9 14.8 13.0 15.4 1.5 Data are means of four (CrCl and CrPic-2) or two oreplicates (all other sources) with two pigs per replicate; average initial BW = 18. i kg. The pigs were fed a com-soybean meal diet without (Basal) or with 200 fig Cr/kg diet as Cr chloride, Cr acetate, Cr oxalate, Cr nioodnate, Cr tripicolinate (CrPic-l;USDA synthesized), Cr nicotinate-glycine-cysteine-glutamate complex, or Cr tripicolinate (CrPic-2; Commercial source). MA*Meant in the same row with uncommon superscripts differ, P < .10.
0 3 fed the basal diet* CrNic, or CrPic-2. Kidney Cr concentration was greater (P <
. 10) in pigs fed CrAc than in pigs fed the basal diet, CrCl, CrNic, CrPic-1, or
CrNGCG. liver Cr concentration was not different (P > .10) between the pigs fed
CrAc or CrOx and both of these forms of Cr resulted in greater (P < , 10) liver Cr
concentration than in pigs fed the basal diet or the other Cr sources. Pancreas Cr
concentration was greater (P < . 10) in pigs fed CrCl than in pigs fed the other Cr
sources. Longissimus muscle Cr concentration was not affected (P > . 10) by dietary
treatment.
Copper. Overall Cr supplementation (basal diet vs all Cr diets) did not affect
(P > .10) tissue copper concentrations. Heart copper concentrations were greater
(P < . 10) in pigs fed CrNic than in pigs fed CrPic-2. Pancreas copper
concentration was greater (P < .10) in pigs fed CrOx than in pigs fed CrNic.
Kidney, liver, and longissimus muscle copper concentrations were not affected
(P > . 10) by dietary treatment.
Zinc. Chromium supplementation (basal diet vs all Cr diets) did not affect
(P > . 10) heart, kidney, liver, pancreas, or longissimus muscle zinc concentrations.
Heart zinc concentration was greater (P < . 10) in pigs fed CrNic than in pigs fed
CrAc, CrNGCG, or CrPic-2. liver zinc was lower (P < .10) in pigs fed CrNic or
CrPic-2 than in pigs fed CrAc, CrNGCG, or CrPic-2. Longissimus muscle zinc concentration was greater (P < .10) in pigs fed CrAc than in pigs fed CrPic-2.
Pancreas zinc concentration was lower (P < . 10) in pigs fed CrNic than in pigs fed 35
the basal diet, CrCl, CrAc, or CrPic-1. Kidney zinc concentration was not different
(P > . 10) among dietary treatments.
. Icon. Heart iron concentration was greater (P < . 10) in pigs fed CrPic-2 than
in pigs fed CrPic-1. Kidney iron concentration was greater (P < . 10) in pigs fed
CrCl, CrNic, or CrPic-2 than in pigs fed the basal diet, CrAc, or CrNGCG. liver
iron concentration was greater (P < .10) in pigs fed CrAc or CrNic than in pigs fed
CrOx or CrNGCG. Pancreas iron concentration was greater (P < .10) in pigs fed
CrNic or CrPic-2 than in pigs fed CrNGCG. Longissimus muscle iron
concentration was not affected (P > .10) by dietary treatments. In addition, there
was no main effect of Cr (P > .10; basal diet vs all Cr diets) on tissue iron
concentrations.
Discussion
Growth Perfnmiflncp.
In general the supplementation of diets for pigs with various Cr sources had
minimal effects on gain, feed intake, and gain:feed. For example, in the grower phase, the decrease in growth due to CrCl, CrAc, and CrOx was overcome in the finisher phase (with the exception of CrCl). It appeared that the overall decrease in growth (the basal diet vs all Cr sources) during the grower phase traded to be compensated for during the finisher phase.
The effect of different Cr sources on growth performance of animals
(especially domestic livestock) has not been evaluated to any extent. Most studies 36
have evaluated either CrCl or CrPic. For example, Page et al. (1993b) investigated
either increasing dietary concentrations of CrPic or individual additions of CrCl,
picolinic acid, or the combination of CrCl and picolinic acid. In one experiment,
CrPic decreased ADG and ADFI in a dose dependent manner from 0 to 800 ppb of
dietary Cr as CrPic (Page et al., 1993b). In another experiment ADG was
decreased in a cubic manner, although the decrease was primarily at one dietary
concentration of Cr (100 ppb as CrPic). A third experiment found no effect on growth performance of pigs fed singular additions of either CrCl or picolinic acid or
the combination of CrCl and picolinic acid. Lindcmann et al. (1995) reported that
250 and 500 ppb Cr as CrPic tended to reduce ADFI without affecting ADG, resulting in an increase in gain:feed. However, Evock-Clover et al. (1993) repoited no effect of 300 ppb Cr as CrPic on ADG, ADFI, or gainrfeed. Other authors have reported either decreased (Boleman et al., 1995) or increased (tendency vs CrCl,
P < . 11; Mooney and Cromwell, 1994) growth of pigs due to dietary CrPic supplementation. Smith et al. (1994) reported no effect of 200 ppb Cr from CrNic on gain, feed intake, or gain:feed.
The conditions under which Cr supplementation results in changes in growth performance have not been defined. Some investigators have suggested that "stress” is necessary to induce a response to supplemental Cr (Anderson, 1994; Mowat,
1994). We believe that the pigs in the current investigation experienced minimal production stress. The pigs were housed in a new facility during the finisher phase 37
and were allowed pen space well in excess of that required (Whittemore, 1993).
Other experiments from this institution (Page et al.( 1993b) were conducted in older
facilities, which may have been more stressful to the pig. This may explain the
difference in results obtained from dietary Cr supplementation.
Ca rrass Traits
The lack of an effect of supplemental Cr (especially CrPic) on carcass traits in
this investigation is hard to explain. Many investigators have reported
improvements in carcass traits of pigs fed Cr (Page et al., 1993a, b; Renteria and
Cuarrin, 1993; Smith et al., 1994; Lindemann et al., 1995). However, not all
reports have been positive regarding the effect of supplemental Cr on linear carcass
measurements. Boleman et al. (1995) reported that supplemental CrPic increased
TRF when it was fed during the growing and finishing phases but decreased TRF
when it was fed only during the finishing phase. Mooney and Cromwell (1994)
reported no effect of dietary Cr as CrCl or CrPic on TRF but an increase in LMA
due to either CrCl or CrPic supplementation. However, Page et al. (1993a)
reported no effect of CrCl on carcass measurements. Boleman et al. (1995) and
Mooney and Cromwell (1995) both reported that pigs fed supplemental CrPic did not have measurable differences in linear carcass measurements (i.e., LMA and
TRF). However, carcass dissection and chemical analysis revealed that pigs fed
CrPic had increased carcass lean and decreased carcass fat compared with unsupplemented controls. Other investigators also have reported variable responses 38
to dietary Cr supplementation of swine diets. Lindemann et al. (1995) reported no
effect of CrPic on carcass traits in one experiment and increased LMA and decreased TRF in another experiment. Evock-Clover et al. (1993) reported no effect of dietary CrPic on carcass measurements in pigs with or without exogenous pituitary porcine somatotropin (ppST) administration. Some investigators have postulated that the genetic potential for lean growth may influence the response obtained in pig carcass composition (Roger Campbell, Prince Nutrition Conference,
1993). Pigs with increased potential for lean gain, and thereby less fat, may not be as responsive to dietary Cr supplementation. The genotype used in this experiment is considerably different than the genotype first used by Page et al. (1993b) at this institution. The genetic potential for lean gain of the pigs from the Louisiana State
University Agricultural Center Swine Unit has continually increased over the years and thus may be affecting the responses we are able to obtain in pig carcass composition. In addition, as noted previously, this study was conducted in a new finishing facility. If stress was responsible for the response to Cr supplementation in previous studies, then perhaps these new facilities reduced the amount of stress to which these pigs were exposed. Another proposed reason for a lack of response to
Cr is that over time we may have contaminated our feed mixing equipment at the
Louisiana State University Agricultural Center Swine Unit. We do not have evidence to support this hypothesis; the diets have not been analyzed for Cr content.
More importantly, some researchers believe that as Cr stores of a population (i.e., a 39
herd) are repleted (assuming they were depleted initially) then it may not be possible
to demonstrate a response to supplemental Cr (Merlin Lindemann, personal
communication). This hypothesis is in line with the belief that Cr is acting as an
essential nutrient and not as a pharmacological agent.
Serum Mfttnhnlites and Hormones
After 21 days on the treatments CrNic, CrNGCG, and CrPic-2 reduced serum
glucose. In fact there was a main effect of Cr in reducing serum glucose (the basal diet vs all Cr sources). However, none of the Cr sources affected serum glucose after 77 days on the experimental diets. Most reports of reduced serum glucose levels have been in the early stage of Cr supplementation. For example, Kitchalong et al. (1995) reported a reduced response to an IVGTT for sheep fed CrPic during the first 2 wk of an experiment but lack of a response after 10 wk of supplementation. Other investigators have reported increased glucose clearance rates due to CrPic supplementation (Bunting et al., 1994; Amoikon et al., 1995) in relatively short-term experiments. Page et al. (1993b) reported no difference in serum glucose of pigs from a single blood sample taken 3 h after initiation of feeding after 73 d of CrPic supplementation. However, Evock-Clover et al. (1993) reported that CrPic supplementation “normalized" plasma glucose concentrations in pigs injected with ppST in an experiment of relatively shorter duration (39 to 50 d).
Lindemann et al. (1995) reported a decrease in the insulin:glucose ratio of sows fed
CrPic, 40
Hie variable response of serum cholesterol and NEFA concentrations to different Cr compounds was not surprising. These two metabolites have been reported to be decreased (cholesterol, Page et al., 1993; NEFA, Amoikon et al.,
1995), increased (cholesterol, Evock-Clover et al., 1993; Amoikon et al., 1995), or unchanged (cholesterol, Lindemann et al., 1995) in pigs fed supplemental CrPic.
However, the reduction in serum NEFA from CrPic-1 supplementation but not
CrPic-2 is surprising since these were identical chemical compounds differing only in their source.
The variability in other metabolites and hormones also has been reported in pigs (Evock-Clover et al., 1993; Page et al., 1993; Lindemann et al., 1995), sheep
(Gentry et al., 1995; Kitchalong et al., 1995; Ward et al., 1995) and cattle (Bunting et al., 1994; DePew et al., 1995). The reason(s) for these variable responses is not known.
Tigsiift Minerals
The lack of an increase in muscle Cr content from supplemental dietary Cr is in agreement with other research (Anderson et al., 1989, 1994). Increased tissue chromium concentrations could become a regulatory issue. However, because most
Americans and others from highly developed countries do not consume the recommended daily safe and adequate intake of Cr (Anderson, 1989), some scientists consider increased Cr content of animal protein as a means of supplementation of human diets with Cr an acceptable method of increasing Cr intake of the population (Anderson et al., 1989). Nonetheless, mineral deposition
data are scarce and necessary. Overall, organic sources of Cr resulted in higher
tissue Cr levels than the basal diet or CrCl. The exception is pancreas Cr. Pancreas
Cr levels were dramatically higher in pigs fed CrCl than in pigs fed the other dietary
treatments. Berggren and Flatt (1985) reported endocrine and exocrine pancreas Cr
concentrations five to sixty-fold higher than liver, kidney, skeletal muscle, cardiac
muscle, spleen, pituitary, testes and ovaries in rats fed CrCl. While it is generally
accepted that inorganic sources of Cr are not absorbed as readily as organic sources
(Mertz, 1969; Votava et al., 1973), the pancreatic Cr pool does not exchange Cr
with the blood as rapidly as other tissues (Jain et al., 1981). Other investigators also
have reported increased Cr in kidney and liver but not in heart or muscle of pigs fed
supplemental Cr as CrPic (Anderson et al., 1994). Anderson et al. (1994) also
reported no effect of CrPic supplementation on the concentration of zinc and iron in muscle tissue of pigs. Schrauzer et al. (1986) reported that mice fed Cr-rich brewer’s yeast had increased Cr levels in the liver and heart. Chromium supplementation also increased liver iron and decreased liver zinc concentrations. In addition, mice fed Cr-supplemented diets had decreased manganese, iron, copper, and zinc concentrations in the heart (Schrauzer et al., 1986). 42
Implications
Chromium addition to swine diets may not result in improvements in body composition. Even though absorption and tissue retention of chromium seemed to occur in the current investigation, changes in body composition were not observed. CHAPTER IV
EFFECT OF DIETARY CHROMIUM SOURCE ON GLUCOSE TOLERANCE AND INSULIN SENSITIVITY IN GROWING PIGS
Introduction
Chromium is most often associated with carbohydrate metabolism, being
necessary for insulin function for glucose uptake by cells (Anderson, 1985).
Chromium deficiency leads to glucose intolerance, and Cr supplementation of the
diets of elderly humans has resulted in improved glucose tolerance (Mertz, 1993).
Total parenteral nutrition patients also have been reported to have reduced diabetic
signs when given supplemental Cr (Jeejeebhoy et al., 1977; Brown et al., 1986).
Steele et al. (1977) reported that a synthetic glucose tolerance factor containing
Cr enhanced the hypoglycemic action of insulin in pigs. Dietary Cr as Cr tripicolinate (CrPic) also has been shown to increase glucose disappearance rate (k) and decrease glucose half-life (TI/a) in calves (Bunting et al., 1994) and pigs
(Amoikon et al., 1995). In pigs, CrPic has been shown to increase loin eye area and decrease fat thickness (Lindemann et al., 1993; Page et al., 1993a,b; Renteria and Cuardn, 1993), and to increase the rate of lean and decrease the rate of fat deposition (Mooney and Cromwell, 1993; Boleman et al., 1994). However, Cr chloride hexahydrate (CrCl) did not affect carcass traits of pigs (Page et al.,
1993a,b). Smith et al. (1994) reported that Cr nicotinate (CrNic) decreased backfat thickness but did not affect loin eye area of pigs. Thus, the form of Cr (inorganic or
43 44 organic) also may affect the glucose responses that have been reported for livestock species. Therefore, these experiments were conducted to determine the effect of Cr as CrCl, CrNic, or CrPic on glucose tolerance and insulin sensitivity in growing pigs.
Materials and Methods
Animals anH nifttary TreatmpJits
Two identical trials were conducted. Crossbred (Yorkshire x Landracc females bred to Hampshire boars) barrow pigs were used [Trial 1, n=36, avg. initial body weight (BW)=20.2 kg; Trial 2, n=36, avg. initial BW=23.0 kg]. The pigs were allotted to treatment on the basis of weight in a randomized complete block design; ancestry was equalized across treatments. Each treatment was replicated three times in each trial with three pigs per replicate. Three pens of three pigs each per trial were fed a control diet without supplemental Cr. Trial 1 was conducted from mid-November through mid-December 1994. Trial 2 was conducted from mid-January through mid-February 1995.
The pigs were fed a com-soybean meal basal diet (Table 4.1) formulated to provide 120% of the NRC (1988) lysine requirement. The dietary treatments consisted of supplementing the basal diet with either 0 or 400 /tg Cr/kg diet from
CrCl (Sigma Chemical Co., St. Louis, MO), CrNic (provided by Dr. R. A.
Anderson, USDA, Beltsville, MD), or CrPic (CHROMAX™, Prince Agri Products, 45
Table 4.1. Composition of the basal diet* as-fed basis
Item %
Com 73.01
Soybean meal, 44% CP 24.74
Limestone 1.06
Dicalcium phosphate .64
Salt .25
Vitamin premix* .25
Trace mineral premixb .05
‘Provided the following per kilogram of diet: riboflavin, 4.4 mg; d-pantothenic acid,
22 mg; niacin, 22 mg; vitamin Bw, 22 ^g; d-biotin, 220 fig; choline chloride, 440 mg; vitamin A, 4400IU; vitamin D}, 440IU; vitamin E, 11IU; menadione dimethylpyrimidinol bisulfite, .25 mg; Se, .1 mg.
‘’Provided the following per kilogram of diet: Zn, 75 mg; Fe, 87.5 mg; Mn, 30 mg;
Cu, 8.75 mg; I, 1 mg; Ca, 9 mg. 46
Quincy, IL). The pigs (three replicates of three pigs each) were allowed to consume
the treatment diets on an ad iihihim basis during the first 21 d of the trials during
which time the pigs were group-penned in 1.8-m x 2.4-m pens in an enclosed
facility with totally slatted floors. After this growth period, five pigs per treatment
were randomly selected and moved into .6-m x 1.2-m plastic metabolism crates and
penned individually. Hie pigs were fed (feed mixed with water) twice daily an
amount approximating one-half their ad Iihihim intake. Growth performance data
were collected for the period prior to placing the pigs in the metabolism crates.
(Tathftterizatinn
After approximately 5 d in the metabolism crates, the pigs were anesthetized i.m. with Telazol® (4.4 mg/kg BW, contains 2.2 mg tiletamine/kg BW as tiletamine
HC1 and 2.2 mg zolazepam/kg BW as zolazepam HC1; Fort Dodge Laboratories,
Inc., Fort Dodge, IA) plus Rompun® (4.4 mg xylazine/kg BW; Miles Inc., Shawnee
Mission, KS). A 12-gauge trocar was inserted into the right jugular fossa. A catheter (Norton Tygon tubing No. AAQ04127; 1.02 mm i.d., 1.78 mm o.d.;
Fisher Scientific, Pittsburgh, PA) was passed through the trocar and into the jugular vein. The catheter was filled with a 6% sodium citrate solution, sutured, and glued to the skin of the pigs at the point where the catheter exited the skin, routed to the top of the neck and held in place with surgical and duct tape. The total length of the catheter was approximately 120 cm; approximately 10 to IS cm of catheter was inserted inside the jugular fossa, 40 cm was required to route the catheter to the top 47
of the neck, and approximately 70 cm of catheter was stored in a small pouch at the
top of the neck for ease in blood collection.
Metabolic Challenges
Two to three d after catheterization, an intravenous glucose tolerance test
(TVGTT) was conducted followed 3 h later by an intravenous insulin challenge test
(IVICT) as outlined by Bunting et al. (1994). In both experiments, feed was
withheld for 15 to 18 h prior to the rVGTT. In brief, for the IVGTT and IVICT,
respectively, glucose (500 mg/kg BW) and insulin [. 1 U of crystalline porcine
insulin (Sigma #I-3505)/kg BW administered in a .9% NaCl solution containing .2%
bovine serum albumin (Fraction V Powder, Cat# 810-1018, Lot#A090767, Grand
Isle Biological Co., Grand Isle, NY) were administered via the jugular vein catheter
as bolus doses. Blood samples were collected via syringe and placed into 7-mL
tubes containing 17.5 mg sodium fluoride and 14.0 mg potassium oxaJate
(Monoject®, Sherwood Medical, St. Louis, MO) at -10,0,5, 15, 30, 45, 60,90,
and 120 min relative to dosing for the IVGTT. For the IVICT, additional samples
were taken at 10, 20, 25, 150, 180, 210, and 240 min. Glucose k (%/min) and Tll7
(min) were calculated between 15 and 30 min for the IVGTT, and between 0 and 30
min for the IVICT as a modification of the method outlined by Kaneko (1989).
Insulin k and T1/2 were calculated between 15 and 30 min for the IVGTT and the
IVICT. 48
rhftmiral Analyses
Blood metabolites and insulin were measured in plasma from the 0 min blood
samples collected before the IVGTT test. All IVGTT and IVICT samples were analyzed for insulin and glucose concentrations. Plasma was analyzed for glucose
(Sigma, 1990) and cholesterol (Sigma, 1989) concentrations, using spectrophotometric procedures outlined in commercially-available kits (Sigma
Chemical Co.). Plasma urea N concentrations were determined spectrophotometrically using the urease procedure outlined by Fernandez et al.
(1988), and plasma NEFA concentrations were determined using a commercial enzymatic procedure (NEFA-C Kit, ACS-ACOD Method; Wako Chemicals USA,
Inc., Richmond, VA). All samples were assayed in duplicate and measurements resulting in errors greater than 5% were re-analyzed.
Plasma insulin was assayed by the RIA method of Amoikon et al. (1995).
Guinea pig anti-bovine insulin antiserum (Code No. 65-101, Lot No. GP616; ICN
ImmunoBiologicals, Lisle, IL) was used at a 1:60,000 final dilution. Purified porcine insulin (26.1 jiU/ng; Sigma Chemical Co.) and bovine 12SI-insulin (ICN
Biomedicals, Inc., Costa Mesa, CA) were used as the standard and radioligand, respectively. Sheep anti-guinea pig antiserum produced in our laboratory was used at a 1:4 dilution as the precipitating antibody. The intraassay and interassay CV for 49 the insulin RIA as determined by pooled bovine plasma samples were 14% and
13%, respectively.
Statistical Analy.w
Data from these experiments were analyzed by ANOVA (Steel and Torrie,
1980) using the GLM procedure of SAS (1985). The growth performance data were analyzed as a randomized complete block design with initial weight as blocks and the pen of pigs was the experimental unit. The metabolite and insulin data were analyzed as a completely random design and the individual pig was the experimental unit. Common criteria for Trials 1 and 2 were found not to contain trial by treatment interactions, therefore the data were pooled for statistical analyses and presentation.
Results and Discussion
Average daily gain (ADG), average daily feed intake (ADFI) and gain/feed were not affected (P > .10) by dietary treatment (Table 4.2). These results are in agreement with other research conducted using pigs (Amoikon et al., 1995; Evock-
Clover et al., 1993; Lindemann et al., 1993; Page et al., 1993a,b; Renteria and
Cuardn, 1993), lambs (Kitchalong et al., 1995), and calves (Bunting et al., 1994).
Plasma cholesterol, NEFA, urea N, and insulin were not affected (P > .05) by treatment (Table 4.3). Reduced levels of circulating total cholesterol have been reported in pigs (Page et al., 1993a) and calves (Bunting et al., 1994) fed CrPic.
However, Evock-Clover et al. (1993) reported that supplemental dietary CrPic 50
Table 4.2. Effect of dietary chromium source on daily gain, feed intake, and gain/feed in growing barrows (Trial 1 and Trial 2 combined)* Treatment Pooled
Criterion CrCl CrNic CrPic SEMControl
ADG, kg .84 .83 .86 .84 .02
ADFI, kg 1.98 2.01 1.98 2.00 .06
Gain/feed .43 .41 .44 .42 .01
■Data are means of six replicates with three pigs per replicate; avg. initial BW =
21.6 kg (Trial 1, 20.2; Trial 2, 23.0). The pigs were fed a com-soybean meal diet without (Control) or with 400 /ig Cr/kg diet as Cr chloride hexahydrate (CrCl), Cr nicotinate (CrNic), or Cr tripicolinate (CrPic). The growth trial period was 21 d. 51
Table 4.3. Effect of dietary chromium source on plasma metabolites and hormones in growing barrows* Treatment Pooled
Criterion Control CrCl CrNic CrPic SD
Glucose, mmol/L 4.72* (11) 4.56' (9) S.Otf (11) 4.77* (8) .39
Cholesterol, mmol/L 2.31 (11) 2.38 (9) 2.31 (11) 2.36(11) .34
Urea N, mmol/L 6.04 (11) 5.81 (9) 5.39 (11) 5.97 (8) 1.28
NEFA, mEq/L •59 (9) .61 (9) .57 (9) •71 (5) .29
Insulin, /zU/mL 4.18(11) 4.13(9) 4.39(11) 4.77 (8) 2.46
*The pigs were fed a com-soybean meal diet without (Control) or with 400 ftg Cr as
Cr chloride hexahydrate (CrCl), Cr nicotinate (CrNic), or Cr tripicolinate
(CrPic)/kg diet. The number of barrows is shown in parentheses. Plasma samples were collected after 15 to 18 h without feed.
‘“Means within a row without a common superscript differ, P < .05. 52
increased total cholesterol and high-density lipoprotein(HDL)-cholesterol in pigs.
Amoikon et al. (1995) also reported increased total cholesterol and decreased
NEFAin pigs fed CrPic. Chromium supplementation in humans also has yielded
variable effects on cholesterol components (Riales and Albrink, 1981; Anderson et
al.* 1983; Evans, 1989; Lefavi etal., 1993). Chromium compounds have been
reported to lower serum triacylglycerol (Ranhotra and Gelrotb, 1986) and NEFA
(Mirsky, 1993) in rats.
During the IVGTT, glucose k and glucose Tl/2 were not affected (P > . 10) by
treatment (Table 4.4). In a similar manner, during the IVICT, glucose k and
glucose TI/2 were not affected (P > .10) by dietary treatment. The response curves
depicting mean glucose concentrations over time during the IVGTT and I VICT are
shown in Figures 4.1 and 4.3, respectively. The response curves depicting mean
insulin concentrations over time during the IVGTT and IVICT are shown in Figures
4.2 and 4.4, respectively. Amoikon et al. (1995) reported that pigs fed 200 jug Cr as CrPic per kg diet had increased glucose clearance and decreased glucose half-life during IVGTT. The reason(s) for the lack of agreement between the data of
Amoikon ct al. (1995) and the current experiment are not known. During the
IVICT (Table 4.5), insulin k and insulin T,^ were not affected (P > .10) by dietary treatment. Amoikon et al. (1995) reported similar findings in pigs fed CrPic. 53
Table 4.4. Effect of dietary chromium source on glucose kinetics in growing barrows* ______Treatment Pooled
Criterion Control CrCl CrNic CrPic SD
Intravenous Glucose Tolerance Test (IVGTT), 15-30 min
Clearance, %/min 3.82(11) 4.02 (9) 3.40 (11) 3.05 (8) 1.28
Half-life, min 20.32(11) 19.23 (9) 23.60(11) 24.80 (8) 7.33
Intravenous Insulin Challenge Test (IVICT), 0-30 min
Clearance, %/min 2.84 (11) 2.71 (9) 2.64 (11) 2.25 (8) .81
Half-life, min 27.27(11) 27.82 (9) 29.31 (11) 33.73 (8) 10.45
“The pigs were fed a com-soybean meal diet without (Control) or with 400 ng Cr as
Cr chloride hexahydrate (CrCl), Cr nicoiinate (CrNic), or Cr tripicolinate
(CrPic)/kg diet. All data are means of individually fed pigs. Number of barrows is shown in parentheses. 54
Table 4.5. Effect of dietary chromium source on insulin kinetics in growing barrows* ______Treatment Pooled
Criterion Control CrCl CrNic CrPic SD
Intravenous Glucose Tolerance Test (IVGTT), 15-30 min
Clearance, %/min 7.72(11) 9.05(9) 6.57(9) 5.55 (5) 4.35
Half-life, min 15.76(11) 10.44(9) 14.22(9) 13.46 (5) 7.60
Intravenous Insulin Challenge Test (IVICT), 15-30 min
Clearance, %/min 6.45(11) 6.77(9) 6.95 (11) 5.12(8) 2.44
Half-life, min 11.74(11) 10.73(9) 11.09(11) 17.76(8) 5.29
The pigs were fed a com-soybean meal diet without (Control) or with 400 /*g Cr as
Cr chloride hexahydrate (CrCl), Cr nicotinate (CrNic), or Cr tripicolinate
(CrPic)/kg diet. All data are means of individually fed pigs. Number of barrows is shown in parentheses. 55
16 -
14 - C ontrol C rC l CrNic e 12 H aS C rPic
u 10 - •a9 a* « 8 H
6 -
4 -
2 I i ~ r~ —r~ — I— 0 20 40 60 80 100 120
Tim e, min.
Figure 4.1. Effect of dietary chromium on glucose tolerance in pigs during an intravenous glucose tolerance test. Pigs were fed a com-soybean meal control diet without (•) or with 400 fig Cr as CrCl (■), CrNic (A), or CrPic (T)/kg diet. For glucose disappearance rate: pooled SD = 1.27; and for glucose half-life; pooled S D = 7.32. 56
60
50 -
1 3 4 0 - C ontrol a C rC l CrNic .a 30 - C rPic S 3 20 -
10 -
0 20 40 60 80 100 120
Tim e, m in.
Figure 4.2. Effect of dietary chromium on insulin clearance in pigs during an intravenous glucose tolerance test. Pigs were fed a com-soybean meal control diet without (•) or with 400 fig Cr as CrCl (■), CrNic (A), or CrPic (Y)/kg diet. For glucose insulin disappearance rate: pooled SD = 4.35; and for insulin half-life; pooled SD= 7.60. 57
6
5
B 4 B
3 C ontrol ■ a C rC l CrNic 8 CrPic 2
1
0 0 40 80 120 160 200 240
Tim e, m in.
Figure 4.3. Effect of dietary chromium on glucose clearance in pigs during an intravenous insulin challenge test. Pigs were fed a com-soybean meal control diet without (•) or with 400 /*g Cr as CrCl (■), CrNic (A), or CrPic (T)/kg diet. For glucose disappearance rate: pooled SD = .81; and for glucose half-life; pooled SD = 10.45. 58
200 -
!= a C o ntrol a 150 - C rC I s = 3 CrNic •s 9 .3 C rPic ats J 100 -
50 -
40 80 120 160 200 240
Tim e, m in.
Figure 4.4. Effect of dietary chromium on insulin clearance in pigs during an intravenous insulin challenge test. Pigs were fed a com-soybean meal control diet without (•) or with 400 /xg Cr as CrCI (■), CrNic (A), or CrPic (▼)/kg diet. For glucose insulin disappearance rate: pooled SD — 2.44; and for insulin half-life; pooled SD= 5.29. 59
fip.np.ral nisrnw inn
Steele et al. (1977) demonstrated that Cr-containing GTF increases insulin sensitivity in pigs. These authors concluded that GTF was biologically active in swine and that it potentiated the insulin-insulin receptor interaction. Beneficial effects of Cr-rich yeast on glucose tolerance and insulin sensitivity in elderly people have been reported (Offenbacher and Pi-Sunyer, 1980). Riales and Albrink (1981) reported that supplementation with Cr as CrClj‘6H20 improved insulin sensitivity in subjects with insulin resistance, but in not subjects with normal glucose tolerance.
Abraham et al. (1992) also has reported no effect of dietary Cr supplementation (as
CrCI) on the glucose status of patieuts with or without non-insulin-dependent diabetes. However, the most prominent data regarding Cr supplementation is (hat glucose intolerance has been associated with Cr deficiency (Schioedcr, 1966;
Stoecker and Oladut, 1985; Anderson, 1987). Chromium supplementation has been found to be essential for normal glycemia and insulin function in human patients receiving long-term total parenteral nutrition (Jeejeebhoy et al., 1977; Brown et al.,
1986).
Bunting et al. (1994) reported that Cr as CrPic increased glucose k in cattle; however, both Kitchalong et al. (1995) and Fomea et al. (1994) reported no effect of dietary CrPic on glucose clearance rates in lambs. Evock-Clover et al. (1993) reported that dietary CrPic "normalized” plasma glucose and insulin in pigs injected 60
with somatotropin. Thus, the conditions which affect the glucose response to Cr
supplementation remain unknown.
The current study was conducted in the same facilities as those used by
Amoikon et al. (1995). Thus a reduction in stress due to better facilities as
hypothesized in the previous chapter of this dissertation is probably not valid in this
case. However, it is possible that genotypic changes or the Cr status of the pigs at
the Louisiana Agricultural Center Swine Unit have changed and thus may be
responsible for the discrepant responses.
Implications
In this report, we have shown that chromium as chromium chloride, chromium
nicotinate, and chromium tripicolinate did not affect glucose tolerance or insulin
sensitivity in pigs. These results are not in agreement with other studies in which
both organic and inorganic sources of chromium have been used to demonstrate the
importance of chromium in insulin sensitivity in humans, pigs, and other animal
species. The reasons for the discrepant results of chromium supplementation for pigs
and other livestock species remain unknown. Some speculate that the level of environmental stress, genotype differences, or Cr status may account for the differences in response to supplemental dietary chromium. The precise conditions under which chromium benefits livestock must be characterized if chromium is to be recognized as an essential feed additive. CHAPTER V
INTERACTIVE EFFECTS OF DIETARY CHROMIUM TRIPICOLINATE AND CRUDE PROTEIN LEVEL IN GROWING-FINSHING PIGS PROVIDED INADEQUATE AND ADEQUATE PEN SPACE: EFFECTS ON GROWTH PERFORMANCE, CARCASS CHARACTERISTICS, AND PLASMA METABOLITES AND HORMONES
Introduction
Page et al. (1993a,b) reported that Cr tripicolinate (CrPic) decreased 10th rib
backfat thickness and increased longissimus muscle area in pigs. Other authors have
also reported improvements in body composition of swine from supplemental dietary
CrPic (Renteria and Cuardn, 1993; Mooney and Cromwell, 1994; Boleman et al.,
1993; Lindcmann et al., 1995). Our laboratory (Boleman et al., 1995) as well as
Mooney and Cromwell (1995) has obtained equivocal results in improved linear
carcass measurements of pigs fed supplemental, dietary Cr. Some investigators have
suggested that animals have to be under some form of stress to elicit a response to
Cr supplementation (Anderson, 1994; Mowat, 1994). Recently, we have conducted
several trials without obvious improvement in the linear carcass measurements in
response to supplemental Cr (unpublished data, and Ward et al., 1995; these experiments were conducted in new finishing facilities). We hypothesized that the production stress the pigs experienced was decreased over that of pigs in earlier studies in which responses to Cr supplementation were found. One means of subjecting pigs to a chronic production stress would be to reduce the pen space allowance per pig. Undemann et al. (1993) reported that the response to
61 6 2
supplemental dietary Cr may be dependent on the lysine (or CP) content of the diet.
Therefore, we conducted the following investigation to investigate the interactive
effects of dietary Cr, CP, and pen space allowance in growing-finishing pigs.
Materials and Methods
Animals and nletary Treatments
Crossbred (Yorkshire x Landrace females bred to Hampshire boars) pigs were
used [avg. initial body weight (BW)=27.2 kg]. The pigs were allotted to treatment
on the basis of BW in a randomized complete block design; ancestry was equalized
across treatments. Each treatment was replicated four times with four pigs per
replicate pen. Four pens of four pigs each were fed a control diet without Cr
supplementation. Sex (two gilts and two barrows) was equalized within pen.
A 2 x 2 X 2 factorial arrangement of treatments was used. The pigs were fed
a com-soybean meal basal diet formulated to provide 80 or 120% of the lysine
requirement (NRC, 1988) during both the grower and finisher phases (Table 5.1).
The additional dietary factor was two levels of Cr (none or 400 Mg Cr/kg diet as
CrPic; Chromax™, Prince Agri Products, Inc., Quincy, IL). The pigs were
allowed to consume the treatment diets on an adiihitum basis. The third treatment
factor was two areas of pen space (mVpig; adequate, .035 BW,(i7 kg, or inadequate,
.025 BW 67 kg; Whittemore, 1993). The pigs were weighed weekly and pen space was adjusted at 10 kg of BW gain increments on an individual pen basis. The pigs were penned in a curtain-sided facility with totally slatted concrete floors. Each pen 63
Table S .l. Composition of the basal diets, as-fed basis
Grower Finisher
Percentage of Lysine Requirement
80 120 80 120
Item %
Com 84.12 73.01 89.15 80.27
Soybean meal, 44% CP 13.44 24.74 8.87 17.91
Limestone 1.05 1.06 .99 .99
Dicalcium phosphate .84 .64 .44 .28
Salt .25 .25 .25 .25
Vitamin premix' .25 .25 .25 .25
Trace mineral premixb .05 .05 .05 .05
'Provided the following per kilogram of diet: riboflavin, 4.4 mg; d-pantothenic acid,
22 mg; niacin, 22 mg; vitamin B)2, 22 ng; d-biotin, 220 u%\ choline chloride, 440 mg; vitamin A, 4400IU; vitamin I>3. 440 IU; vitamin E, 11 IU; menadione dimethylpyrimidinol bisulfite, .25 mg; Se, .1 mg. Treatment diets were fed without and with 400 //g Cr as Cr tripicolinate/kg diet. bProvided the following per kilogram of diet: Zn, 75 mg; Fe, 87.5 mg; Mn, 30 mg;
Cu, 8.75 mg; I, 1 mg; Ca, 9 mg. 64
contained a single-hole feeder and a single nipple waterer. The pen space allowance
calculations accounted for the space occupied by the feeder. Pigs were switched to
finisher diets when the average BW of a pen was approximately 55 kg. The pigs
were removed from the experiment when die average BW of a pen was
approximately 104 kg. This experiment was conducted from the end of August
1994 to the first wk of January 1995.
Blnnd Sampling
All pigs were bled via the anterior vena cava following 20 h without access to
feed and again 2 h postfeeding on d 21 of the experiment. Blood for plasma
metabolites was placed into 7-mL tubes (Monoject®, Sherwood Medical, St. Louis,
MO) containing 17.5 mg sodium fluoride and 14.0 mg potassium oxalate. Blood for
plasma hormones was placed into 7-mL tubes (Monoject®) containing 10.5 mg
KjEDTA. All blood samples were placed in an ice bath immediately after
collection. The blood samples were centrifuged for 20 min at 1,600 X g at 4°C.
Plasma was collected and frozen (-20°C) for later analyses.
P-amass Traits
Pigs were killed via electrical shinning followed by exsanguination at the
Louisiana State University Agricultural Center Department of Animal Science Meats
Laboratory. Hot carcass weights were taken with heads off the carcass and leaf fat in the carcass. Longissimus muscle area (LMA) at the 10th rib interface and 10th rib fat (TRF) thickness three-fourths the distance from the midline were measured 65
after approximately 20 h at 2°C. Longissimus muscle area and TRF were adjusted
to 104.3 kg (NSIF, 1987), and percentage of muscling (PM) was calculated by
methods described by the NPPC (1991). Dressing percentage (DP) was calculated
by dividing hot carcass weight by live BW times 100.
Chemical Analyses
Plasma was analyzed for glucose (Sigma, 1990; Sigma Chemical Co.; St.
Louis, MO), total cholesterol (Sigma, 1989), and NEFA (NEFA-C Kit, ACS-
ACOD Method; Wako Chemicals USA, Inc., Richmond, VA) concentrations using
spectrophotometric procedures outlined in commercially-available kits. Plasma was
analyzed for urea N by the urease method outlined by Fernandez et al. (1988).
Plasma insulin was assayed by the R1A method of Amoikon et al. (1995).
Guinea pig anti-bovine insulin antibody (Code No. 65-101, Lot No. GP616; ICN
ImmunoBiologicals, Lisle, IL) was used at a 1:60,000 final dilution. Purified porcine insulin (26.1 /xU/ng; Sigma Chemical Co.) and bovine 123I-insulin (ICN
Biomedicals, Inc., Costa Mesa, CA) were used as the standard and radioligand, respectively. Sheep anti-guinea pig antibody produced in our laboratory was used at a 1:4 dilution as the precipitating antibody. The intraassay CV for the insulin RIA as determined by a pooled bovine plasma sample was 12.9%.
Plasma GH was measured by a double-antibody RIA technique based on antibody (AFP-10318545; A.F. Parlow, Pituitary Hormone and Antisera Center,
Harbor-UCLA Medical Center, Torrance, CA) against porcine GH (pGH). Highly 66
purified pGH was radioiodinated via the chloramine-T method (Greenwood et al.,
1963; 2.5 fig of chloramine-T/ftg of GH, 0 °C for 30 s). The antiserum was diluted
1:150,000 in PBS containing .033 M EDTA, .112% normal rhesus monkey plasma
(Calbiochem, San Diego, CA), and 33% noninhibitory horse plasma; 300 /iL of this
solution was used per tube. The horse plasma was added to minimize and to
stabilize nonspecific binding of radioiodinated pGH to the assay tubes. Antiserum
(Calbiochem) against rhesus monkey immunoglobulin-G was diluted 1:21 and was
used at 200 ftL per tube to precipitate the primary antibody. Cross-reactivities of other porcine pituitary hormones in the assay were as follows (percentage relative to pGH): prolactin, .08; FSH, .12; LH, .001; and thyroid-stimulating hormone, .004
(A.F. Parlow, personal communication). Inhibition curves produced by serial dilutions of porcine sera and pituitary extracts were parallel to those produced by the reference standard (USDA-pGH-Bl; National Hormone and Pituitary Agency,
Baltimore, MD). Sensitivity of the assay averaged . 1 ng; the sample size was 200
/*L in a typical assay. Intraassay CV in the GH RIA was 7.1%.
Statistical Analyses
Data from this experiment were analyzed by ANOVA (Steel and Torrie, 1980) using the GLM procedure of SAS (1985). The data were analyzed as a randomized complete block design with initial BW as the blocking factor. The pen of pigs was the experimental unit for all data. Statistical analysis of DP included final BW in 67
the model as a covariate. Orthogonal single-degree-of-freedom contrasts were used
to evaluate the main effects and interactions.
Results
fimwth Performance
During the grower phase of production, pigs fed 120% of the lysine
requirement had greater (P < .01) average daily gain (ADG) and gain/feed and
lower (P < .03) average daily feed intake (ADF1) than pigs fed 80% of the lysine
requirement (Table 5.2). Pigs provided inadequate pen space (.025 BW67 kg) had lower ADG (P < .01), ADFI (P < .01), and gain/feed (P < .03) than pigs provided adequate pen space (.035 BW 67 kg). Average daily gain was decreased to a greater extent by inadequate pen space in pigs fed 80% of the lysine requirement than in pigs fed 120% of the lysine requirement (CP x pen space effect, P < .01).
Gain/feed was decreased by inadequate pen space in pigs fed 80% of the lysine requirement; however, in pigs fed 120% of the lysine requirement inadequate pen space increased gain/feed (CP X pen space effect, P < .08). Chromium supplementation did not affect (P > .10) ADG, ADFI, or gain/feed during the grower phase.
During the finisher phase, pigs fed 120% of the lysine requirement liad less
(P < .10) ADFI but similar (P > .10) ADG compared with pigs fed 80% of the lysine requirement. Gain/feed was higher (P < .10) in pigs fed 120% of the lysine requirement than in pigs fed 80% of the lysine requirement. Average daily gain and Table 5.2. Effect of dietary chromium, crude protein level, and pen space on growth performance and carcass characteristics of growing-finishing pigs" ______Percentage of lysine requirement 80 120 80 120 80 120 80 120 Cr, Mg/kg diet 0 0 400 400 0 0 400 400 Pot space allowance, m2/pigb Pooled £ n .m s .035 .035 .035 .025 _ .025 __ .025 .025 SKM Grower phase ADG, kg*-1* .685 .750 .678 .728 .594 .715 .598 .696 .019 ADFI, kgv 2.101 2.006 2.077 1.982 1.951 1.898 1.935 1.853 .049 Gain/feed'4'5 .326 .374 .327 .368 .304 .376 .307 .376 .004
Finisher phase ADG, kg^ .833 .839 .859 .826 .732 .767 .781 .756 .019 ADFI, kg?'* 3.472 3.115 3.283 3.367 3.112 2.878 3.147 2.974 .136 Gain/feetf* .240 .269 .261 .249 .238 .267 .250 .255 .010
Overall ADG, kg*** .774 .805 .789 .788 .674 .747 .701 .734 .016 ADFI, k g ^ 2.915 2.689 2.811 2.802 2.617 2.521 2.612 2.536 .080 Gain/feede,h .265 .299 .280 .283 .258 .297 .268 .289 .007 (table con'd) Carcass characteristics LMA, cm26* 30.4 35.0 30.0 33.5 31.4 35.7 32.1 35.7 .8 TRF, cme,w 3.1 2.5 2.9 2.5 2.6 2.0 2.7 2.3 .1 PM, %e>f 45.9 49.1 45.8 49.0 47.7 51.6 47.7 50.9 .5 DP. % 74.6 75.2 75.0 74.9 75.3 75.4 75.0 75.5 .5 'Data are means of four replicates of four pigs per replicate; average initial BW = 27.2 kg ; LMA=longissimus muscle area, TRF=tenth rib fat thickness, PM=percentage of muscling, DP= dressing percentage. bWhittemore, 1993. Adequate pen space, m2/pig = .035 BW-*7, kg; Inadequate pen space (crowded), m2/pig = .025 BW'67, kg. *CP effect, P < .01. *0? effect. P < .03. *CP effect, P < .10. 'Pen space effect, P < .01. *Pen space effect, P < .03. hCP x Cr effect, P < .05. *CP x Cr effect, P < .09, jCP x pen space effect, P < .01. ‘‘CP x pen space effect, P < .08. 'Cr x pen space effect, P < .07. 70
ADFI were lower (P < .01) in pigs provided inadequate pen space than in pigs
provided adequate pen space. Gain/feed was not affected (P > . 10) by pen space.
In pigs fed 80% of the lysine requirement, Cr supplementation increased ADG and
gain/feed; however, in pigs fed 120% of the lysine requirement Cr supplementation
decreased ADG and gain/feed (CP x Cr effect, P < .09 and P < .05,
respectively).
During the overall combined grower and finisher phases pigs fed 120% of the
lysine requirement had higher (P < .01) ADG and gain/feed and lower (P < . 10)
ADFI than pigs fed 80% of the lysine requirement. Pigs provided inadequate pen
space had lower (P < .01) ADG and ADFI, but similar gain/feed (P > .10), than
pigs provided adequate pen space. In pigs fed 80% of the lysine requirement, Cr
supplementation increased gain/feed; however, in pigs fed 120% of the lysine
requirement Cr supplementation decreased gain/feed (CP x Cr effect, P < .05).
Carcass Traits
Pigs fed 120% of the lysine requirement had greater (P < .01) LMA and PM and less (P < .01) TRF than pigs fed 80% of the lysine requirement (Table 5.2).
Pigs provided inadequate pen space had greater LMA (P < ,03) and PM (P < .01) and less TRF (P < .01) than pigs provided adequate pen space. Chromium supplementation increased TRF in pigs provided inadequate pen space; however, Cr supplementation decreased TRF in pigs provided adequate pen space (Cr Xpen space effect, P < .07). 71
Plasma Mrtahnlitre and Hnrmnnea
Prefeeding. In pigs that were fed 120% of the lysine requirement, plasma glucose (P < .11), NEFA (P < .03), and urea N (P < .01) were greater, but plasma cholesterol was lower (P < .01) than in pigs fed 80% of the lysine requirement (Table S.3). Chromium supplementation decreased plasma NEFA concentration of pigs fed 80% of the lysine requirement but increased plasma NEFA concentration of pigs fed 120% of the lysine requirement (CP x Cr, P < .10). In pigs provided inadequate pen space, increasing dietary lysine (120% of requirement) increased plasma glucose; however, in pigs given adequate pen space, dietary lysine did not affect plasma glucose concentration (CP x pen space effect, P < . 11). In pigs provided inadequate pen space, Cr supplementation decreased plasma insulin of pigs fed 80% of the lysine requirement but did not affect plasma insulin of pigs fed
120% of the lysine requirement. However, in pigs provided adequate pen space, Cr supplementation increased plasma insulin of pigs fed 80% of the lysine requirement but decreased plasma insulin of pigs fed 120% of the lysine requirement (CP X Cr x pen space effect, P < .08). Plasma GH was not affected (P > . 10) by treatment.
Pnstfeeding. In pigs that were fed 120% of the lysine requirement, plasma glucose (P < .11), NEFA (P < .11), cholesterol (P < .01), and GH (P < .01) were lower, but plasma urea N and insulin were higher (P < .01) than in pigs fed
80% of the lysine requirement (Table 5.3). In pigs provided adequate pen space, Cr Table 5.3. Effect of dietary chromium, crude protein level, and pot space on plasma glucose, NEFA, insulin, and growth hormone concentrations of growing-finishing pigs1 ______Percentage of lysine requirement 80 120 80 120 80 120 80 120 Cr. ue/kp diet 0 0 400 400 0 0 400 400 Pen soace allowance. m2/meb Pooled Criterion .035 .035 .035 .035 .025 .025 .025 .025 SEM Glucose, mmol/L Unfed* 4.5 4.5 4.6 4.5 4.3 4.6 4.3 4.5 .1 Fed* 5.8 5.5 6.4 5.8 6.0 5.9 6.0 5.7 .3
NEFA, mEq/L Unfed44 .64 .73 .57 .80 .74 .72 .59 .76 .07 Fed* .i6 .12 .14 .13 .16 .14 .17 .13 .02
Insulin, pU/mL Unfed" 6.0 4.4 4.1 4.5 3.8 5.6 7.0 5.1 1.1 Fed*4 19.1 28.0 17.5 38.4 15.4 41.8 21.3 32.1 5.7
Growth hormone, ng/mL Unfed 9.5 7.7 6.6 4.7 7.4 4.4 6.4 6.8 1.6 Fed* 11.4 9.7 11.9 8.5 13.8 7.7 12.2 8.9 1.8 Urea nitrogen, mmol/L Unfed* 5.5 7.9 5.5 7.9 5.2 8.3 4.9 8.4 .5 Fed* 5.8 9.5 5.6 8.0 6.0 8.5 5.7 8.5 .5 (table con’d) Cholesterol, mmol/L Unfed6 2.6 2.2 2.5 2.1 2.6 2.2 2.7 2.2 .1 Fed6______2.7 2.3 2.5 2 3 2.6 2.3 2.7 2.4 .1 'Data are means of four replicates with four pigs per replicate; average initial BW = 27.2 kg; Unfed = 20 h without access to feed; Fed - 2 h postfeeding. bWhittemore, 1993. Adequate pen space, m2/pig = .035 BW 67 kg; Inadequate pen space (crowded), m2/pig = .025 BW 67 kg. 6CP effect, P < .01. "CP effect, P < .03. 'CP effect, P < .11. fCP X Cr effect, P < .10. *CP x pen space effect, P < .11. bCP x Cr x pen space effect, P < .08. *CP x Cr x pen space effect, P < .10. 74
supplementation decreased plasma insulin of pigs fed 80% of the lysine requirement
but did not change plasma insulin of pigs fed 120% of the lysine requirement.
However, in pigs provided adequate pen space, Cr supplementation increased
plasma insulin of pigs fed 80% of the lysine requirement but decreased plasma
insulin of pigs fed 120% of the lysine requirement (CP x Cr x pen space effect,
P < .08).
Discussion
The literature shows varied growth performance responses to Cr
supplementation of pig diets. Page et al. (1993b) reported either decreased growth
performance or no effect of Cr supplementation in separate experiments.
Lindemann et al. (1995) reported that 250 and 500 ppb Cr as CrPic tended to reduce
ADFI without affecting ADG, resulting in an increase in gain/feed ratio. However,
Evock-Clover et al. (1993) reported no effect of CrPic on ADG, ADFI, or gain/feed. Mooney and Cromwell (1995) reported that CrPic increased ADG but did not affect ADFI or gain/feed. The current data are consistent with those of lindemann (1993) whereby Cr supplementation of diets that are deficient in lysine
(or at least marginally so) improves feed efficiency (and ADG, finisher phase only).
The mechanism responsible for this effect is not known; however, the involvement of Cr in RNA and DNA synthesis (Okada et al., 1984) may be responsible or an increase in amino acid uptake (Hegsted et al., 1995). 75
The effects of crowding on the growth performance of pigs are known
(Komegay and Notter, 1984; NCR-89, 1986). Crowding previously has been
reported to reduce growth performance of pigs primarily through its effect of
reducing feed intake (Komegay et al., 1993; Moser et al., 1985; NCR-89, 1986).
However, there has been some discussion as to whether increasing the nutrient
density of pigs diets could overcome the effects of some stressor (e.g., crowding)
(Baker, 1995). Our data indicate that increasing the CP content of the diet will
improve growth performance of pigs that have been subjected to crowding.
Carcass Traits
As expected, the pigs fed lysine (and CP) adequate diets had greater LMA and
less TRF than pigs fed inadequate lysine. The presence of adequate or perhaps excess dietary amino acids coupled with the increased feed efficiency of pigs fed
120% of the lysine requirement were probably responsible for this dramatic effect.
However, the improved carcass traits of pigs that were given inadequate pen space was not necessarily expected. The response was probably due not only to decreased
ADFI of crowded pigs but also a generally slower growth rate. Another possible cause of the reduction in TRF may have been a reduction in glucose utilization by the adipose tissue with subsequent reduction in lipogenesis as reported by
Rosebrough and Steele (1986). However, feed efficiency of crowded pigs was decreased or not affected by crowding. One would expect that an increase in feed efficiency would be coupled with increased carcass lean. Zimmerman (1995) 76
reported similar increases in muscling and decreases in fat due to reducing pen space
in pigs fed to 136 kg.
We had originally hypothesized that stressed (i.e., crowded) pigs may respond
to Cr supplementation more readily than unstressed (i.e., uncrowded). However, no
main effects of Cr supplementation were observed for carcass traits. In fact, Cr
supplementation of crowded pigs tended to make the carcasses of those pigs fatter
(i.e., increased TRF). It may be that some other form of stress is necessary or that
the pigs adapted to the crowding situation and thus were not "stressed”. Another
possibility is that the linear carcass measurements were not sensitive enough to
detect differences in carcass composition. Boleman et al. (1995) and Mooney and
Cromwell (1995) have reported that CrPic supplementation of pig diets resulted in
increased carcass lean and decreased carcass fat without measurable differences in
linear carcass measurements. In addition, the rate of lean accretion was increased
and the rate of fat accretion was decreased (Boleman et al., 1995; Mooney and
Cromwell, 1995).
Lindemann et al. (1993) reported that the level of dietary CP may affect the
response obtained from Cr supplementation; pigs fed marginal levels of CP (lysine) had increased muscling and decreased fat in response to Cr supplementation.
However, Harris et al. (1995) reported no effect of dietary CrPic on growth performance or carcass characteristics of pigs fed adequate and low CP diets. Some investigators have hypothesized that pigs that have increased potential for lean gain 77
may not be as responsive to Cr supplementation as pigs with less potential for lean
gain (Roger Campbell, Prince Nutrition Conference, 1993). The pigs in the current
experiment had an average PM of 48.5% even with the low lysine treatments (pigs
with adequate lysine PM — 50.2%). Previous studies that have demonstrated
positive responses to Cr supplementation have typically used pigs with PM in (he
low to mid 40%’s (Page et al., 1993a,b). The specific conditions necessary to elicit
improved carcass characteristics or carcass composition of pigs fed supplemental Cr
are not known. However, as we previously hypothesized (see Chapter 1 and 2), the
level of stress, genotype, or Cr status may affect the response to Cr
supplementation.
Both insulin and glucose have been reported to reduce plasma NEFA
concentrations (Dole, 1956). Therefore, the increased plasma NEFA after 20 h
without access to feed in pigs fed 120% of the lysine requirement compared with pigs fed 80% of the lysine requirement is an enigma. These pigs tended to have higher plasma glucose but lower plasma insulin concentrations than pigs fed 80% of the lysine requirement. One would expect that die lower carcass lipid stores (lower
TRIO would have resulted in less adipose stores for NEFA mobilization. In fact, this seems to be the case in the 2 h postfeeding samples. Pigs tied 120% of the lysine requirement had higher plasma insulin and lower plasma NEFA concentrations. Atinmo et al. (1978) reported that protein-malnourished pigs failed 78
to respond to arginine-induced insulin release. Furthermore, protein-malnourished
pigs had higher NEFA levels than control pigs. These findings support the current
data, in which pigs fed 80% of the lysine requirement had lower plasma insulin and
higher plasma NEFA concentrations 2 h postfeeding than pigs fed 120% of the
lysine requirement. In addition, growth hormone is associated with increased
lipolysis (Keller and Miles, 1991) and may be involved in the higher plasma NEFA
levels of pigs fed 80% of the lysine requirement compared with pigs fed 120% of
the lysine requirement. Plasma urea N was higher in pigs fed diets containing adequate (probably excess) amino acids. These effects have been reported previously (Ward and Southern, 1993).
Lindemann et al. (1995) reported that dietary Cr supplementation diminished the insulin response to feeding in gestating gilts. Evock-Clover et al. (1993) also reported that dietary Cr tended to “normalize" glucose and insulin concentrations in pigs injected with ppST. The changes in plasma insulin may depend on both the nutritional and environmental status of the pig, as well as other factors. For example, dietary Cr appeared to accentuate the protein-induced increase in plasma insulin of uncrowded pigs. However, dietary Cr supplementation somewhat modulated the protein-induced increase in plasma insulin of crowded pigs. Thus the ability of a pig to respond to supplemental Cr may depend on the overall health and nutritional status of the pig. 79
Amoikon et al. (1995) reported that dietary Cr supplementation decreased
plasma NEFA level of growing pigs. In the present investigation, the metabolic
response to dietary Cr supplementation again appeared to depend on the nutritional
status of the pig. Dietary Cr supplementation was effective in reducing plasma
NEFA of pigs fed inadequate lysine, but dietary Cr increased plasma NEFA levels of pigs fed adequate lysine. The differences in nutritional or environmental status of pigs may partially account for the varied responses in the literature.
Implications
The assertion that dietary chromium may alleviate “stress” in animals or that
“stressed” animals may respond in a positive manner to chromium supplementation is not apparent in pigs in which restricted pen size is the stressor. However, under conditions in which pigs are fed diets which arc inadequate or marginally adequate in amino acids (or protein), supplemental dietary chromium may result in improved growth performance. Thus, the economic benefits of supplementing pig diets with chromium may depend on the production environment and availability of adequate dietary protein as well as other factors. CHAPTER VI
EFFECT OF DIETARY CHROMIUM TRIPICOLINATE ON GROWTH PERFORMANCE, BODY COMPOSITION, PLASMA METABOLITES AND HORMONES, AND GLUCOSE TOLERANCE AND INSULIN SENSITIVITY IN BROILERS
Introduction
Chromium is known to influence protein, lipid, and carbohydrate metabolism,
and it has been'shown to promote growth in turkey poults (Steele and Rosebrough,
1979, 1981; Rosebrough and Steele, 1981) and to increase N utilization in lambs
(Britton et al., 1968). Recently, Cr supplementation of swine diets was reported to increase carcass muscling and decrease tenth rib fat thickness (Lindemann et al.,
1993, 199S; Page et al., 1993a,b). Boleman et al. (1995) and Mooney and
Cromwell (1995) also have reported that dietary Cr increased the rate of lean deposition and decreased the rate of fat deposition in pigs.
Consistent reduction of the fat content of broiler carcasses has eluded the poultry industry. Chromium is capable of facilitating the action of insulin (Mertz,
1969); therefore, it may be efficacious in modulating insulin and glucose metabolism. Lower plasma glucose and higher plasma insulin has been associated with increased body fat in growing broilers (Touchbum et al., 1981). Because of the association of Cr with nutrient utilization and improvements in body composition
(increased muscling and decreased fat), it may be possible to supplement broiler diets with Cr and achieve improvements (increased lean and decreased fat) in carcass
80 81
composition. Therefore, these experiments were conducted to determine the effect
of dietary Cr as CrPic on growth performance, carcass composition, and glucose clearance rate in broilers.
Materials and Methods
Three experiments were conducted to determine the effects of dietary Cr as Cr tripicolinate (CrPic) on growth performance and body composition of broilers. An additional experiment was conducted to determine the effect of dietary CrPic on glucose clearance rate and insulin sensitivity of broilers. Unless otherwise indicated, all birds were housed in heated starter batteries during the first 2 weeks (wk) of each experiment and moved to grower batteries during the ensuing experimental periods.
Battery pens were housed in an environmentally controlled building. Starter batteries, as well as the room in which both starter and grower batteries were housed, contained continuous fluorescent lighting.
Experiment 1
An experiment was conducted using unsexed Arbor Acres x Arbor Acres broiler chicks (Sanderson Farms, Laurel, MS) from 5 to 19 days of age. From hatching to 4 days posthatching, all chicks were fed a com-soybean meal diet (Table
6.1). After they were deprived of feed overnight, chicks were weighed and randomly assigned to treatments on the basis of body weight (BW; average initial
BW = 64.2 g). Each treatment consisted of four replicates of five chicks each. A control treatment (four pens of five chicks each) without Cr supplementation was 82
Table 6.1. Composition of the basal diet, as-fed basis (Exp. 1) I n g r e d i e n t ______%
Com 47.14 Soybean meal, 44% CP 42.S0 Com oil 5.00 Defluorinaled rock phosphate 2.1.0 Alfalfa leaf meal 2.00 Oyster shell flour .40 Salt .40 Vitamin-mineral premix1 .25 DL-Methionine . 15 M nS04'H 20 .05 Z nC 03 .01
Total 100.00
Nutrient Calrnlatort Crude protein 23.06 Calcium .99 Total phosphorus .79 Available phosphorus .53 Methionine + cystine .87 Lysine 1.28 Metabolizable energy, kcal/lcg 3,038 'Roche Chemical Division, Nutley, NJ 07110. Provided the following per kilogram of diet: vitamin A (retinyl acetate), 6,614 IU; cholecalciferol, 1,653 IU; dl-a-tocopheryl acetate, 7 IU; menadione, 1.5 mg; thiamine, 1.1 mg; riboflavin, 6.6 mg; niacin, 33.1 mg; d-pantothenic acid, 11.0 mg; folic add, .7 mg; pyridoxine, 1.1 ing; d-biotin, .055 mg; vitamin Bw, .011 mg; choline (as chloride), 551 mg. 83 included. The basal diet was supplemented with 0, 200, or 400 Atg Cr/kg diet as
CrPic (12% Cr). Chicks were allowed ad lihitum access to feed and water.
Total fecal collections were taken from day 10 to 14 of the experiment at 24 h intervals. The feces were pooled by replicate, lyophilized, and ground. Kjeldahl nitrogen (N) analysis was conducted on the feed and fecal samples. At the termination of the experiment (day 14), individual chicks were weighed and pen feed consumption was determined. All chicks were killed via cervical dislocation.
Three chicks per replicate were randomly selected and nmscle samples of the breast and leg were collected. The other two chicks per replicate were placed in plastic bags and frozen for later whole-body compositional analyses. Moisture percentage of the muscle samples was determined after lyophilizing. Ether extract was conducted by Soxhlet extraction (ethanol and diethyl ether, 20 h each). Kjeldahl N analysis of the meat samples also was conducted. For whole-body composition, chicks were defrosted, weighed, placed in a stainless-steel blender container, and autoclaved for 90 min (121 °C and 1.27 kg/cm2). Samples were cooled for 15 min, water was added (10% of the chick weight), and the sample was blended until smooth. Samples of the slurry were dried in a forced-air oven (100°C for 16 h) for dry matter (DM) determination. After drying the samples were placed in a muffle furnace (600°C for 8 h) for determination of ash. An additional sample was lyophilized, ground, and analyzed for Kjeldahl N. Fat analysts of the slurry was conducted by rapid analysis methodology using a CEM Model AVC 80 analyzer 84
(AOAC, 1990). All whole-body analyses were conducted in duplicate and were corrected for moisture content (i.e., expressed on a dry matter basis).
Experiment 2
Male Ross x Peterson broilers (Sanderson Farms, Laurel, MS) were allowed a 4-d adaptation period as in Exp. 1. The chicks (average initial BW — 81 g) were then randomly assigned to four dietary treatments: 1) com-soybean meal basal
(Table 6.2), 2-4) the basal diet plus 400, 1,600, or 16,000 g Cr as CrPic/kg diet.
Each treatment was replicated three times with five chicks per replicate pen. A control treatment (three pens of five chicks each) without Cr supplementation was included. The chicks were weighed weekly and pen feed intake was determined weekly. The chicks were bled (approximately 3 mL) via wing vein puncture following 12 h without access to feed (fasted) and again 2 h postfeeding on day 25 of the experiment. The blood was placed into tubes (Monoject®, Sherwood
Medical, St. Louis, MO) containing 8 mg potassium oxalate and 10 mg sodium fluoride and immediately placed on ice. The blood was centrifuged (1,600 x g for
20 min at 4°C) and the plasma was collected and frozen for later hormone and metabolite analyses. The chicks were killed via CQj inhalation on day 28 of the experiment. The digestive tract of each chick was removed and cleaned of contents.
In addition liver, heart, and ventriculus weights were recorded. The digestive tract and organs were then placed into plastic “ziplock" bags with the carcass and frozen for subsequent proximate analyses methods as in Exp. I. 85
Table 6.2. Composition of the basal diet, as-fed basis (Exp. 2) Ingredient %
Soybean meal, 44% CP 46.18 Com 41.99 Com oil 6.00 Alfalfa leaf meal 2.00 Dicalcium phosphate 1.53 Oyster shell flour 1.35 Vitamin-mineral premix* .25 Salt .40 DL-Methionine .20 Selenium premixb .10
Total 100.00
Nutrient Calculated Crude protein 23.00 Calcium 1.00 Total phosphorus .69 Available phosphorus .45 Methionine + cystine .93 Lysine 1.36 Metabolizable energy, kcal/kg 3,039 'Provided the following per kilogram of diet: vitamin A (retinyl acetate), 11,000 IU; cholecalciferol (deactivated animal sterol), 1,650 IU; dl-a-tocopheryl acetate, 8 IU; menadione (menadione sodium bisulfite), .72 mg; thiamine (mononitrate), 1 mg; riboflavin, 4.4 mg; niacin, 33 mg; d-pantothenic acid (d-calcium pantothenate), 8.1 mg; folic add, .33 mg; d-biotin, .055 mg; vitamin B13, .011 mg; choline (as chloride), 382 mg; manganese (oxide), 60 mg; zinc (sulfate), 44 mg; iron (ferrous sulfate), 20 mg; copper (oxide), 2 mg; iodine (ethylenediamine dihydriodide), 1.2 mg; cobalt (carbonate), .2 mg; ethoxyquin, 125 mg. '’Provided .1 mg Se per kilogram of diet as NajSeOj. 86
E x p erim e n t
Male and female (two replicates of five chicks of each sex; average initial
BW = 79 g) were randomly assigned to three dietary treatments. The chicks were
fed one of the following three diets: 1) a com-soybean basal diet (Table 6.3), 2) the
basal diet plus 400 pg Cr as CrPic/kg diet, or 3) the basal diet plus 1,600 pg C r as
CrPic/kg diet. The diets were reduced in crude protein (CP) content as
recommended by the NRC (1994) at wk 3 and wk 6 of the experiment (Table 4.5).
The cliicks were weighed weekly and feed intake was determined weekly.
The chicks were bled (approximately 5 mL) via wing vein puncture
following 12 h without access to feed (fasted) and again 2 h postfeeding on day 45 of the experiment. The blood was placed into tubes (Monoject®) containing 14 mg potassium oxalate and 17.5 mg sodium fluoride and immediately placed on ice. The blood was centrifuged (1,600 x g for 20 min at 4°C) and the plasma was collected and finozen for later hormone and metabolite analyses. The birds were killed via
CQt inhalation on day 49 of the experiment. The digestive tract of each chick was removed and cleaned of contents. Liver, heart, spleen, proventriculus, small and large intestine, and ventriculus weights were recorded. Both breasts and the right leg and thigh of each bird were dissected of lean tissue. The lean tissue weight and the abdominal fat pad weight of each bird were recorded. The digestive tract, organs, and dissected tissues were then placed into plastic “ziplock* bags with the carcass and frozen for subsequent proximate analyses. 87
T^ble6;3;iCom^sitiOT^sAe^^d^eteiias^^!^M is_^^=3^ Wlf of P.yppriment Ingredient ______(K3______3^>______6-7
Soybean meal, 4496 CP 46.18 54.95 61.04 Com 41.99 33.40 27.64 Com oil 6.00 6.00 6.00 Alfalfa leaf meal 2.00 2.00 2.00 Dicalcium phosphate 1.53 1.19 1.00 Oyster shell flour 1.35 1.51 1.37 Vitamin-mineral premix1 .25 .25 .25 Salt .40 .40 .40 DL-Methionine .20 .20 .20 Selenium premixb .10 .10 .10
Total 100.00 100.00 100.00
Nutrient Calculated Crude protein 23.00 20.00 18.00 Calcium 1.00 .90 .80 Total phosphorus .69 .63 .56 Available phosphorus .45 .40 .35 Methionine + Cystine .93 .85 .79 Lysine 1.36 1.13 .97 Metabolizable energy, kcal/kg 3,039 3,141 3,217
•Provided the following per kilogram of diet: vitamin A (retinyl acetate), 11,000 IU; cholecalciferol (d-activated animal sterol), 1,650 IU; dl-a-tocopheryl acetate, 8 IU; menadione (menadione sodium bisulfite), .72 mg; thiamine (mononitrate), 1 mg; riboflavin, 4.4 mg; niacin, 33 mg; d-pantothenic acid (d-calcium pantothenate), 8.1 mg; folic acid, .33 mg; d-biotin, .055 mg; vitamin B,a, .011 mg; choline (as chloride), 382 mg; manganese (oxide), 60 mg; zinc (sulfate), 44 mg; iron (ferrous sulfate), 20 mg; copper (oxide), 2 mg; iodine (ethylenediamine dihydriodide), 1.2 mg; cobalt (carbonate), .2 mg; ethoxyquin, 125 mg. '’Provided . 1 mg Se per kilogram of diet as Na2Se03. 88
Proximate Analyses
In Exp. 2 and 3, three birds per replicate were selected at random for carcass
compositional analyses. The carcass, digestive tract, and tissues of each bird were
thawed, separated, and weighed. In Exp. 2 the digestive tract plus organs (Gut) and
in Exp. 3 the leg plus thigh meat (Dark), and breast meat (White), and Gut were
ground individually in a Hobart (Model No. A-200) grinder equipped with a .64 cm
die and then further ground in a food processor. Samples of the Gut, Dark, and
White were placed into 7.6-cm x 12.7-cm plastic "ziplock" bags. The remainder of
the carcass was placed into a stainless-steel container, enclosed with a plastic
cooking bag (Reynolds Metal Co., Richmond, VA), and autoclaved for 120 min
(121°C and 1.27 kg/cm2). The carcass was immediately blended until smooth and a
sample of the resultant slurry was placed into a 7.6-cm x 12.7-cm plastic "ziplock”
bag. Ten percent of the Gut, Dark, and White tissues from each bird within a
replicate were included in the pooled sample. One percent of the weight of each
carcass within a replicate was used for pooling. Pat and moisture analyses of each
of the pooled samples (Gut, Dark, White, and carcass) were conducted by rapid
analysis methodology using a CEM Model AVC 80 analyzer (AOAC, 1990).
Duplicate samples of the slurry were dried in a forced-air oven (100° C for 12 h) for
DM determination. After drying, the samples were placed in a muffle furnace
(5S0°C for 12 h) for determination of ash. An additional sample was lyophilized, ground, and analyzed for Kjeldahl N. 89
Experiment A
An experiment was conducted to determine the effect of dietary Cr as CrPic
on glucose tolerance and insulin sensitivity in mature broilers. Twenty-eight-day-old
unsexed Arbor Acres x Arbor Acres broilers (Sanderson Farms, Laurel, MS) were
used. From hatching to 28 days posthatching, all broilers were fed a com-soybean
meal diet (Table 6.1). After they were deprived of feed overnight, the broilers were
weighed and randomly assigned to treatments on the basis of BW (average initial
BW= 976 g). Chicks were provided continuous fluorescent lighting and caged in
starter batteries with raised wire floors. The battery heaters were not used and every
other battery level was used to allow adequate space for the broilers. Each treatment
consisted of four replicate pens of five chicks each. The basal diet was
supplemented with 0 (a control treatment) or 200 ^g Cr/kg diet as CrPic. Chicks
were allowed ad lihitnm access to feed and water. The diets were changed at 6 wk
of age as recommended (Table 6.4; NRC, 1994).
Growth performance [average daily gain (ADG), average daily feed intake
(ADFI), and gain/feed] was measured from 28 to 58 days of age. On day 58, two broilers per replicate were randomly selected to remain on treatment diets; the other three broilers were killed via C02 inhalation. The remaining broilers were fed the treatment diets until approximately 11 wk of age. After being without feed overnight, the broilers were fitted with brachial vein catheters. This was accomplished by inserting a 16-gauge needle into the brachial vein and passing 90
Table 6.4. Composition of the basal diets, as-fed basis (Exp. 4) ______Wk o f Experiment Ingredient ______3^6______6-11
Soybean meal, 44% CP 57.04 64.80 Com 33.97 27.95 Com oil 6.04 4.62 Defluorinated nock phosphate 1.40 1.17 Oyster shell flour .90 .88 Vitamin-mineral p remix* .25 .25 Salt .24 .24 Selenium premixb .10 .10 DL-Methionine .07
Total 100.00 100.00
Nutrient f ’fllfinlateri Crude protein 20.00 18.00 Calcium .90 .80 Total phosphorus .63 .57 Available phosphorus .40 .35 Methionine + Cystine .72 .65 Lysine 1.13 .97 Metabolizable enerfiVT kcal/kg 3.200 3,200
"Provided the following per kilogram of diet: vitamin A (retinyl acetate), 11,000 IU; cholecalciferol (d-activated animal sterol), 1,650 IU; dl-a-tocopheryl acetate, 8 IU; menadione (menadione sodium bisulfite), .72 mg; thiamine (mononitrate), 1 mg; riboflavin, 4.4 mg; niacin, 33 mg; d-paiitothenic acid (d-calcium pantothenate), 8.1 mg; folic acid, .33 mg; d-biotin, .055 mg; vitamin B12l .011 mg; choline (as chloride), 382 mg; manganese (oxide), 60 mg; zinc (sulfate), 44 mg; iron (ferrous sulfate), 20 mg; copper (oxide), 2 mg; iodine (ethylenediamine dihydriodide), 1.2 mg; cobalt (carbonate), .2 mg; etlioxyquin, 125 mg. '’Provided . 1 mg Se per kilogram of diet as Na2Se03. 91 catheter tubing (i.d., .58 mm; o.d., .965 mm; Intramedic®, Becton Dickinson &
Co., Parsippany, NJ) through the needle. The needle was then removed and the tubing remained in the vein. The tubing was glued with "super glue” to the skin under the wing, passed behind the wing, and taped to the back of the bird. Birds were held in individual pens during blood sampling.
After at least 1 h rest, the broilers were given a bolus dose of .88 g glucose/kg
BW intravenously (TVGTT) as a 50% solution in .9% NaCl. Blood samples were collected at -10, 0, 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, 180 min relative to glucose infusion. The blood was placed into tubes containing 10 mg sodium fluoride and 8 mg potassium oxalate and immediately placed in an ice bath. The blood samples were centrifuged (1,600 x g for 20 min at 4°C) and the plasma wits collected and frozen for later hormone and metabolite analyses. At least 2 h after the last sample was collected in the IVGTT, an intravenous insulin challenge test
(TVTCT) was conducted. The broilers were administered i.v. 1.5 U bovine insulin
(Sigma Cat.# 1-5500, Lot# 112H0353, Sigma Chemical Co., St. Louis, MO)/kg BW as a bolus dose. The insulin was administered as a solution containing 13.5 U/niL in .9% NaCl containing .2% bovine serum albumin (Fraction V Powder, Cat.# 810-
1018, Lot# A090767, Grand Isle Biological Co., Grand Isle, NY). Blood samples were collected in the same manner as the IVGTT and at the same time points relative to i.v. infusion of insulin. 92
Glucose clearance rate (k, %/rain) and half-life (min) were calculated between
IS to 45 min and 0 to 60 min for the IVGTT and IVICT, respectively, as a modification of the procedure outlined by Kaneko (1989). Insulin clearance rate (k,
%/min) and half-life (min) during the IVGTT were calculated from 15 to 30 min.
Approximately 2 d after the IVGTT and IVICT, a least sampling minimal model glucose tolerance test (LSMM) was conducted (Ader et al., 1985; Bergman,
1989). This procedure amounts to a combination IVGTT and IVICT. The basis of such a procedure is to separate, via computer analysis, the insulin and glucose effects on glucose clearance from the plasma. The techniques have been used successfully in mammals (Ader et al., 1985; Yang et al., 1987; Bergman, 1989).
The methodology is similar to the IVGTT and IVICT previously conducted. The broilers were administered i.v. .528 g glucose/kg BW at time 0. Blood samples were collected as described previously except at -1,2, 4, 8, 19,22, 30, 40, 50, 70,
90, and 180 min relative to glucose administration. In addition, at 20 min postinfusion of glucose, .75 IU bovine insulin/kg BW was administered i.v.
Chemical Analyses
Plasma cholesterol (Sigma, 1989), glucose (Sigma, 1990), and NEFA (TVGTT
-10 min samples only, NEFA-C Kit, ACS-ACOD Method; Wako Chemicals USA,
Inc., Richmond, VA) were analyzed using spectrophotometric procedures outlined in commercially-available kits. 93
Insulin was measured in plasma samples with a homologous radioimmunoassay previously validated for chicken tissues (McMurtiy et al., 1983). Antiserum against chicken insulin (supplied by J.P. McMurtiy, USDA, Beltsville, MD) was diluted
1:20,000 in normal guinea pig serum (1:400), and 200 #*L of this solution was used per tube. Highly purified chicken insulin (obtained from D.J. Bolt, USDA,
Beltsville, MD) was radioiodinated via the chloramine-T method (Greenwood et al.,
1963), and was used at approximately 40,000 cpm/tube. An aliquot of the highly purified hormone was diluted to 10 ng/mL and used as the reference standard. For assay, samples (between 100 and 200 /iL) and standards were mixed with the first antibody and were incubated *24 h. All incubations were at S°C. After addition of labeled hormone, the tubes were vortexed and then incubated *24 h. Diluted (1: IS) antiserum generated in a sheep against guinea pig gamma-globulin was added, and the tubes were vortexed and incubated *48 h. The precipitated insulin-antibody complexes were harvested by centrifugation after addition of 2 mL of .01 M phosphate-buffered .155 M saline. Serial dilution of chicken plasma produced an inhibition curve in the assay parallel to the reference standard, and recovery of purified chicken insulin added to a low-insulin plasma pool was 115%. Sensitivity of the assay was .02 ng; intra- and interassay coefficients of variation averaged 8 and 10%, respectively. Due to the crossreactivity of bovine insulin with the chicken insulin assay, insulin analysis was not conducted on the IVICT or LSMM samples. 94
Plasma immunoreactive glucagon was assayed as outlined by Gaskins et al.
(1991). Rabbit anti-porcine glucagon antibody (Unger 04A, Pool 1, Lot #22; R.H.
Unger, Southwestern Medical Center, The University of Texas, Dallas, TX) specific for small molecular weight (3,500 mol. wt.) glucagon was used at a final dilution of
1:112,500. Purified porcine-bovine glucagon (Product No. G-2450; Sigma
Chemical Co.) was the standard. Porcine 125I-glucagon (Product No. 07-152126;
ICN Biomedicals, Inc.) was the radioligand, and goat anti-rabbit immunoglobulin G prepared in the laboratory of Donald L. Thompson, Jr. (Louisiana State University
Agricultural Center, Baton Rouge, LA) was used at a 1:12 dilution as the precipitating antibody. The intraassay CV as determined by pooled turkey plasma samples was 5.8%.
Statistical Analyse
The growth performance, organ and tissue weight, metabolite and hormone, and carcass composition data were analyzed as completely random designs using the pen as the experimental unit. In Exp. 3, the data were analyzed as a 3 x 2 factorial arrangement of treatments in a completely random design. The two factors were three levels of Cr and two sexes. In Exp. 4, the glucose and insulin clearance data were analyzed as a completely random design using the individual bird as the experimental unit. The LSMM data was analyzed as a split-plot in time with replicate within treatment as the error term in the model to test for treatment effects. Results
Experiment 1
Supplemental dietary Cr did not affect (P > .10) ADG, ADFI, gain: feed
ratio, or N balance fTable 6.5). Supplemental Cr did not affect (P > . 10) the
percentage of CP, moisture, or ether extract in breast and leg muscle samples. In
addition, Cr supplementation did not affect (P > .10) whole-body CP, fat, or ash
percentage. However, dietary Cr supplementation decreased percentage of DM
(quadratic effect, P < .09). In addition, 200 Mg Cr/kg diet tended to decrease
(P < . 13) fat percentage compared with the control diet.
Experiment 2
Dietary Cr supplementation did not affect (P > .10) ADG, ADFI, or
gain/feed (Table 6.6). Chromium supplementation did not affect (P > .10) liver,
heart, or abdominal fat pad weights; however, ventriculus weight was increased (Cr
cubic effect, P < .07) in broilers fed supplemental Cr. Dietary Cr supplementation
increased carcass ash percentage (Cr quadratic effect, P < .06); however, carcass and Gut moisture, fat, CP, and Gut ash percentage were not affected (P > .10) by dietary Cr supplementation. Dietary Cr supplementation did not affect (P > .10) plasma NEFA, cholesterol, glucose, insulin, or glucagon concentrations in the fed or fasted state. 96
Table 6.5. Effect of dietary chromium on growth performance, N balance, and breast and legjnusd^n^wh^^bod^com^sition^^toJQ^^^^l^^lSJ^^EiJil Dietary Cr, jug/kg ------Pooled Item______0______200______400______SEM Growth performance ADG, g/chick 26.8 26.8 26.6 .5 ADFI, g/chick 36.7 36.6 36.7 .5 Gain:feed .731 .732 .725 .009 N balance, g/chick/d 1.40 1.35 1.40 .04
Breast CP, % 23.4 23.6 23.6 .4 M oisture, % 75.2 75.7 75.3 .3 Ether extract, % 2.3 2.2 2.5 .3
Leg CP, % 19.6 20.6 18.5 1.4 M oisture, % 74.5 72.3 73.0 1.0 Ether extract, % 7.6 10.6 10.4 1.1
Whole-Body DM, %b 30.3 28.9 29.7 .4 CP, % of DM 54.3 55.9 54.4 1.0 Fat, % o f DM 31.3 28.6 29.2 1.1 Ash. % of DM 7.9 8.3 8.1 .2 “Growth performance data are means of four replicates of Eve broilers each. Breast and leg composition data are means of four replicates of three chicks each and whole-body composition data are means of four replicates of two chicks each. bCr quadratic effect, P < .09. 97
Table 6.6. Effect of dietary chromium on growth performance, organ weights, body
Dietary Cr, //g/kg Pooled 0 400 1.600 16.000 SEMItem Growth performance ADG, g/chick 49.3 49.2 49.9 49.1 .8 ADFI, g/chick 80.1 81.5 82.0 80.5 1.2 Gain:feed .615 .604 .608 .610 .010
Organ weights, % BW Liver 2.82 2.89 3.03 2.89 .11 Heart .64 .62 .66 .63 .03 Ventriculusb 1.57 1.78 1.84 1.83 .05 Abdominal fat pad .53 .56 .55 .59 .04
Body Composition Carcass Moisture, % 68.8 68.9 69.7 68.3 .7 Fat, % 8.3 8.5 7.9 8.9 .4 CP, % DM 61.7 63.2 64.0 63.4 1.5 Ash, % DM* 9.2 9.4 9.7 8.9 .2
Gut M oisture, % 71.8 72.8 73.9 72.5 1.0 Fat, % 6.2 4.7 5.0 5.9 1.0 CP, % DM 68.4 68.1 65.4 64.5 3.3 Ash, % DM 5.0 5.0 4.9 4.7 .2
Metabolites & hormones Fed NEFA, mEq/L .393 .397 .378 .389 .012 Cholesterol, mmol/L 2.8 2.9 2.7 2.6 .1 Glucose, mmol/L 11.32 11.79 11.59 11.96 .20 Insulin, ng/mL 1.35 1.13 1.26 1.07 .19 Glucagon, pg/mL 515.2 621.1 547.5 489.0 48.8 (table con’d) 98
Fasted NEFA, mEq/L .620 .588 .581 .547 Cholesterol, mmol/L 2.6 2.7 2.4 2.6 Glucose, mmol/L 9.91 10.21 9.98 10.35 Insulin, ng/mL .90 .96 .79 .82 Glucagon, pg/mL 736.7 794.5 668.9 810.1 94.1 •Data are means of three replicates of five broilers each; avg. initial wt. = 81 g. bCr cubic effect, P < .07. *Cr linear effect, P < .04, quadratic effect, P < .06. 99 Experiment 3
During the first three wk, dietary Cr supplementation did not affect (P > .10)
ADG; however, gain/feed was increased (Cr quadratic effect, P < .06) in birds fed
supplemental Cr (Table 6.7). Males grewer faster and ate more feed than females
(Sex effect, P < .OS). During wk 0 to 3, males ate the most feed when fed the diet
containing 400 ppb Cr; however, females ale the least feed when fed the diet
containing 400 ppb Cr (Sex X Cr quadratic effect, P < .06). During wk three to
six, neither dietary Cr supplementation nor sex affected (P > .10) ADG, ADFI, or gain/feed. During the last wk of tlie experiment, ADG and gain/feed were higher in males than in females (Sex effect, P < .05). Dietary Cr supplementation increased
ADG and gain/feed (Cr linear effect, P < .04); however, ADFI was not affected
(P > .10) by dietary Cr supplementation. Males had the highest ADG when fed the diet containing 400 ppb Cr; however, females had the highest ADG when fed the diet containing 1,600 ppb Cr (Sex x Cr quadratic effect, P < .06).
Dietary Cr supplementation did not affect (P > .10) liver, heart, ventriculus, pancreas, spleen, or small intestine weights (Table 6.8). Ventriculus weight as a percentage of BW was greater in males than in females (Sex effect, P < .07).
Proventriculus weight was the lowest in males when fed 400 ppb Cr; however, females had the highest proventriculus weight when fed 400 ppb Cr (Sex x Cr quadratic effect, P < .05). Large intestine weight was greatest in males fed the diet Table 6.7. Effect of dietary chromium on growth performance of 5 to 54- Dietary Cr. u s fk z Pooled Item______0______400______1,600______0______400______1,600 SEM Growth performance 0-3 wk ADG, g* 42.2 43.8 42.5 39.7 37.9 39.9 .9 ADFI, gW 66.0 66.2 64.3 62.9 58.7 60.1 1.3 Gainrfeed6*4 .639 .662 .660 .631 .646 .663 .008 3-6 wk ADG, g 54.4 62.2 64.5 56.9 53.3 52.6 5.9 ADFI, g 128.4 138.7 140.0 128.2 126.3 131.6 9.5 Gaimfeed .419 .449 .461 .444 .422 .401 .027 6-7 wk ADG, ^ 46.9 60.2 57.4 46.7 49.3 50.6 1.2 ADFI, g 149.4 166.3 163.0 131.1 153.7 155.3 10.9 Gain:feedb,c ______314______363______346______305______321______326______.007 'Data are means of two replicates of five broilers each; avg. initial wt. = 79 g. *Sex effect, P < .05, cCr linear effect, P < .04. dCr quadratic effect, P < .06. 'Sex x Or linear effect, P < .05. fSex x Cr quadratic effect, P < .06. 1 0 1 containing 1,600 ppb Cr; however, large intestine weight was greatest in females fed 400 ppb Cr (Sex x Cr quadratic effect, P < .01). Dietary Cr supplementation decreased leg meat as a percentage of BW in birds fed 400 ppb Cr but increased leg meat as a percentage of BW in birds fed 1,600 ppb Cr (Cr quadratic effect, P < .10; Table 6.8). Abdominal fat pad weight as a percentage of BW was decreased more in females fed 1,600 ppb Cr than males fed 1,600 ppb Cr (Sex X Cr linear effect, P < .09). Neither sex nor dietary Cr supplementation affected (P > .10) breast or thigh meat as a percentage of BW. During the fed state, males had higher plasma NEFA levels than females (Sex effect, P < .08, Table 6.9). However, during the fasted state, females had higher plasma NEFA levels than males (Sex effect, P < .03). Plasma cholesterol was increased by Cr supplementation during the fed (Cr linear effect, P < .07) and fasted (Cr quadratic effect, P < .02) states. During the fasted state, plasma cholesterol was higher in males than in females (Sex effect, P < .08). Plasma insulin, in the fasted condition, was higher in females than in males (Sex effect, P < .03). Plasma insulin, in the fasted state, was increased by Cr supplementation (Cr quadratic, P < .02). In the fed state, plasma insulin concentration was not affected (P > .10) by treatment. Plasma glucose and glucagon concentrations were not affected (P > .10) by treatment in either die fed or fasted state. Carcass moisture, in males, decreased as dietary Cr level increased; however, in females, carcass moisture increased as dietary Cr level increased (Sex x Cr linear Table 6.8. Effect of dietary chromium on organ and tissue weights of 5 to 54-dav-old broilers (Exp. 3)* Male Female Dietary Cr. uzPes. Pooled Item 0 400 1,600 0 400 1,600 SEM Organ weight, % BW Liver 2.82 2.46 2.41 2.11 2.37 2.55 .11 Heart .50 .51 .52 .53 .55 .55 .02 Proveotriculusf .27 .25 .30 .27 .29 .29 .01 Ventriculusb 1.21 1.07 1.22 1.30 1.38 1.35 .06 Pancreas .16 .18 .19 .20 .20 .17 .01 Spleen .19 .17 .14 .15 .15 .22 .02 Small intestine 1.86 1.88 1.98 2.03 2.05 1.85 .05 Large intestine' .47 .51 .61 .49 .57 .49 .01 Tissue weight, % BW Breast meat 11.94 12.07 12.36 12.82 11.70 13.49 .57 Leg meaf 2.92 2.82 3.36 3.00 2.74 2.93 .10 Thigh meat 3.73 3.75 3.84 3.57 3.34 3.91 .14 Abdominal fat pad4 1.49 1.65 1.19 1.82 1.79 1.25 .15 'Data are means of two replicates of five broilers each; avg. initial wt. = 79 g. bSex effect, P < .07. 'Cr quadratic effect. P < .10. dSex x Cr linear effect, P < .09. 'Sex x Cr quadratic effect, P < .01. fSex x Cr quadratic effect, P < .05. Table 6.9. Effect of dietary chromium on plasma metabolites and hormones of 5 to 54-day-old broilers (Exp. 3)* Male Female Dietary Cr. Mg/kg Pooled Item 0 400 1,600 0 400 1,600 SEM Fed Cholesterol, mmol/Ld 3.0 3.2 3.0 3.0 2.9 3.0 .1 NEFA, mEq/L* .33 .33 .32 .32 .29 .28 .02 Glucose, mmol/L 12.6 12.0 12.5 12.0 11.4 11.8 .5 Insulin, ng/mL .72 .98 .92 .98 1.36 1.13 .19 Glucagon, pg/mL 225.6 220.8 229.7 485.9 145.8 358.8 118.3 Fasted Cholesterol, mmol/Lc,c 3.2 3.4 3.6 3.4 3.2 3.1 .1 NEFA, mEq/Lb .49 .50 .51 -55 .59 .58 .03 Glucose, mmol/L 10.6 10.8 10.8 10.9 10.5 10.8 .3 Insulin, ng/mL**-* .15 .24 .36 .44 .34 .34 .04 Glucagon. pg/mL 664.6 836.6 861.6 1162.2 1095.4 714.0 232.2 “Data are means of two replicates of five broilers each. bSex effect, P < .03. *Sex effect, P < .08 dCr linear effect, P < .07. 'Cr quadratic effect. P < .02. 104 effect, P < .04) (Table 6.10). In males, carcass fat percentage increased as dietary Cr level increased; however, in females, carcass fat percentage decreased as dietary Cr level increased (Sex x Cr linear effect, P < .04). Carcass ash and CP, as a percentage of DM, were not affected (P > .10) by treatment. Gut moisture was higher in males than in females (Sex effect, P < .06) but was not affected (P > .10) by dietary Cr supplementation. Gut fat percentage in males was the highest when fed 400 ppb Cr; however, in females, dietary Cr decreased Gut fat percentage (Sex x Cr quadratic effect, P < .03). Gut ash as a percentage of DM was decreased as dietary Cr level increased (Cr linear effect, P < .01). Gut CP as a percentage of DM was not affected (P > .10) by treatment. White meat moisture was higher in males than in females (Sex effect, P < .06). White meat moisture, in males, decreased when fed 400 ppb Cr; however, in females, white meat moisture increased when fed 400 ppb Cr (Sex x Cr quadratic effect, P < .03). White meat fat and CP were not affected (P > .10) by treatment. White meat ash percentage was higher in females than in males (Sex effect, P < .06). Dark meat moisture and CP were higher in females than in males (Sex effect, P < .06). Dark meat CP as a percentage of DM was higher in females than in males (Sex effect, P < .06). Dark meat fat and ash percentage were not affected (P > . 10) by treatment. I Male Female *™^"“ — ^ — — — m h — « » » _ _ m ^ _ Dietary Cr. ^g/kg Pooled Item 0 400 1,600 0 400 1,600 SET Carcass Moisture, %c 62.2 61.2 61.6 59.3 60.1 62.8 .6 Fat, %e 15.2 17.4 16.7 18.5 18.3 14.5 .7 CP, % DM 50.1 47.1 51.8 46.4 45.2 48.8 2.1 Ash, % DM 8.8 8.4 8.5 7.8 7.9 8.6 .3 Gut Moisture, %h 70.6 68.6 70.6 65.5 67.0 66.8 .8 Fat, %bAf 8.3 10.7 8.3 12.5 9.6 9.4 .7 CP, %DM 61.7 56.4 59.2 51.5 58.1 57.0 3.7 Ash, % DM0 5.5 4.1 5.2 4.7 5.3 4.2 .2 White meat Moisture, %kAf 71.8 71.5 71.8 68.8 70.4 70.4 .3 Fat, % .3 .6 .4 .6 .3 .8 .4 CP, % DM 87.5 87.7 83.5 90.6 92.0 92.3 4.3 Ash, % DM* 4.9 5.0 5.1 5.3 5.5 5.4 .2 (table con’d) I Dark meat Moisture, %b-eAe’f 71.7 75.7 79.7 73.7 77.7 81.7 .5 Fat, % 5.8 5.6 5.8 5.4 6.8 6.5 .8 CP, %DM* 63.8 66.1 67.2 73.0 69.7 72.7 2.6 Ash, % DM 3.8 3.5 3.8 3.8 3.7 3.7 .1 *Data are means of two replicates of three broilers each. bSex effect, P < .06. eCr linear effect, P < .01. dCr quadratic effect, P < .06. °Sex x Cr linear effect, P < .04. fSex x Cr quadratic effect, P < .03. o CJ\ 107 Experiment’ 4 Supplemental dietary Cr did not affect (P > .10) growth performance (Table 6.11). Dietary Cr also did not affect (P > .10) glucose clearance during IVGTT or IVICT or insulin clearance during IVGTT. The concentrations of glucose and insulin across time for the IVGTT are shown in Figures 6.1 and 6.2, respectively. The plot of glucose concentrations during IVICT is shown in Figure 6.3. Plasma glucose was not different (P > .10) between dietary treatments during the LSMM. However, plasma glucagon was lower (P < .04) in broilers fed CrPic during the LSMM. The concentrations of glucose and glucagon during LSMM are shown in Figures 6.4 and 6.5, respectively. Discussion Supplemental dietary Cr affected ADG, ADFI, and gain:feed ratio inconsistently in broilers in this investigation. Steele and Rosebrough (1979) reported increased growth rate and feed efficiency of turkey poults fed diets supplemented with 20 ppm Cr as Cr+J. Steele and Rosebrough (1981) also reported increased growth rate, as well as increased lipogenesis, of poults fed 20 ppm Cr. However, the increase in lipogenesis did not increase liver lipid content. Rosebrough and Steele (1981) reported that 20 ppm dietary Cr as CrCl3 increased in vitro incorporation of 14C-glucose into glycogen. Thus, apparently CrCl3 also is an active source of Cr in poultry, as opposed to data from the pig (Page et al., 1990). 108 Table 6.11. Effect of dietary chromium on growth performance, and glucose and insulin clearance in broilers (Exp. 4) Dietary Cr. ue/ke. Pooled Item______0 200_____ SD Growth Performance ADG, g 50.6 46.7 4.5 AD FI.g 135.1 134.0 6.2 Gain: feed .374 .348 .023 Intravenous Glucose Tolerance Test Glucose, 15 to 45 min Clearance, %/min 2.59 (6) 2.22 (7) .37 Half-life, min 27.2(6) 32.2(7) 5.0 Insulin, 15 to 30 min Clearance, %/min 10.92 (4) 8.43 (5) 2.96 Half-life, min 6.96(4) 9.37(5) 4.05 Intravenous Insulin Challenge Test. Glucose, 0 to 60 min Clearance, %/min 1.50(5) 1.32(6) .16 Half-life, min 47.0(5) 53.4(6) 5.6 *Growth performance data are from four replicates of five birds each (28 to 58 d of age; avg. initial wt. = 976 g). Clearance rate and half-life data are from 11 wk old broilers; number of broilers is shown in parentheses. 109 30 Control 25 CrPic 15 10 50 T im e , m in Figure 6.1. Effect of dietary chromium on glucose clearance in broilers during an intravenous glucose tolerance test. Broilers were fed a com-soybean meal diet without (O) or with (□) 200 ng Cr as chromium tripicolinate (CrPic)/kg diet. For glucose clearance rate: pooled SD = .37; and for glucose half-life; pooled SD = 5.0. 110 4.0 3.5 Control 3.0 CrPic i a 2.5 a s 2.0 a 1.5 1.0 0.5 0.0 0 25 50 75 100 125 150 175 200 T im e , m in Figure 6.2. Effect of dietary chromium on insulin clearance in broilers during an intravenous glucose tolerance test. Broilers were fed a com-soybean meal diet without (O) or with (□) 200 Mg Cr as chromium tripicolinate (CrPic)/kg diet. For insulin clearance rate: pooled SD = 2.96; and for glucose half-life; pooled SD = 4.05. I ll 14 12 Control i CrPic B «r 10 tfi © © 3 3 8 6 4 25 50 75 100 125 150 175 200 T im e , m in Figure 6.3. Effect of dietary chromium on glucose clearance in broilers during an intravenous insulin challenge test. Broilers were fed a corn-soybean meal diet without (O) or with (□) 200 f*g Cr as chromium tripicolinate (CrPic)/kg diet. For insulin clearance rate: pooled SD = . 16; and for glucose half-life; pooled SD = 5.6. 1 1 2 25 20 Control CrPic 0 25 50 75 100 125 150 175 200 T im e , m in Figure 6.4. Effect of dietary chromium on plasma glucose concentrations in broilers during an intravenous least-sampling minimum model (LSMM) glucose tolerance test. Broilers were fed a com-soybean meal diet without (O) or with (□) 200 t+g Cr as chromium tripicolinate (CrPic)/kg diet. 113 900 800 Control 700 CrPic S i 500 3 400 300 200 100 0 25 50 75 100 125 150 175 200 T im e , ra in Figure 6.5. Effect of dietary chromium on plasma glucagon concentrations in broilers during an intravenous least-sampling minimum model (LSMM) glucose tolerance test. Broilers were fed a com-soybean meal diet without (O) or with (□) 200 Mg Cr as chromium tripicolinate (CrPic)/kg diet. Page et al. (1993a) reported increased, decreased, and no change in ADG of pigs fed CrPic. Other investigators have reported inconsistent (yet primarily a lack of) effects of dietary Cr on growth performance of pigs (Boleman et al., 1995; Page et al., 1993a,b; Renteria and Cuaron, 1993), lambs (Gentry et al., 1995; Kitchalong et al., 1995), calves (Bunting et al,, 1994) and steers (Claeys et al., 1994). However, it may be inappropriate to compare the results of other investigators with our data because of the dramatic differences in length of treatment, physiological and chronological age of the animals, as well as species differences. These same factors may partially explain why we were not able to detect an increase in N balance, in Exp. 1, as reported by Britton et al (1968) in lambs. Kitchalong et al. (1993) also reported no effect of CrPic on N balance of lambs. Supplemental dietary Cr did not affect the percentage of CP, moisture, or ether extract in breast and leg muscle samples in Exp. 1. This lack of effect on meat characteristics is in agreement with other data from pigs (Page et al., 1992b). However, Data from Exp. 2 and 3 indicate that Cr is affecting the composition of body components. The increased weight of tire ventriculus, a highly muscular organ, follows the increased muscling reported by other investigators in pigs (Boleman et al., 1995; Lindemann et al., 1995; Mooney and Cromwell, 1995; Page et al., 1993). Furthermore, in Exp. 3, dietary Cr supplementation increased CP in the carcass. Recall that the carcass did not contain the breast meat or the right leg and thigh meal. This suggests that dietary Cr may have subtle effects on multiple carcass components or that Cr is affecting some individual carcass component that was not measured. In contrast to Exp. 1, in Exp. 3 the percentage of moisture in the dark meat was increased by dietary Cr supplementation. The positive association of moisture content with protein content and negative association of moisture content with fat content suggest that Cr may be increasing total carcass protein. In addition, Cr supplementation in Exp. 3 resulted in a reduction in abdominal fat pad weight, which is correlated with total carcass fat content. Thus, Cr may be affecting fat deposition and(or) protein deposition. The data of Mooney and Cromwell (1995) suggests that both protein and fat deposition arc affected simultaneously. Similarly, the increased carcass ash (Exp. 2) is in agreement with data from pigs (Boleman et al., 1995; Mooney and Cromwell, 1995). Thus, it appears that CrPic may be influencing the total amounts of body fat (less) and body CP (more) in broilers. The current data suggest that long-term feeding is more beneficial than short-term feeding. This is the opposite response that has been reported for one study with pigs (Boleman et al., 1995). However, it is wise to use caution when comparing these results. To more accurately describe the response to length of feeding Cr, broilers should be fed Cr different lengths of time, but all birds should be fed to a conventional finished broiler end point. The influence of Cr on insulin function and glucose status of mammals is well documented; however, the importance of insulin in the metabolism of the chicken is not well established. The effects of Cr on the body composition of broiler chicks 116 may implicate the importance of insulin in avian metabolism or it is possible that Cr is mediating its effects via other and/or additional mechanisms from insulin. Touchbum et al. (1981) reported a relationship between plasma glucose and insulin and body fat content of broilers. That is, broilers that have been selected for increased body fat content have lower fasting glucose and higher fasting insulin concentrations (Touchbum et al., 1981). However, in commercial broilers, such as those we used, that historically have been selected for increased rate of BW gain (and incidentally increased fat), it is not clear if the relationship between insulin and glucose and body tat remains. In Exp. 3, in the fasted condition, broilers had increased plasma insulin due to Cr supplementation, but no change was seen in glucose concentration. If the relationship as described by Touchbum et al. (1981) is true then it would follow that the broilers given supplemental Cr would contain more fat. However, the data do not support this conclusion. Due to the lack of effect of dietary Cr on glucose and insulin clearance in this investigation, it is not possible to draw unequivocal conclusions regarding the relationship between dietary Cr supplementation and plasma glucose and plasma insulin and body composition. It is not possible to draw many conclusions from the LSMM data. However, it is interesting that the dramatic rise in glucagon that occurred in both treatment groups following the insulin infusion was not capable of inducing a concomitant increase in blood glucose. In addition, the overall lower level of plasma glucagon observed in the broilers fed Cr is perplexing. Glucagon is highly lipolytic in the 117 avian species. Lower plasma glucagon would imply that the birds fed Cr may deposit more fat; however, the carcass composition data do not support this. The trend toward increased protein and decreased fat in the carcass of broilers indicates that Cr as CrPic may be of economic importance in the poultry industry. Providing the human population a healthier (less fat) product is a desirable goal from both a human and animal nutritionist viewpoint Implications Dietary chromium may affect metabolism of broilers and thereby also affect body composition. Increased protein and decreased fat content of chicken meat would be of considerable benefit to the world human population that consumes the meat. However, conduction of conventional floor pen studies and industry carcass measurements would better quantify the effect of dietary chromium on the body composition of chickens. Dietary chromium supplementation may be a means of decreasing fat content and increasing protein content of broilers. CHAPTER VII OVERALL SUMMARY AND CONCLUSIONS The results of the first experiment indicate that the puiported effects of dietary Cr supplementation on body composition are not always evident. Dietary Cr, in both inorganic and organic forms, did not decrease tenth rib fat thickness nor increase longissimus muscle area, as has been reported by other investigators. However, other researchers have reported increased carcass protein and decreased carcass fat in pigs fed supplemental Cr when linear carcass measurements (i.e., tenth rib fat thickness and longissimus muscle area) were not affected. Only linear carcass measurements were taken in experiment one; therefore, subtle changes in carcass composition may have occurred. The reasons for a less dramatic effect, perhaps, on carcass composition are not known. Some investigators have hypothesized that the level of stress, genotype differences, or Cr status may be responsible for the different results obtained from dietary Cr supplementation. The second experiment suggested that dietary Cr supplementation as Cr chloride hexahydrate, Cr nicotinate, or Cr tripicolinate did not affect glucose and insulin clearance in pigs given intravenous glucose tolerance tests or intravenous insulin challenge tests. Thus, again the reason for the discrepant results between experiment two and other experiments with Cr tripicolinate from the same laboratory are not known. In experiment three, decreased pen size and decreased dietary crude protein concentration were not effective in inducing a Cr effect on 118 119 linear carcass traits of growing-finishing pigs. The following are possible reasons: 1) the chronic effect of decreased pen size and decreased dietary crude protein were not effective as stressors, 2) the pigs were able to adapt to the stress, or 3) the effects of dietary Cr are not mediated through stress-related mechanisms. In experiment four, body composition of broiler chickens was affected by dietary Cr supplementation. Although the data are inconclusive, they suggest that dietary Cr supplementation as Cr tripicolinate is effective in increasing carcass crude protein and decreasing carcass fat content. 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FASEB J. 9:A615 (Abstr.). Zimmerman, D. R. 1995. Effects of space allowance for pigs fed to 300 pounds body weight. 1994 Swine Research Report, Iowa State University, Ames. AS-629:41. Zook, E. G., F. E. Greene, and E. R. Morris. 1970. Nutrient composition of selected wheats and wheat products. VI. Distribution of manganese, copper, nickel, zinc, magnesium, lead, tin, cadmium, chromium, and selenium as determined by atomic absorption, spectroscopy and colorimetry. Am. Ass. Cereal Chem. 47:720. VITA Terry Lynn Ward was bom on August 25, 1965 to Alton and Lynette Ward in Grapevine, Texas. In 1973 the Wards and their six children moved to Gould, Oklahoma where they were involved in animal agriculture and crop farming. Terry attended Gould High School until his graduation in May of 1983. Terry’s agricultural background and interest in swine production led him to obtain nn Associate of Science degree in Agriculture from Western Oklahoma State College at Altus, Oklahoma in May of 1985. He then transferred to Oklahoma State University where he obtained a Bachelor of Science degree in Agriculture with a major in Animal Science in December of 1987. On January 2, 1988 Terry was married to Donnita Newell of Earisboro, Oklahoma. Also in January of 1988, Terry accepted an assistantship offer from the Louisiana State University Animal Science Department to study nonruminant nutrition under the direction of Dr. L. Lee Southern. In the the spring of 1989 he became a Research Associate at Louisiana State University working for Dr. Southern. In December of 1989, Terry received his Master of Science in Animal Science from Louisiana State University. Terry and Donnita had two children while living in Baton Rouge. Cody Lee Ward was bom on June 1, 1990 and Leah Kaye Ward was bom on October 9, 1992. Terry continued to pursue a Ph.D. in Animal Science in the area of nonrumiuant nutrition at Louisiana State University. Terry completed his doctoral degree requirements in the Fall of 1995. 155 DOCTORAL EXAMINATION AND DISSERTATION REPORT Candidate: Te rrv Lvnn Ward Major Field: Animal Science Title of Dissertation: p ie ta rv Chromium Supplem entation fo r Piqs and Cnickens Approved: Major Professor and Chairman ean of the Graduate School EXAMINING COMMITTEE: tff kQ *-. j} 'JUit m :A C. Date of Bxamination; August 2 , 1995