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DIETARY FIBER AND ACTIVE IMMUNIZATION AGAINST CHOLECYSTOKININ
(CCK) DURING GESTATION INCREASES LACTATION
FEED INTAKE AND PRODUCTIVITY OF sows
By

Daniel Arthur Nelson

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1 996

ABSTRACT
DIETARY FIBER AND ACTIVE IMMUNIZATION AGAINST CHOLECYSTOKININ
(CCK) DURING GESTATION INCREASES LACTATION
FEED INTAKE AND PRODUCTIVITY OF SOWS
By

Daniel Arthur Nelson

Three experiments were conducted to evaluate gestation (G) management
practices employed to enhance the average daily feed intake (ADFI) of sows in lactation
(L). In Experiment 1, thirty York x Landrace (YxL) gilts were assigned to a com-
soybean meal (control), a com-soybean meal-wheat straw (WS), or a com-soybean meal-
soybean hull (SBH) G diet for three parities. Animals within each treatment were offered
the same daily amount of metabolizable energy, crude protein, lysine, calcium, and
phosphorus. During L, all sows had ad libitum access to the same diet. The ADF I of
SBH and WS sows was greater (P<.10) than control sows in L. The WS sows farrowed
more (P<.05) live pigs than SBH sows, and weaned more (P<.05) pigs than either the
SBH or control sows. The SBH sows were less constipated at d 109 of G (P<.001) and d
10 of L (P<.05). In Experiment 2, thirty three YxL gilts were assigned to either the
control or SBH diet in G. In L, all sows had ad libitum access to the same diet containing
.13% flavor. The ADF I of SBH sows was greater (P<.10) in parity 1, but not in parity 2
or overall. During G, SBH sows digested less (P<.001) dietary gross energy and crude
protein and less (P<.10) ether extract, but more (P<.05) neutral detergent fiber and more

(P<.001) acid detergent fiber than control sows. The SBH sows were less (P<.001)

constipated at d 110 of G. In Experiment 3, twenty four gilts immunized on d 64 of G
farrowed litters; 20 were immunized with the desulfated C-terminal octapeptide of
cholecystokinin (CCK) conjugated to Keyhole limpet hemocyanin (KLH) and 4 control
females with KLH alone. Booster doses of immunogen (B1, B2, B3) were administered
at 14 (1 intervals. Prior to each vaccination and on d 7, 14 and 21 of L (LD7, LD14, LD21
respectively), blood samples were taken from all sows. Colostrum and milk samples
were collected from all sows at farrowing and on LD7, LD14, and LD21. Blood samples
were collected from 2 piglets of each litter at LD7. Mean log CCK-antibody (CCK-AB)
titers of sow serum at BZ, B3, LD7, LD14, and LD21 was .27, 1.10, 1.77, 1.67, and 1.58
respectively. Mean log CCK-AB titer of colostrum was 2.42, and that of milk was .50, -
.003, and -.20 at LD7, LD14 and LD21 respectively. These log titer were highly (P<.005
in all cases) interrelated to each other. Mean log CCK-AB titer of piglet serum (n=30) at
LD7 was 1.22. The ADFI of sows over the 21 d L increased (r2=.37, P<.05) with the
average of the seven samples taken from the sow during L (ALTSS). Total litter gain
also increased with the ALTSS (r2=.41, P<.05) and with the ADF I of sows in week 3
(r2=.28, P<.05). Nutrient digestibility was not impaired as a result of CCK immunization,
and increased with CCK-AB titer in G. Dietary fiber from WS or SBH fed to sows in G
increased L ADFI. The L ADFI of sows vaccinated against CCK increased with the anti-

CCK-AB log titer of the sow.

ACKNOWLEDGMENTS

"Any one man really owes no more than an exceedingly small amount to his own
efforts. To over emphasize the importance of a persons own efforts is to belittle
the contributions of other".

From:
J. Wilson. 1954. How legitimate are names on papers? Science. 120:276.

I know this to be true and acknowledge everyone who helped me attain this
degree. Thank you Dr. Josep Garcia-Sirera for countless "bleeds", infusions,
vaccinations, weigh-days, surgeries, clean-ups, computer help. For days that began at
4:00 A.M. and for nights that lasted until 2:00 A.M. You are truly a friend and I thank
you from the bottom of my heart. This would never have gotten done without your help -
of that I am certain. Thank you Ross Santell for your friendship and support. Thank you
fellow graduate students: Kim Howard, Dan Jennings, Mark Edwards, Scott Krammer ’
and Michell Mater for help, even when I seemed unappreciative. Thank you Ph.D.
committee: M.G. Hogberg, D.W. Rozeboom, J .C. Pekas, M.S. Allen, R. F ogwell, and M.
Bennink for guidance and patience. Thank you for sharing your wisdom and friendship
E.R. Miller and DE. Ullrey. Thank you Pau Ku, Sharon DeBarr, David Main and Jane
Link for your laboratory help and instruction. Thank you Ellie, Emily, Adam and Abbey
for the time we gave up together then, for something better now. Thank you God.

Without you I am, and can do, nothing.

iv

TABLE OF CONTENTS

LIST OF TABLES ...................................................................................................... viii
LIST OF FIGURES .................................................................................................... x
INTRODUCTION ...................................................................................................... 1
CHAPTER 1
DIETARY FIBER IN SWINE DIETS ....................................................................... 5
Introduction .................................................................................................... 5
Dietary Fiber .................................................................................................. 6
What is fiber? ............................................................................. . ....... 6
Methods of fiber analysis ................................................................... 7
Fiber digestion ................................................................................... 8
Dietary fiber reduces nutrient digestibility of the diet ................................... 10
Stage of gestation affects nutrient digestibility .............................................. 1 1
Use of soybean hulls in swine diets ............................................................... 12
Experiments with high-fiber ingredients and performance of sows ............... 12
Dietary fiber and constipation ........................................................................ 14
Dietary fiber during gestation and subsequent lactation feed intake .............. 14
Summary ......................................................................................................... 17
CHAPTER 2
DIETARY WHEAT STRAW OR SOYBEAN HULLS DURING GESTATION
AND SUBSEQUENT LACTATION PERFORMANCE .......................................... 19
Abstract .......................................................................................................... 19
Introduction .................................................................................................... 20
Materials and Methods ................................................................................... 21
Data analysis .................................................................................................. 24
Results and Discussion ................................................................................... 24
ADF I ................................................................................................... 24
Constipation, sow body weight and weight changes .......................... 27
Conclusions ........................................................................................ 32

CHAPTER 3
EFFECT OF FEEDING SOYBEAN HULLS DURING GESTATION ON
SOW PERFORMANCE, NUTRIENT DIGESTIBILITY AND CHANGES
IN BODY COMPOSITION ......................................................................................
Abstract ..........................................................................................................
Introduction ....................................................................................................
Materials and Methods ...................................................................................
Data analysis ..................................................................................................
Results and discussion ...................................................................................
ADF I ...................................................................................................
Constipation, body weight, body weight change and body composition
changes ...............................................................................................
Reproductive data ..............................................................................
Apparant digestibility .........................................................................
Conclusions ........................................................................................

CHAPTER 4

REVIEW OF THE LITERATURE PERTAININ G TO CHOLECYSTOKININ

(CCK) .........................................................................................................................
Introduction ....................................................................................................
Regulation of voluntary feed intake ...............................................................
Cholecystokinin: structure, function and sites of release ...............................
Mechanism of CCK as a satiety factor ...........................................................
Role of cholecystokinin in lactation ...............................................................
Immunization against endogenous CCK ........................................................
Summary .........................................................................................................
Implications ....................................................................................................

CHAPTER 5
ACTIVE IMMUNIZATION OF GILTS AGAINST CHOLECYSTOKININ
(CCK) IMPROVES LACTATION PERFORMANCE .............................................
Abstract ..........................................................................................................
Introduction ....................................................................................................
Objectives .......................................................................................................
Materials and Methods ...................................................................................
Vaccine Preparation ...........................................................................
Sows ...................................................................................................
Piglets .................................................................................................
Lab Analysis .......................................................................................
Data Analysis .....................................................................................
Results and Discussion ...................................................................................
Anti-CCK titers of serum, colostrum, and milk .................................
Anti-CCK titers of piglet serum .........................................................

vi

33
33
34
35
39
40
4O

42
45
47
50

52
52
53
54
55
56
58
6O
60

61
6 1
63
64
64
64
64
69
69
70
7O
70
75

Feed intake and response to CCK immunization ...............................
Body weight loss and response to CCK immunization ......................
Response of body composition and backfat thickness to CCK
immunization ......................................................................................
Litter performance ..............................................................................
Digestibility response to CCK immunization ....................................
Other observations ..............................................................................
Ensuing data analysis .........................................................................
Implications of Experiment 3 .........................................................................

APPENDIX ................................................................................................................

LABORATORY PROCEDURES ..................................................................
Determinations of Antisera Titer Against Cholecystokinin ...............
Sequential Fiber Analysis ...................................................................

Neutral Detergent Fiber (N DF) ..............................................

Acid Detergent Fiber (ADF) ..................................................

Acid Detergent Lignin (ADL) and Ash ..................................
Determination of ether extract (EE) ...................................................
Determination of crude protein (CP) ..................................................
Determination of starch ......................................................................
Preparation of macro amounts of ground corn stalk
chromium mordant .............................................................................
Determination of chromium (Cr) concentration .................................
Determination of gross energy (GE) ..................................................

LITERATURE CITED .........................................................

vii

77
81

82
86
91
95
96
96

98
98
98
102
102
102

103

105
106
108

109
111
112

113

LIST OF TABLES

Table 1. Lactation feed intake (kg'hd"'d") of sows fed
fibrous diets during gestation ................................................................... 15

Table 2. Composition and calculated analysis of diets used in Experiment 1 and 2. 23

Table 3. ADFI and constipation scores for sows in Experiment] ............................ 26
Table 4. Lactation ADF I by parity for Experiment 1 ............................................... 27
Table 5. Sow weights and weight changes in Experiment 1 .................................... 29
Table 6. Sow backfat depths and backfat depth changes in Experiment 1 ............... 30
Table 7. Sow reproductive performance in Experiment 1 ........................................ 31

Table 8. Composition and calculated analysis of lactation diet used in
Experiment 2 and 3 ..................................................................................... 36

Table 9. Lactation ADF I for Experiment 2, kg ........................................................ 41

Table 10 Sow weights, weight changes, and constipation scores in Experiment 2.. 43

Table 11 Changes in sow body composition in Experiment 2, kg ............................ 44
Table 12. Changes in sow body composition by stage in Experiment 2, kg .............. 45
Table 13 Sow reproductive performance in Experiment 2 ........................................ 46
Table 14 Nutrient digestibility coefficients (DC) for Experiment 2 .......................... 48

Table 15 Comparison of digestibility coefficients (DC) by stage
for Experiment 2 ......................................................................................... 49

viii

Table 16. Endogenous inhibitory and stimulatory agents of appetite ........................ 53

Table 17. Matrix table of correlation coefficients, r, between all pairs of log
titer values for sow serum (SS), colostrum (C) and milk (M) .................... 75

Table 18. Correlations, r of ADFI with the log titer of sow serum and defatted
colostrum .................................................................................................... 79

Table 19. Summary of sow body weight changes during lactation by weekly
periods and correlations, r with the log titer of serum, colostrum and
milk ............................................................................................................. 81

Table 20. Body weight change and empty body (EB) composition changes of
CCK-immunized sows during gestation and lactation, kg ......................... 83

Table 21. Sow backfat depth and change, mm and correlations, r with the log titers
of serum, colostrum and milk ..................................................................... 84

Table 22. Summary of litter weight gains during lactation by weekly periods and
correlations, r with the log titer of serum, colostrum and milk .................. 87

Table 23. Regression coefficients, r2 for CCK-antibody titer and dietary DC
in gestation ................................................................................................. 92

Table 24. Digestibility coefficients measured in gestation and lactation for CCK
immunized sows ....... 93

ix

LIST OF FIGURES

Figure 1 - Indicator method equation used to calculate digestibility coefficients ........ 38
Figure 2. Experimental time line ................................................................................ 67
Figure 3. CCK-AB titers for sow and piglet serum, colostrum and milk .................... 72
Figure 4. Relationship between log titer of sow serum and colostrum ...................... 73
Figure 5. Relationship between log titer of colostrum and piglet serum ..................... 76
Figure 6. Relationship between log titer of sow serum and piglet serum ................... 77
Figure 7. Relationship between average titer of seven sow samples (ALTSS)

and ADFI week 3 ......................................................................................... 80
Figure 8. Relationship between average log titer of seven sow samples (ALTSS)

and backfat depth change ............................................................................ 85
Figure 9. Relationship between average log titer of seven sow samples (ALTSS)

and litter weight gain .................................................................................. 88
Figure 10. Relationship between log titer of piglet serum and litter gain .................... 89
Figure 11. Relationship between sow ADFI and litter weight gain, kg ....................... 89

INTRODUCTION

Current swine production practices mandate that sows farrow and wean large
litters, return to estrus promptly, conceive and repeat the process again. Herd averages of
2.5 litters/sow/year and 25 pigs/sow/year weaned, unheard of a decade ago, are now
being achieved. Maximum lactation feed intake is essential to maintain this level of
production. However, the producer is in a catch-22. Consumer demand for lean pork has
forced the swine industry to select for animals with reduced fat reserves. Inadvertently,
there has been a concurrent reduction in voluntary feed intake of sows of lean-type
genetics (Riley, 1989). Although daily voluntary feed intake increases as lactation
progresses, most lactating sows do not eat enough to maintain body weight during the
average 3 to 4 week lactation period. Lactation weight loss is most rapid during the latter
stages of lactation as milk output peaks. Excessive weight loss can lead to delayed return
to estrus (> 10 days) following weaning, especially in the primiparous sow. In lactating
sows (Matzat, 1990) and growing pigs (Pekas, 1985), voluntary feed intake limits
productivity. Lactating sows which were superalimentated via a gastric fistula and
received 20% more feed than those sows allowed ad libitum access to feed were able to
preserve a greater amount of body mass and were also able to synthesize greater
quantities of milk (Matzat, 1990). Gastric fistulation revealed the potential of swine to

l

2
utilize more feed, but it's implementation as a production practice has no practical merit.

Several researchers (Pollmann et al., 1979; Pond et al., 1985; Holzgraefe et al.,
1986; Mroz et al., 1986; Carter etal., 1987; Hagen, 1988; Nelson et al., 1992) have fed
different fiber sources to sows and gilts during gestation and monitored their subsequent
lactational performance. All observations indicate that moderate levels of dietary fiber do
not adversely affect sow performance. Some research groups have even reported
enhanced litter size and lactation feed intake of sows fed fibrous gestation diets.
Additionally, fibrous feed ingredients are usually available and are not expensive.

Fibrous ingredients do have some disadvantages. The low bulk density of fibrous
ingredients often makes them difficult to handle with conventional feed-mixing
equipment. Due to their lower digestibility, fibrous ingredients can increase manure
volume. Additionally, the insolubility of the resulting manure may decrease the degree
of solid removal from pits and lagoons, and reduce the effectiveness of manure handling
practices that employ water flush systems.

Certainly other techniques that enhance the feed intake of lactating sows must be
explored. In young growing swine, feed intake has been consistently increased by
immunoneutralization of cholecystokinin (CCK). In a series of studies (Pekas and Trout,
1990; Pekas, 1993; Pekas, 1996) with growing pigs, active immunization against CCK
stimulated feed intake and growth in proportion to the anti-CCK antibody (CCK-AB)
titer elicited by the animal. Recently, superior CCK immunogens have been developed
and reported by Pekas (1996), which elicited greater antiserum titers in swine.

Immunoneutralization of CCK to enhance the feed intake of lactating swine has not been

3
reported. If gestating sows were vaccinated against CCK, the resulting CCK-antibodies
may increase voluntary feed intake during lactation. If so, sows may respond similarly
to super-alimentated sows which synthesized greater quantities of milk while
simultaneously preserving a greater amount of body mass (Matzat, 1990). Elevated milk
production may aid in piglet survival and enhance litter weaning weights. Preservation of
body mass during lactation may contribute to timely rebreeding (< 10 d post-weaning)
following weaning.

Enhancing lactation feed intake through gestation management practices such as
increasing dietary fiber intake and vaccinating sows so they develop their own active
titers against CCK are two methods, the feasibilities of which need to be further explored.

Due to the inherent differences in the chemical composition of fiber, two fibrous
ingredients may be vastly different even though they may possess similar concentrations
of crude fiber. This characteristic makes it difficult for the nutritionist to make
generalized recommendations as to their use. Recommended inclusion rates of most
fibrous ingredients within swine gestation diets have yet to be published. Active
immunization against CCK has been observed to increase the voluntary feed intake of
growing pigs, but it's use in sows has not been published.

This dissertation is a composite of three experiments conducted to evaluate these
two methods of increasing sow lactation feed intake. The duration of these experiments
lasted over four years, using over 90 sows and more than 130 litters. Experiment 1 and 2
(Chapter 2 and 3 respectively), investigated the effects of including two dietary fiber

sources in gestating swine diets; wheat straw and soybean hulls. Experiment 3 (Chapter

4
5), evaluated active immunization of sows against CCK as a means of increasing lactation

feed intake.

CHAPTER 1

DIETARY FIBER IN SWINE DIETS

Muslim

The majority of sows in the United States are housed in confinement units.
However, prior to the popularity of swine confinement units most sow herds were
maintained on grass and legume pastures throughout gestation. Even today it is not
uncommon to find sow herds on pasture during gestation that forage for the majority of
their nutrient demands. In other parts of the world (Southeast Asia) where grains are
relatively expensive, fibrous feedstuffs are used exclusively to maintain the gestating sow
herd. In these situations, sow performance (ie; litter size, litter birth weight, rebreeding
interval) is seldom compromised.

When poor weather conditions cause grain prices to rise sharply, producers often
seek alternative ingredients to serve as partial grain replacements. Many of these
potential grain replacements contain higher amounts of fiber. Gestating sows have the
potential to utilize more dietary fiber than is currently being incorporated into most swine
gestation diets. Incorporation of high-fiber ingredients into gestation diets is also
attractive as the practice increases feeding volume without increasing nutrient intake. As
feed intake is generally restricted during gestation, this procedure may appease sow

5

6
appetite resulting in increased animal comfort. AS world population continues to grow
and place more pressure on grain supply for direct use, swine producers may need to
reformulate conventional high-grain diets for animals.

This chapter will review the literature pertaining to the use of dietary fiber in
swine diets. It will address the topics of fiber digestibility by pigs and the influence that
dietary fiber exerts on the digestibility of other nutrients within the diet. This chapter will
also review the results of studies in which fiber was included in high amounts in swine
gestation diets and the effects of dietary fiber on sow performance and subsequent
lactation feed intake.

Dim

W
Dietary fiber is defined as that portion of plant material that is not digested by enzymes
of the mammalian digestive system (Schneeman and Gallaher, 1990). The majority of
the fiber that is found in plant material originates in the cell wall where it gives rigidity
and strength to the plant. Dietary fiber is composed of non-starch polysaccharides which
include cellulose, B- glucans, hemicellulose, pectins, and gums. Cellulose and B- glucans
are polymers of glucose with B 1-4 bonds holding the sugar residues together
(Schneeman and Gallaher, 1990). Within a cellulose fiber, long chains of glucose
polymers line up parallel to one another to form sheets (glucan) which are held together
by hydrogen bonds between oxygen atoms and the hydrogen atoms of hydroxyl groups
(Albershein, 1975). These glucan Sheets can also line up one above the other, also held in

place by hydrogen bonds, in this instance, between the sheets. It is the large number of

7

hydrogen bonds that gives cellulose it's strength and resistance to degradation
(Albershein, 1975). The backbone of hemicellulose is composed of xylans, galactans,
and mannans while side-chains are usually arabinose and galactose (Schneeman and
Gallaher, 1990). Galacturonic acid is the principal sugar comprising the backbone of
pectin, and galactose and arabinose form it's side chains (Schneeman and Gallaher, 1990).
Within a typical cell wall, hemicellulose and pectin may bind to one another and serve as
the "glue" which holds the cellulose fibers together (Albershein, 1975). Lignin is also a
constituent of the cell wall of plants. Lignin is classified as fiber but is actually a non-
carbohydrate constituent composed of phenylpropane units arranged in a complex three-
dimensional structure.

Methcduiflizemnalms

Crude fiber is defined as that portion of feed that remains after boiling in a weak
acid and then in a weak alkali solution (Ensminger et al., 1990). The assay was originally
developed to imitate the digestive processes that occur in the mammalian gastrointestinal
(GI) tract, and to serve as an indicator of the relative indigestibility of the sample.
However, this method gives no indication as to the type and amounts of the non-starch
polysaccharides within the sample. The Van Soest (1967) sequential fiber analysis
technique allocates the fibrous portion of the feed into its different components. Neutral
detergent fiber (NDF) is that portion of sample remaining after boiling in neutral
detergent and is composed of cellulose, hemicellulose and lignin. Acid detergent fiber
(ADF) is that portion of sample remaining after boiling with acid detergent, and is

composed of cellulose and lignin. The amount of hemicellulose in a sample can be

calculated by difference (N DF - ADF).

Dietary fiber can be partially digested by mammals. The enzymes responsible for
the degradation of dietary fiber are synthesized by the huge microbial population of the
GI tract. Savage (1977) estimated that the normal human organism is composed of over
10” cells, of which only about 10% are animal cells! The rest are microbial in nature and
largely reside in the GI tract. A similar situation may exist in pigs. Two of the most
significant cellulolytics found in ruminants; Bacteroides succinogenes and Ruminococcus

flavefaciens are present in the large intestines of pigs (Varel et a1, 1984).

Pigs can adapt to diets high in fiber. A greater amount of hemicellulose is
digested by pigs after an 80-day adaptation period to a high fiber diet (Pollmann et al.,
1979), and the extent of wood cellulose digestibility by pigs increases with time
(Longland et al., 1993). Growing-finishing pigs adapt to high fiber diets by increasing
the number of cellulolytic bacteria in the large intestine (Varel and Pond, 1985), as do
sows and gilts (Varel and Pond, 1986). The cellulolytic population increase at the
expense of other microbial species as the total density of microorganisms changes little
with the amount and type of polysaccharide being fed to pigs (Bach Knudsen et al.,
1993a). The total number of a given specie of microflora inhabiting the gastrointestinal
tract may also be influenced by the genetics of the animal. Lean pigs have been observed
to have a trend toward larger numbers of cellulolytic bacteria in their gastrointestinal tract

than obese pigs (Varel, 1987).

9

Carbohydrates are the principle substrate for GI tract microflora in pigs (Bach
Knudsen et al., 1991; 1993a). Within the GI tract, the process of fiber fermentation
occurs primarily in the cecum and colon (Zebrowska, 1988). Bach Knudsen et al.
(1993a) confirmed that more than 92% of all dietary structural carbohydrates were
degraded in the cecum, proximal colon and ascending colon. Microbial degradation of
fiber within the gastrointestinal tract generates volatile fatty acids (V FA), including
acetic, propionic and butyric, lactic acid, and various gases (hydrogen, carbon dioxide
and methane) (Bach Knudsen et al., 1991). It has been estimated that the VFA arising
from microbial degradation of fiber within the cecum and colon of growing pigs may
provide up to 30% of pigs maintenance energy requirements (Varel, 1987). A more
conservative range of 10 to 24% was published by Bach Knudsen and Hansen (1991).
Nitrogen compounds (ammonia) absorbed from the hind gut are not utilized by pigs
(Fernandez et al., 1986).

The extent of degradation of dietary fiber is a function of the rate of passage and
the rate of microbial fermentation (Ehle et al., 1982; Fernandez et al., 1986) . Microbial
fermentation rates will not be maximized until microbes and their enzymes are attached
or are in close proximity to fiber digestion sites. Microbial attachment is dependent on
the number of available attachment sites, the mass of fiber digesting microbes in the hind
gut, the species composition of the microbial population and the ability of the different
species to attach to and colonize the fiber (Allen and Mertens, 1988). Additionally, a
coarsely ground fibrous ingredient that exhibited a longer retention time would have a

greater digestibility than the same material ground finer that had a shorter retention time.

1 0
When comparing growing pigs and sows, the differences that exist in ability to digest
different ingredients are largest when comparing fibrous materials (Fernandez et al.,
1986). Sows have a larger intestinal capacity and a longer ingesta transit time than
growing pigs. These two factors increase the extent of microbial fermentation of
structural carbohydrates of fiber by sows as compared to growing pigs (Varel and Pond,
1986). The stage of maturity of the fiber source being fed to sows will influence the
degree of microbial fermentation. Allee (1981), reported that third-cutting alfalfa has
higher digestibility coefficients (DC) for both energy and protein than more mature first-
cutting alfalfa . This observation is probably the result of the greater degree of
lignification of the more mature plant. It has been reported that the nonstructural
polysaccharides (N SP) of unligrrified cell walls from the endosperm and aleurone layers
of wheat and oats are much more extensively degraded than those NSP of the secondary

lignified cell walls from pericarp or testa (Bach Knudsen and Hansen 1991).

 

The inclusion of a fibrous ingredient into diets fed to pigs reduces the digestibility
of other nutrients within the diet (Pollmann et al., 1979; Komegay, 1981; Frank et al.,
1983; Calvert et al., 1985; Bray et al., 1986; Mroz et al., 1986; Pond et al., 1986; Everts
and Smits, 1987; Moore et al., 1987; Bach Knudsen and Hansen, 1991; Bach Knudsen et
al., 1993b).

Dietary alfalfa meal or corn cobs reduced the DC for dry matter, cell contents, and
crude protein of the diet (Pond et al., 1986). Alfalfa inclusion rates of 50 and 90% of

gestating sow diets lowered the DC for energy, protein and fiber constituents (Calvert et

l 1
al., 1985). Similarly, the DC for nitrogen, dry matter, and digestible energy decreased
linearly with increasing amounts of dietary corn cobs (Frank et al., 1983). The DC for
nitrogen, energy and DM were also lower when oat hulls were added to sow gestation
diets (Mroz et al., 1986). Everts and Smits (1987) reported that for every 1% increase in
DM dietary fiber, the DC for dry matter and crude protein were reduced by 1.8 and 1.1%
respectively.

The overall reduction in diet DC due to fiber inclusion may be the cumulative
result of several factors. If the fiber within a feed ingredient is not soluble when they
pass through the small intestine, they may act as a barrier against digestive enzymes and
potentially reduce the digestibility of starch and other nutrients in the small intestine
(Bach Knudsen et al., 1993a). Pectins within fiber are capable of forming a viscous gel-
matrix that may similarly reduce exposure of nutrients to digestive enzymes (Mitaru et
al., 1984). This pectin gel may also encapsulate the products of digestion and reduce
their access to sites of absorption within the intestine (Mitaru et al., 1984). Due to its
ability to hydrophobically bind amino acids, lignin may contribute to reduced protein and
amino acid digestibilities when fiber is added to the diet (Mitaru et al., 1.984).

The extent of nutrient digestibility by sows is influenced with the stage of
gestation. Bray et a1. (1986) reported that the DC for nutrients in both the control and
test diet tended to increase from early gestation to mid gestation while the lowest DC
values were consistently recorded for late gestation. Similarly, nitrogen balance and

nitrogen retention decreased from the first to the third trimester of gestation (N utzback et

l 2

al., 1984). Nutrient utilization of sows fed high levels of alfalfa was lowest when
measured during the last 10-15 (1 of gestation (Calvert et al., 1985).
II E l 1 ll . . 1

Moore et a1. (1987) observed that the addition of soybean hulls at the rate of 15%
to a com-soybean meal diet reduced the DC for DM, but increased the DC for all fibrous
components. Similarly, as soybean hulls were substituted for a com-oat-soybean meal
diet at the rate of 7.5, 15.0 and 30.0%, the digestibility of DM, energy, CP, cell content
and ADF nitrogen decreased, while digestion coefficients for NDF, ADF, cellulose and
lignin increased (Komegay, 1981). However, when soybean hulls replaced 50% of the
basal diet, digestibility of DM and energy were reduced as were the DC for NDF and
hemicellulose (Bray et al., 1986). Perhaps the difference between these results and those
reported by Komegay (1981) were due to the larger amount of soybean hulls used by
Bray et a1 (1986). In agreement with Komegay (1981), the DC for ADF and cellulose
were higher for those sows consuming a 50% soybean hull diet than sows consuming the
basal diet (Bray et al., 1987). Mineral utilization of growing pigs was not influenced by

substituting 15% soybean hulls in the basal diet (Moore and Komegay, 1987).

 

One of the earliest reports of the use of fiber in diets for sows concluded that
breeding efficiency was increased by the addition of dietary alfalfa meal (Fairbanks et al.,
1945). In the years since, other scientific reports have shown similar results. Sows
consuming a diet containing 18% alfalfa had more live pigs and weaned more pigs/litter

than those on a legume-free diet, and when examined early in gestation, contemporary

l 3
sows on the alfalfa diet possessed a greater number of corpora lutea than those fed the
legume-free diet (Teague, 1955). Similarly, sows fed gestation diets balanced for similar
daily ADF intakes from either alfalfa or sunflower hulls have a greater number of pigs
born alive/litter (Carter et. al., 1987). Gilts were observed to have a higher farrowing rate
when consuming a gestation diet containing 96.5% alfalfa, than those consuming diets
with less alfalfa (Danielson and Noonan, 1975). Additionally, it was observed that
animals fed higher levels of alfalfa during gestation are more docile and easier to handle
(Danielson and Noonan, 1975). Allee (1981) suggested that a diet consisting of three
pounds of alfalfa haylage dry matter, two pounds of grain, and a vitamin and mineral
premix is more than adequate for gestating sows.

Not all reports have shown an improvement in sow performance from dietary
fiber. When alfalfa was included at 50 and 90% of the diet of gestating sows, the number
of live pigs farrowed and weaned per sow, and the average birth weights of piglets were
not affected by dietary treatment (Calvert et al., 1985). However, dietary alfalfa inclusion
rates of greater than 95% has been associated with reduced birth weights (Calvert et al.,
1985; Pond et al., 1985). In a large trial with 567 sows over five parities, there was no
clear evidence that either the inclusion of roughage or higher dietary crude fiber improved
reproductive performance (Everts, 1991).

The inconsistencies in the literature may be explained partially by the fact that in
many instances, fiber was substituted at the expense of other ingredients resulting in
differences in dietary nutrient intakes. In experiments where alfalfa was substituted for

the basal diet, B-complex vitamins and protein concentrations may have been elevated.

14
In experiments where oat hulls or soybean hulls were substituted, protein and energy
concentrations may have been reduced. Additionally, fiber composition of an ingredient
(alfalfa) changes with plant genetics, maturity, and growing conditions. It is not
surprising then, to find some slightly contradictory results.
D. 111 l . .

The laxative effects of dietary fiber have long been confirmed. Fiber may reduce
the incidence of constipation through more than one mechanism. In one study, the GI
tract transit time was reduced for those sows receiving gestation diets containing oat hulls
(Mroz, et al., 1886). As removal of water from ingesta is a major function of the large
intestine, a shorter transit time would result in feces with a higher water content (less
constipated). This was observed when soybean bulls were included in the basal diet of
gestating sows; fecal output increased as fecal DM decreased (Komegay, 1981). The
dietary fiber in wheat was reported to increase fecal bulk by virtue of its water-holding
capacity, while the dietary fiber in oats did so by increasing microbial biomass (Bach
Knudsen et al., 1991). Similarly, the fecal bulking effect of oat bran is mainly caused by
an increase in the excretion of protein and fat of bacterial origin (Bach Knudsen et al.,
1993a)

,D‘A.‘ 0L Q--lt°" .1011... .0 ‘!-'.l a .201 "1

Many factors can influence the ADFI of lactating sows. Data from 3,559

lactations revealed that an increase in lactation ADFI was associated with nursing larger

litters, increasing parity and longer lactations (O'Grady et al., 1985).

15

Table 1. Lactation feed intake (kg hd"'d") of sows fed fibrous diets during gestation

 

 

 

 

 

 

 

 

 

Authors Daemon Significance
9.9mm! Alfalfa iunflmhylls
Carter et al., 5.7 5.4 Alf.+S.F.hulls>Control
1987.
#1; #2; #3; #4;
46% Alf.+ #2+ #1+
Control Orchardgrass Lasalocid Lasalocid
Holzgraefe 4.7' 5.3”“ 5.7" 5.0‘c P<.05
et al., 1986.
97% 66%
9.9111191 Alfalfam W
Pollrnan et al., 4.7 4.8 NS.
1979.
40% Dehydrated 96% Dehydrated 20% Corn
Central _al.fal£a__ ml 42.9113.
Pond et al., 43' 4.5" 4.7" 47" P<.01
1985.
67% of control 33% of control
Control M as r teatime Allhasture
Prince et al., 7.7 7.6 8.4 8.5 No Stats
1986.
30% of ME. 60% of ME. 90% of ME.
from alfalfa from alfalfa from alfalfa
Control haylage ML haylage
Hagen, 1988. 5.3 5.9 6.2 6.4 Linear P<.01
30% of ME.
Qantml ficmalfaliahaxlage
Nelson et al., Wk 1; 5.2 5.8 P=.11
1992. Total; 6.1 6.3 NS.

 

l 6
An inverse relationship has been reported between lactation ADF I and gestation weight
gain (O'Grady et al., 1985; Weldon et al., 1992), and lactation ADF I and ambient
temperature of the farrowing room (O'Grady et al., 1985; Matzat, 1990).

Several research groups have observed increased lactation ADF I of sows fed diets
containing "high fiber" during the gestation period (Table l). The results of some of
these studies may have been confounded because nutrient intake per animal per day was
different among treatments during gestation. However, in every experiment, those
animals that were fed fiber during gestation ate more of a common diet during lactation.
Reasons for the elevated lactation ADFI are not known. In growing pigs it has been
observed that pigs fed "high fiber" diets have heavier stomach weights (Anugwa et al.,

fi—vr

1989) and heavier GI tract weights (Pekas et al., 1983; Anugwa et al., 1989). Similarly,
N

 

the weights of liver, heart, stomach, small intestine, cecum and colon expressed as a
percent of body weight are greater for young adult pigs fed diets high in alfalfa than those
fed a diet which is low in alfalfa (Pond et al., 1988). Although it has not been
documented, heavier stomach weights of pigs fed diets high in fiber may indicate a
greater stomach volume or capacity. Perhaps this is why sows fed "high fiber" gestation
diets can eat more in lactation.

Stretch receptors within the stomach (Gonzalez and Deutch, 1981) and within the
duodenum (Davis and Campbell, 1978) are reported to limit intake via the vagus nerve.
Perhaps these receptors from sows on high fiber gestation diets have become acclimated
to the increased volume of feed. Sows fed fiber in gestation would come into lactation

with the ability to immediately consume more feed than those sows which consumed the

l 7
normal 4 to 5 1b com-soybean meal diet throughout gestation.

Varel (1987) and Anugwa et a1. (1989) found that fibrolytic bacteria increase and
replace other bacteria in the intestines of pigs fed ”high fiber" diets. The production of
tyrosine and tryptophan decrease as the number of proteolytic bacteria decrease. These
two amino acids are precursors for the anoretic agents dopamine and serotonin and
therefore, it has been hypothesized that pigs fed fiber eat more feed due to lower brain
levels of dopamine and serotonin (Bergner, 1981). Anarerobic degredation of tyrosine in
the intestine also leads to the formation of volatile phenols such as p-cresol (Scheline,
1968). Para cresol and other volatile phenols are potential toxins (Lumanta, 1987)..
Dietary fiber has been shown to reduce the production of p-cresol in rats (Scheline,
1968), and may have similar effects in pigs fed fiber . The residual time effect on feed
intake after withdrawal of fibrous material from the diet has not been reported.

Summaut

As the trend for world population growth continues, the demand for feed grains to
be used for direct human consumption will also continue to increase. As feed costs
represent approximately from 60 to 70% of the total cost of swine production, and the
conversion of grain to animal products is an inefficient process. Alternative feedstuffs
must be investigated. Diets high in fiber are not well suited for growing/ finishing pigs or
for lactating sows where energy intake often limits productivity. However, the literature
is convincing that, in moderation, fiber can be added to the diets of gestating sows. As
most gestating sows are fed only 1.8 to 2.7 kg-hd"'d" they may be able to use fibrous

diets more effrcaciouSIy than full-fed animals due to both a slower GI tract transit time

l 8
and smaller digesta volume. Both of which may facilitate the extent of microbial
fermentation of the fibrous component.

Before nutritionists can make recommendations as to the inclusion rates of these
alternative feedstuffs, better scientific experimentation must explore and elucidate the
repercussions of including the feedstuffs in the diet. To ascertain if the observed results
are actually due to the dietary fiber components, the daily intake of other dietary nutrients
needs to be balanced during gestation. To establish which fiber component is responsible
for the observed effects, the NDF or ADF (not crude fiber) content of the dietary
treatments needs to be considered. Chapter 2 and 3 of this dissertation investigate the
consequences of the use of two dietary feedstuffs in gestating swine diets: wheat straw

and soybean hulls.

CHAPTER 2

DIETARY WHEAT STRAW OR SOYBEAN HULLS DURING
GESTATION AND SUBSEQUENT LACTATION PERFORMANCE

Abstract

Thirty crossbred (Yorkshire x Landrace) gilts were randomly assigned to a com-
soybean meal (control), a com-soybean meal-wheat straw (WS), or a com-soybean meal-
soybean hull (SBH) gestation diet for three consecutive parities. Gestation diets were
formulated so that animals within each treatment were offered the same daily amount of
metabolizable energy, crude protein, lysine, calcium, and phosphorus. The WS and SBH
diets were formulated to provide similar daily intakes of NDF. During lactation, all sows
consumed the same com-soybean meal diet. Sows were weighed at breeding, within 12
hours post-parturition, at d 21 of lactation and at weaning. The total number of pigs born,
born alive, and stillborn was recorded. Individual piglet weights were recorded at birth,
21 d of age and at weaning. Fecal hardness of sows was determined on d 109 of gestation
and on d 3 and 10 of lactation. The SBH sows gained less (P<.05) weight in gestation
and lost less (P<.10) weight in lactation than WS sows. The weight changes of SBH and
control sows were similar. Both SBH and WS sows consumed more (P<.10) feed in
lactation than the control sows. Control sows lost more (P<.05) backfat than either SBH

or WS sows. The WS sows farrowed more (P<.05) live pigs than SBH sows, and weaned

l9

20

more (P<.05) pigs than either the SBH or control sows. Consequently, litter weaning
weights of the WS sows were greater (P<.05) than for control or SBH sows. The SBH
sows had a lower incidence of constipation at d 109 of gestation (P<.001) and d 10 of
lactation (P<.05) than the control or WS sows . The WS sows had stools that were
Significantly firmer than the SBH sows at each time point. These results Show that both
WS and SBH can increase lactation feed intake when incorporated into gestating swine
diets. Dietary SBH can also reduce the incidence of constipation.
Introduction
Several researchers (Pollmann et al., 1979; Pond et al., 1985; Holzgraefe et al.,

1986; Mroz et al., 1986; Carter et al., 1987; Hagen, 1988; Nelson et al., 1992) have fed
different fiber sources to sows and gilts during gestation and monitored their subsequent

lactational performance. All indicate that moderate levels of dietary fiber in gestation
I does not adversely affect sow performance. Some research groups have even reported
enhanced litter size and lactation feed intake of sows fed fibrous diets during gestation.

To study the influence of dietary fiber in gestation on subsequent sow

performance, the current experiment was in part undertaken by the North Central Region
Committee on Swine Nutrition (N CR—42). This committee's protocol included corn-
soybean meal (control), and com-soybean meal-wheat straw (WS) gestation diets. Wheat
straw was chosen as a fiber source due to its availability among research stations in the
Midwest. As wheat straw is a fairly non-fermentable fiber source, this experiment also
included a diet containing the more fermentable fiber of soybean hulls.

The objectives of this study were to determine the effects of dietary fiber in

21

breeding and gestating diets on the lactation ADFI, weight change and return to estrus
interval of sows and the weight gain of their litters, and to determine if fiber source
(wheat straw vs soybean hulls) and composition (measured by neutral detergent fiber
[NDF]) distinctly modify these performance variables.

MaterialaauiMflthS

Thirty Yorkshire X Landrace gilts were‘assigned to either a corn-soybean meal
(control), a com-soybean meal-wheat straw (WS), or a com-soybean meal-soybean hull
(SBH) gestation diet for three consecutive parities. Gilts were assigned to dietary
treatments in the order they initially expressed estrus (ie; control, WS, SBH, control, WS,
SBH).

The, amount of each treatment diet was calibrated in gestation so that sows among
treatments were offered the same daily amount of metabolizable energy, crude protein,
lysine, calcium, and phosphorus (Table 2); The WS and SBH diets provided similar daily
intakes of NDF. During gestation in parity 2 and 3, sows were offered 1.8 (Control), 2.1
(WS), or 1.9 (SBH) kg of feed-sow'd". The amount of each treatment diet offered to
parity 1 gilts was 25% greater to allow for continued body growth. The nutrient content
of soybean hulls used in diet formulation were fi'om the U.S.-Canadian Feed Tables
(1982). Wheat straw was assigned a value of zero for all nutrient contributions when
formulating the experimental diets. The wheat straw used in this experiment was ground
by hammer mill twice through a one-half inch screen. Resulting wheat straw length
ranged from between 6 and 12 mm. Gilts were fed their assigned diets beginning

approximately one week prior to breeding and throughout each gestation. Throughout

22

lactation all sows were fed twice daily an amount of feed to provide constant ad libitum
access to the diet which control sows consumed throughout gestation. Sows were fed
from individual pre-weighed amounts of feed stored in containers in front of each sow.
Feed consumption was determined by difference (initial feed weight - [unused feed
weight + orts]) and recorded weekly.

Within each parity during the period from weaning to breeding, sows were housed
in their respective treatment groups in an outdoor unit. After supervised mating, sows
were moved inside a gestation building and were placed in individual gestation crates
over partially slotted flooring. Sows were weighed at breeding, d 109 of gestation, within
24 hours post-farrowing, at d 21 of lactation and at weaning. Backfat depths of sows
were measured at breeding, d 109 of gestation, at d 21 of lactation, and at weaning with
an ultrasonic backfat probe (Renco Lean Meater,R Serial # 4458; Mpls., MN). Backfat
depths were measured approximately 3 cm off midline at the last posterior rib and the last
posterior lumbar vertebrae, and the average of the two readings was recorded. The
number of days from weaning to estrus was recorded throughout the study. Sows that
failed to establish or maintain pregnancy were removed from the experiment. An
assessment of the effects of wheat straw and soybean hulls on the incidence of
constipation was made on d 109 of gestation and on d 3 and 10 of lactation. This was
done via tactile appraisal of fecal hardness, and the following subjective scoring system

was used;

1. Very loose 3. Normal
2. Moderately loose 4. Moderately hard
5. Very hard

 

'I

 

 

 

23

Table 2. Composition and calculated analysis of diets used in Experiment 1 and 2.

 

 

a Wheat Soybean
Diet Control straw‘ hull
i "/ 1
Corn 79.50 69.20 66.60
Soybean meal (44%) 15.50 13.50 10.00
Soybean oil 1.00 .85 .85
Wheat straw - 13.05 -
Soybean hulls - ~ - 19.10
Dicalcium phosphate 1.85 1.75 1.69
Limestone 1.08 70 .76
Salt .40 .35 .37
Vitamin TM mixb .50 .45 .45
Folic acid supplement .02 .02 .02
Biotin supplement .15 .13 .13
L-lysine HCI - - .03
lat i t ' k
Metabolizable energy, Kcal/d 5960.0 5960.0 5960.0
Crude protein, g/d 246.0 246.0 246.0
Lysine, g/d 11.7 11.7 11.8
Calcium, g/d 16.0 16.0 16.0
Phosphorus, g/d 12.0 12.0 12.0
Crude fiber, g/d 49.0 162.0 196.0
NDF, g/d 172.0 402.0 403.0
ADF, g/d 71.0 218.0 249.0
E .1 . l l
Gilts 2.3 2.6 2.4
Sows 1.8 2.1 2.0

 

‘Used in Experiment 1 only.

bVitamin-TM premix, and F olic acid and Biotin supplements were supplied free of charge by
Feed Specialties Co., Inc., Des Moines, IA. VTM supplied the following amounts per kg of diet:
6600 IU vit A; 1210 IU vit D; 22 IU vit E; 3.37 mg menadione; 22 mg pantothenic acid; 33 mg

_ niacin; 1 mg folic acid; 5.5 mg riboflavin; 28 meg B”; 577 mg choline; 122 mg Zn; 126 mg Fe;
61 mg Mn; 12.2 mg Cu; .52 mg l; .3 mg Se. Biotin supplement supplied .33 mg biotin/kg diet.
Folic acid supplement supplied 1 mg folic acid/kg of diet.

24

The total number of pigs born, born alive, and stillborn was recorded. Individual
piglet weights were recorded at birth, 21 d of age and at weaning. Cross fostering of pigs
was not practiced in this study unless a sow had more pigs than functional teats (and then
only within treatment). Creep feed was not offered to piglets.
2313mm

Data were analyzed with the General Linear Models (GLM) procedure of SAS to
determine significant relationships between main effects (treatment, group and parity)
and all interactions. When group, parity or an interaction was not significant, it was
removed from the model, and the analysis was performed again. Means were separated
by the PDIFF procedure within SAS.

W

Lactation length was not standardized, but was not different among treatments
(mean i SE = 27.1 i .6, 26.6 i .7, 26.4 i .6 d for control, WS and SBH respectively).
As 21 d is a typical lactation length, 21 d and total lactation length means will be
presented.

ADFI

Both the WS and SBH sows ate more (P<.05) feed during the first 21 d of
lactation and more (P<.10) total lactation feed than control sows (Table 3). The WS sows
ate 14.7 kg/sow more feed, while SBH sows consumed 18.9 kg/sow more feed than
control sows during the first 21 d of lactation. When analyzed by parity (Table 4), the
treatment differences in lactation ADFI were not apparent until parities 2 and 3. During

parity 2, the lactation total ADF I of SBH sows was greater (P<.05) than control sows,

25

while WS and control sows consumed similar amounts. However, during parity 3
lactation, both the WS and SBH sows ate more (P<.Ol) than control sows. In parity 3
lactation, WS and SBH sows consumed and average 18.9 kg/sow more than control sows
during the first 21 d of lactation. These results imply that older sows may benefit from
dietary fiber more than primiparous sows. The influence that dietary fiber may have on
GI tract capacity or function may be time-dependent and may not have had time to exert

an influence in the primiparous animals of this experiment.

26

Table 3. ADFI and constipation scores for sows in Experiment 1‘

 

 

Wheat Soybeazl-nB

Treatment Control straw hull

MeanADELkg
Gestation 2.0 : .01b 2.2 i .01° 2.1 i .01d
Tod21 lactation 5.2:.15‘ 5.9i.18f 6.1 j;.l6f
Lactation 5.0 j; .12g 5.3 1.14h 5.6 : .12h

ngthnfnhwfi
Gestation 115.0 :1; .4 116.0 : .5 114.7 1 .4
Lactation 27.1 i .6 26.6 i .7 26.4 i .6
Wean to estrus 6.2 1: .7 5.8 i .8 5.1 i .7

i i c ‘

d109ofgestation 4.4;r_.1b 4.633.]b 3.4_-I;.1c
d 3 of lactation 3.9 :1; .2jk 4.3 i .2j 3.5 i .2k
(1 10 oflactation 3.3:.1" 3.21:.1‘ 3.0i.lf

 

‘ Least square means 1 SE.
bodMeans in a row with a common or no superscript do not differ (P<.001).
“Means in a row with a common or no superscript do not differ (P<.05).
ghMeans in a row with a common or no superscript do not differ (P<.10).
i1 to 5: 1=diarrhea; 2=loose; 3=normal; 4=firm; 5=constipated

jkMeans in a row with a common or no superscript do not differ (P<.01).

27

Table 4. Lactation ADFI by parity for Experiment 1“

 

Treatment Control straw hull
We

Parity 1 lactation total 4.1 i .2 4.2 i .2 4.5 i .2

Parity 2 to d 21 of lactation 4.8 i .3 5.1 i .3 5.6 i .3

Parity 2 lactation total 4.9 1 .2b 5.4 i .3"“ 5.8 i .2“

Parity 3 to d 21 of lactation 5.7 i .l“ 6.6 i .2“ 6.6 i .1“

Parity 3 lactation total 5.8 i .2d 6.6 : .2c 6.6 i .1“

 

' Least square means t SE.
b“Means in a row with a common or no superscript do not differ (P<.05).
d“Means in a row with a common or no superscript do not differ (P<.01).

t' w ’ w i e

Soybean hulls in the gestation diet decreased the incidence of constipation (Table
3). Constipation scores of SBH sows were lower (P<.001) than control and WS sows at d
109 of gestation and lower than WS sows at d 3 (P<.Ol) and d 10 (P<.05) of lactation.
Although sows from both fiber treatments consumed similar NDF daily, the cellulose
content of the SBH diet was greater. The cellulose within the SBH treatment may have
provided additional energy for the microbial population, and the reduction in constipation
was actually the result of an increase in fecal biomass (Bach Knudsen et al. 1993a). The
lower incidence of constipation in late gestation and early lactation of SBH sows may

have contributed to their ability to consume larger quantities of feed in lactation. Wetter

feces also indicate that the digesta transit time through the GI tract is shorter, allowing

 

28

more feed to be consumed over a given amount of time. This would explain the greater
ADFI of SBH sows, but is not consistant with the observation that WS sows had firmer
feces than control sows, yet a higher ADFI.

The daily intake of nutrients among sows on different treatments were calculated
to be similar throughout gestation. However, differences did exist among treatments in
sow body weight at several time points throughout the reproductive cycle (Table 5). The
WS sows were heavier (P<.01) than control sows at d 109 of gestation. The water
holding capacity of the dietary fiber consumed by WS sows may have contributed to
these differences. The WS and SBH sows were heavier (P<.10) than control sows at
breeding, post farrowing and at d 21 of lactation. SBH sows were heavier (P<.05) than '
either control or WS sows at weaning. The elevated ADFI of the WS and SBH sows
during lactation augmented these differences. The WS sows gained more (P<.05) weight
in gestation than SBH sows, but lost more (P<.10) weight in lactation than either control
or SBH sows (Table 5). The majority (mean= 76%) of lactation weight loss by all sows
occurred during the last 5 to 6 d of lactation. In light of these data it is easy to see how
the currently popular production practice of segregated early weaning (SEW) at 10 to 14
d of lactation can help sows maintain body condition and contribute to greater
performance in successive parities. The WS sows also lost more (P<.05) subcutaneous
backfat in lactation than did the SBH sows (Table 6). These observations may be
explained partially by the fact that WS sows farrowed more live pigs (P<.05) than SBH

sows, and weaned more pigs (P<.05) per litter than control and SBH sows (Table 7).

29

Table 5. Sow weights and weight changes in Experiment 13

 

 

Wheat Soybean
Treatment Control straw hull
n 24 20 25
W
Breeding 136.3 i 2.1b 143.5 1: 2.4“ 145.5 3; 2.1c
d 109 gestation 179.2 : 2.8d 191.7 i 3.0" 185.2 i 2.8“
Post-farrow 163.6 i 2.4d 175.4 1 2.5c 174.0 1; 2.3"
d 21 of lactation 161.2 1 2.4f 167.7 :1; 2.78 172.9 i 2.3g
Weaning 154.9 j; 2.4b 158.8 :1; 2.7b 166.5 i 2.3c
Mannheim
Breeding to d 109 of 42 .3 : 2.2"c 46.6 : 2.5b 39.6 i 2.1“
Post-farrow to d 21 -l .8 i 2.0fg -5.7 i: 2.3f -.8 i 1.98
Post-farrow to weaning -8.1 i 2.2f -l4.6 : 2.6g ~7.3 : 2.2f
Breeding to weaning 18.4 : 2.3fg 15.2 1; 2.5f 21.2 i 2.33
MW
Post-farrowtod21 -.1:.1fg -.3_«:.1f <-.1i .18
d 21 lactation to weaning -1.3 i .3 -2.0 i .4 -1.4 _t .3
Post-farrow to weaning -.3 i .1" -.5 j: .1“ -.3 :1; .1b

 

aLeast square means 1 SE.

b“Means in a row with a common or no superscript do not differ (P<.05).
dcMeans in a row with a common or no superscript do not differ (P<.01).
ngeans in a row with a common or no superscript do not differ (P<.10).

30

Table 6. Sow backfat depths and backfat depth changes in Experiment 1a

 

 

Wheat Soybean
Treatment Control straw hull
Brim
Breeding 21.6 _+_: .9 22.6 i 1.1 21.6 i .9
d 109 ofgestation 21.8 i .8 22.8 i .9 21.3 i .8
d 21 oflactation 21.0 i .7 21.1 i .8 20.5 i .7
Weaning 20.3 i .7 20.7 i .8 20.7 i .7
BackiaLdepthshanam
Breeding to d 109 gestation .3 i .7 -.1 i .8 -.3 i .6
d 109 gestation to d 21 lactation -.8 i .6 -2.3 i .7 -.6 i .6
d 109 gestation to weaning -1.4 i .6“ -2.5 i .7" -.5 i .6“
Breeding to weaning -1.1 i .9 -2.7 i 1.1 -.9 j: .9

 

‘1 Least square means _+_ SE.
b“Means in a row with a common or no superscript do not differ (P<.05).

The number of liveborn pigs and the total litter weight of the WS sows was greater
(P<.01) than either control or SBH sows. Litter size was not standardized in this trial, but
WS sows maintained their litter size advantage. The litter weights of WS sows were
significantly larger than those of control or SBH sows at each time point measured.
However, as litter weight gains were not different among treatments, the WS litter
weight advantage must be attributed to their larger initial birth weight.

The SBH sows weaned heavier (P<.05) piglets than did control or WS sows.
This difference is attributed to the significantly smaller number of pigs weaned/litter by

SBH sows compared to WS sows. It is assumed that piglets from SBH sows has less

31

Table 7. Sow reproductive performance in Experiment 1a

 

 

Wheat Soybean
Treatment Control straw hull
n 24 20 25
WW
Live born 9.4 i .6“ 10.6 3: .6b 8.5 _t .5“
Stillborn .6: .1b .2: .1c 1: .1c
Live at d 21 8.3 i .4b 9.6 : .4° 7.4 i .4b
Weaned 8.3 :t .4“ 9.6 i .4“ 7.4 i .4“
MW
Live born 13.6 : .8d 17.1 i .9“ 12.9: .8d
Total at birth 14.0 i .9b 17.1 i 1.0c 12.8 1: .8b
(1 21 47.3 : 2.0b 54.8 i 2.2“ 48.0 i 2.0b
Weaning 56.5 _+_- 2.4b 64.5 i 2.6“ 56.6 :1; 2.4“
L. w . l . 1
To (1 21 lactation 33.6 i 1.7 37.2 i 1.8 35.3 i 1.6
To weaning 43.1 i 2.1 47.3 i- 2.5 43.8 3: 2.1
W . l I . l
Bomalive 1.5: l 1.6: .1 1.5: .1
d 21 lactation 5.8 1 .2d 5.8 1 .2d 6.8 i .2“
Weaning 7 0 + 3b 6.8 + .3b 8.0 i .3“

 

“ Least square means : SE.

b“Means in a row with a common or no superscript do not differ (P<.05).
d“Means in a row with a common or no superscript do not differ (P<.01).

32

competition from one another and were able to consume greater quantities of milk from
the sow.
Conclusions

Both WS and SBH fed in gestation increased ADFI during lactation. Each test
diet increased the daily intake of NDF from about 172g (control) to about 400g/sow (WS
and SBH) in gestation. The additional volume the NDF provided with its water-holding
capacity may have induced changes in the stomach and GI tract of the sows during
gestation which enabled them to consume greater quantities of feed than control sows
when they entered the lactation period.

The WS increased the number of pigs weaned/litter and litter weaning weights.
Because litter size was not standardized in this experiment, and suckling intensity is
known to drive feed intake, the observed effects of greater ADFI of WS sows cannot be
attributed solely to treatment.

Soybean hulls, but not WS alleviated the incidence of constipation which is
common malady in sows near the time of parturition. The elevated feed intake of the
SBH sows may have been related to their lower incidence rate of constipation.

The results of this three parity study indicate that wheat straw or soybean hulls
can be used in swine gestation diets (at 13.05 and 19.1% respectively) and lactation sow
performance will be similar to or greater than that of sows fed a common com-soybean

meal gestation diet.

CHAPTER 3
EFFECT OF FEEDING SOYBEAN HULLS DURING GESTATION ON SOW

PERFORMANCE, NUTRIENT DIGESTIBILITY AND CHANGES IN BODY
COMPOSITION

Abulmot

Thirty-three York x Landrace gilts (about lO/group) were fed a com-soybean
meal (Control) diet or a corn-soybean meal-sOybean hull (SBH) diet throughout the
gestation periods through two parities. These diets were formulated to provide similar
daily intakes of ME, CP, Lysine, Ca., and Phos. In lactation, sows from both treatments
consumed a com-soybean meal diet containing .13% of a flavoring agent (Kent Feeds).
During lactation, sows were fed twice daily and orts were discarded once daily. Average
daily feed intake (ADFI) was summarized weekly. Sows and piglets were weighed
within 12 hours of parturition, and weekly thereafter. In parity 1, the deuterium dilution
technique was employed to ascertain if the normal changes in body composition that
occur during a parity were influenced by dietary treatments. In parity 1 and 2 lactation
and in parity 2 gestation, dietary nutrient digestibility coefficients (DC) were estimated
by the indicator method.

The ADFI of SBH sows was greater (P<.10) in parity 1, but not in parity 2 or

overall. Sows consuming the SBH diet had a much lower (P<.001) incidence of

33

34

constipation at d 110 of gestation. There were no differences between treatments in any
reproductive parameter measured. There were no statistically significant differences
between treatments in the rate of body weight change for any period over the two parities.
Similarly, data derived from the deuterium-dilution technique revealed no differences in
body composition changes in any period between sows on either treatment in parity 1.
However, ultrasonic measurement revealed that SBH sows lost more (P<. 10)
subcutaneous backfat in lactation than control sows. During gestation, SBH sows
digested less (P<.001) dietary gross energy and crude protein and less (P<.10) ether
extract. However, SBH sows digested more (P<.05) neutral detergent fiber and more
(P<.001) acid detergent fiber than control sows during gestation. In lactation, sows from
both treatments had similar dietary DC. There was no residual effect of the dietary
treatment imposed in geStation, on dietary DC measured in lactation.
hitroduution

Previous research (Chapter 2), indicated that SBH could be incorporated into
swine gestation diets with a concurrent increase in the ADFI in lactation. Litter size was
not standardized in that experiment. However, SBH sows consumed more feed than the
control sows during lactation, even though they tended to wean almost 1 pig/litter less.
To further study the implications of these results, this experiment was initiated. As
suckling intensity is known to influence ADFI, litter size was standardized. Because the
level of feed intake is known to impact changes in body composition, the deuterium
dilution technique was employed to estimate body composition. As dietary fiber has

been reported to influence DM digestibility, digestion coefficients (DC) were measured in

35

late gestation and early lactation. The latter to determine if there may be a residual effect
of the dietary gestation treatment in lactation. The dietary changes that the fiber-fed
sows make as they are switched from a high fiber gestation diet to a com-soybean meal
lactation diet is often radical in terms of physical form, nutrient compositon and taste.-
However, the change that control sows make frOm their com-soybean meal gestation diet
to their corn-soybean meal lactation diet is negligible. It is possible that this difference
contributes to the elevated rate of ADFI inlactation of sows fed high fiber diets during
gestation. In an attempt to reduce this disparity, a flavoring agent was added to the
lactation diet so that all sows had what we perceived to be, a significant change in diet as
they were switched from their gestation to lactation diets.
MotofialaflMethoda

Thirty-three York x Landrace gilts were divided into three groups (about 11
gilts/ group). To synchronize estrus among gilts, each group was moved from inside a
finishing barn to an outdoor unit and were artificially inseminated (A1) with the semen of
purebred Hampshire boar on expression of their second estrus. Gilts were heat-checked
with boars twice daily and inseminated 12 hours after the onset of standing heat, and at
12 hour intervals thereafter for a total of three inseminations/ gilt. After breeding and
throughout gestation gilts were housed indoors in individual gestation crates over
partially slotted floors. During parity 2, sows were housed in individual gestation crates

post-weaning and bred via AI upon expression of estrus.

36

Table 8. Composition and calculated analysis of
lactation diet used in Experiment 2 and 3'.

 

Ingredient % of total
Corn 69.25
Soybean meal (44%) 23.00
Soybean oil 3.00
Monodicalcium phosphate 2.20
Lime 1.15
Vitamin TM mixb .50
Salt .50
Biotin supplement .15
Flavor2 .13
L-lysine HCL .10
Folic acid supplement .02
Coloulatoduualm
Metabolizable energy, kcal/kg 3313.50
Crude protein, % 15.70
Lysine, % .90
Crude fat, % 5.47
Calcium, % .90
Phosphorus, % .77

 

“Flavor was used in Experiment 2 only. In Experiment
3, corn replaced the flavor.
I’Vitamin-TM premix, and Folic acid and Biotin
supplements were supplied free of charge by Feed
Specialties Co., Inc. VTM supplied the following
amounts per kg of diet: 6600 [U vit A; 1210 IU vit D;
22 IU vit E; 3.37 mg menadione; 22 mg pantothenic
acid; 33 mg niacin; 1 mg folic acid; 5.5 mg riboflavin;
28 mcg Bu; 577 mg choline; 122 mg Zn; 126 mg Fe;
61 mg Mn; 12.2 mg Cu; .52 mg I; .3 mg Se. Biotin
supplement supplied .33 mg biotin/kg diet. Folic acid
supplement supplied 1 mg folic acid/kg of diet.

37

Dietary treatments were initiated on the day of breeding and consisted of a com-
soybean meal (control) diet and a com-soybean meal-soybean hull (SBH) diet. These
two diets were also used in Experiment 1 (Chapter 2, Table 2). Gilts were allotted to
treatment as they exhibited estrus with every other gilt receiving the SBH diet. On the
day of farrowing, sows in both treatment groups were switched to a common com-
soybean meal diet (Table 8). This lactation diet contained .13% of a flavoring agent
(Kent Feeds).

Sows were weighed at breeding, d 110 of gestation, within 12 hours post
parturition, at d 7, l4 and 21 of lactation and at weaning. Backfat depths of sows were
measured at breeding, d 110 of gestation and at weaning with an ultrasonic backfat probe
(Renco Lean Meater, Serial #4458; Mpls., MN). Backfat depths were measured
approximately 3 cm off midline at the last posterior rib and at the last posterior lumbar
vertebrae, and the average of the two readings was recorded. Stillbom and live piglets
were individually weighed within 12 hr of birth, at d 7, 14 and 21 of age and at weaning.

Apparant digestibility of dietary nutrients was measured in late gestation and
early lactation. In parity 1 and 2, beginning on the day of farrowing and lasting for 12
days, a chromium-mordant fiber made from ground corn stalks (5.4% Cr) was added to
the lactation diet of all sows at the rate of .28% to provide a ration concentration of 150
ppm chromium. In parity 2 at an average gestation length of 100 days and lasting for 12
days, the same mordant-fiber was again used to supply a similar dietary chromium
concentration. In each lactation and in gestation, a 7 day adjustment period was followed

by a 5 day collection period. In the collection period a grab sample of fresh manure was

38

collected twice daily (AM and PM) from each sow. Daily samples were pooled and
frozen during the collection process. Prior to analysis, samples were dried for 72 hours at
55°C and ground in a blender. Feed and fecal nutrient analysis was done using the
following proceedures: gross energy content by adiabotic bomb calorimetry, starch by
high performance liqued chromatography (HPLC) analysis of free glucose following
incubation in sodium hydroxide and amylase, crude protein by a modified Hach total
nitrogen procedure, ether extract by Soxhelet ether extraction, and fiber fractions by the
Van Sooste detergent sequential fiber analysis technique. For specific protocols detailing
laboratory procedures used for the analysis of feed and fecal nutrient content, see the
Appendices.

Digestibility coefficients for gross energy (GE), crude protein (CP), ether extract
(EE), ash, neutral detergent fiber (N DF), acid detergent fiber (ADF), and lignin were

determined using the indicator method equation, Figure 1.

 

% Digestibility = 100 - (100 * Zamdroatouuksd "‘ W)
of nutrient % indicator in feces % nutrient in feed

 

Figure l. Indicator method equation used to calculate digestibility coefficients.

In parity 1, body composition of gilts was estimated at three different time points

Via the deuterium oxide dilution technique. Body composition was estimated between

39

days 7 and 14 of gestation, at two days post-parturition and at 7 days post-weaning. The
amount of empty body water, empty body fat, empty body protein, and empty body ash
that was estimated at each time point was then used to calculate the change in body
composition during each production phase (ie; gestation and lactation).

With the deuterium oxide dilution technique, gilts were infused with deuterium at
the rate of .25 g/kg body weight. Deuterium was infused through an ear vein following a
24 hour fast and 12 hours without water. Blood samples were collected via external
jugular vein puncture at 2.5 and 3.5 hours post infusion. The deuterium concentration in
the 3.5 hour post-infusion sample was compared to that in the 2.5 hour post-infusion
sample to verify that deuterium equilibrium had occurred within the body. Whole blood
samples were sublimated in liquid nitrogen and the resulting water was analyzed for
deuterium concentration via high performance liquid chromatography. The equations of
Rozeboom et al. (1994) were used to estimate body composition of gilts near breeding.
The equations of Sheilds et al. (1984) were used to estimate body composition of sows
post-farrowing and post-weaning.
DataAnaleis

Data were analyzed with the General Linear Models (GLM) procedure of SAS to
determine significant relationships between main effects (treatment, group and parity)
and all interactions. When group, parity or an interaction was not significant, it was
removed from the model, and the analysis was performed again. Means were separated

by the PDIF F procedure within SAS.

40

Rosebmddisoum

ADFI

In contrast to Experiment 1 where SBH sows had a significantly greater lactation
ADFI, the pooled lactation ADFI for the two parities in Experiment 2 did not differ
(P>.19) between treatments (Table 9). However, the SBH sows did have a numerically
larger ADFI at each time point measured. When ADFI was analyzed by parity, statistical
differences were apparent in parity 1. The SBH sows tended (P<.12) to consume more
feed in week 1 of lactation. The ADFI of the SBH sows was significantly greater (P<.08)
for the period from farrowing to d 21 of lactation and greater (P<.10) for the entire parity
1 lactation. During parity 2 there were no statistical differences between treatments in
ADFI. These observations were different from those observed in Experiment 1, where
differences in ADF I become significant during later parities. The reasons for the
different response between experiments are unclear. Interestingly, sows from both
tr eatrnents in this experiment had an ADFI about 1 kg more/d in each parity, than

contemporary sows in Experiment 1.

41

Table 9. Lactation ADFI for Experiment 2, kg“

 

 

Soybean P

Treatment Control hull value

W
Weekl 4.71.1 4.935.] 19
Week 2 ' 5.8 t .2 5.9 i .2 .62
Week 3 6.0 j; .2 6.1 i .1 .71
Tod21 5.51.1 561.1 .47
To weaning 5.6 i .1 5.8 i .1 37
Days of lactation 26.1 i .6 24.9 i .5 12

Karim
Week 1 4.3 i .2 4.6 i .1 .12
Week 2 5.1 i .2 5.5 i .2 18
Week 3. 5.4 _t .2 5.8 i .2 .14
Tod21 4-9:.1 53:1 .08
Toweaning 5.1:.1 5.4:.1 .10

Purim;
Weekl 5.31.1 3:.1 .87
Week 2 6.3 i .4 6.2 + .93
Week 3 6.6 1; .4 3 i .3 .52
Tod21 60:3 59:. .79
To weaning 6.0 i .3 6.1 :I_- .2 .94

 

“Least square means 1 SE

42

C nor on W‘l't ud wi hane - u u- . rm '01 1.1."

The SBH sows were significantly less (P<.001) constipated than the control sows
at d 110 of gestation (Table 10). Differences in the degree of constipation had
disappeared by d 3 of lactation and were also similar on d 10 of lactation. These results
are nearly identical to what was observed in Experiment 1.

There were no significant differences between treatments in body weight of sows
at any time point (Table 10). Similarly, there were no differences between treatments in
body weight change of sows during any period. However, SBH sows tended (P<.11) to
lose more body weight during week 3 of lactation. Backfat depths of sows on the control
and the SBH diet were similar at every time measured. However, the backfat depth of
SBH sows fluctuated to a greater extent than did that of the control sows. The SBH sows
lost more (P<.09) backfat in lactation and gained more (P<.01) backfat in gestation than
control sows.

During parity 1 there were no significant differences between treatments in the
changes in body composition of any measured parameter in either gestation or lactation
(Table 11). However, the increases during gestation and losses during lactation were
numerically larger for all compartments of the control sows. The inclusion of SBH did
not influence the body compositional changes of gilts consuming the test diet. Although
the ADFI of SBH sows was greater in parity 1 lactation, it was not sufficiently larger to

influence the degree of change in body composition.

43

Table 10. Sow weights, weight changes, and constipation scores in

 

 

Experiment 2“
Soybean P
Treatment Control hull value
Eighth:
Breeding 151.6 i 2.2 153.3 j; 1.8 .55
d 110 gestation 197.9 : 2.9 199.9 1 2.3 .59
Post-farrowing 179.7 1: 2.1 182.5 _t 1.7 .32
d 7 lactation 185.6 : 2.3 186.8 : 1.8 .68
d 14 lactation 183.9 : 2.4 186.0 1; 1.9 .49
d 21 lactation 183.3 i 2.7 183.6 : 2.2 .94
Weaning 181.1 : 2.9 183.9 1 2.4 .44
III . l l l .
Gestation total . 45.4 i 2 3 45.9 i 1.8 .88
Week 1 lactation 6.0 i .9 5.0 i .7 .38
Week 2 lactation ~21 :1; 1.0 -1.5 :t .8 .60
Week 3 lactation -.3 i 1.1 -2.6 i .9 .11
To (1 21 lactation 3.9 i 1.9 1.2 i 1.5 .28
Lactation total 2.3 i 2.1 1.6 i 1.7 .81
More;
Breeding 20.6 i .9 20.5 i .7 .91
d 110 gestation 20.3 j; .8 21.9 i .7 .15
Weaning 19.6 i .8 19.9 i .7 .79
MW
Gestation -.2 j; .5 1.8 1- .4 .01
Lactation -1 .0 i -2.2 i .4 .09
Parity -1.1 1 -.5 i .5 .53
C . .
d 110 gestation _ .1 .1 .001
d 3 lactation i .1 i .1 .90
d 10 lactation : .1 i .1 .85

 

IILeast square means j; SE

44

Table 11. Changes in sow body composition in Experiment 2, kg“

 

 

Soybean P

Treatment Control hull value

D . .
Body weight 39.5 i 1.3 37.3 i 1.2 .21
Empty body weight 32.9 3: 1.2 30.8 i 1.1 .20
Empty body water 14.4 i 1.0 12.5 3; 1.0 .21
Empty body protein ' 1.9 3; .3 1.6 i .2 .36
Empty body fat 10.0 i .8 9.9 i .8 .93
Empty body ash .76 i .1 .72 i .1 .66

mm
Body weight -23.4 _+_ 2.3 -20.2 i 2.1 .31
Empty body weight -21.4 i 2.1 -18.5 i 1.9 .31
Empty body water -8.4 i 1.1 -7.4 1: 1.1 .54
Empty body protein -1.5 i .2 -1.2 i .1 .26
Empty body fat -10.1 i 1.3 -8.4 i 1.2 .35
Empty body ash -.3 i .03 -.2 :t .03 .70

 

“Least square means : SE

45

Table 12. Changes in sow body composition by stage in Experiment 2, kg“

 

 

Mimi 1.2mm
Soybean Soybean

Treatment Control hull Control hull

Body weight 39.5 2: 1.8“ 37.3 i 1.71) -23.4 i 1.8“ .202 i 1.7c
Empty body weight 32.9 : 1.7b 30.8 i; 1.6b -21.4 i 1.7c -18.5 j; 1.6c
Empty body water 14.3 : 1.1b 12.5 : 1.0b -8.4 1: 1.1“ -7.4 i 1.0“
Empty body protein 1.9 1 .2b 1.6 : .2b -1.5 i .2“ ~12 j: .2“
Empty body fat 10.0 i 1.1b 9.9 i 1.0[3 .10.0 _+_ 1.1c -8.6 : 1.0c
Empty body ash .7 i .05b .7 :1; .05b -.3 i .05“ -.2 j; .05“

 

“Least square means : SE
b“Means within a row lacking a common superscript are different (P<.001).

When treatments were compared by stage (gestation verses lactation), the body
composition for both the control and the SBH sows changed similarly (Table 12). All
sows gained weight of every compartment during gestation, and lost weight of every
compartment during lactation.

KW

There were no differences between treatments in any measured reproductive
variable (Table 13). The SBH sows farrowed and weaned a similar number of pigs/litter
as the control sows. Additionally, the litter weights and litter weight gains of SBH litters

were not different than those recorded for the litters from control sows.

46

Table 13. Sow reproductive performance in Experiment 2“

 

 

Soybean P

Treatment Control hull value

Nmber of pigs /litter
Total born 10.4 : .7 10.3 : .6 .90
Live born 9.6 : .7 9.9 : .5 .74
Live at d 21 9.4 : .2 9.7 : .2 .29
Weaned 9.4 : .2 9.7 : .2 .22

Liuermightdce
Total at birth 14.9 : .9 14.4 : .7 .69
Live born 13.7 : .8 14.2 : .6 .67
d2] 54.0:2.1 53.4: 1.6 .81
Weaning 66.0 : 2.5 63.6 : 2.0 .46
Birthtod7 11.2: .6 11.2: 5 .96
d7tod 14 14.6: .8 14.0: .9 .53
d 14tod21 15.6: .9 15.4: .7 .86
d 21 to weaning 14.1 : 1.4 13.7 :1.1 .79
Birth to weaning 52.8 : 2.3 49.3 : 1.9 .25

 

“Least square means : SE

47
i 1' .1 '1'

During parity 1 lactation when sows from both treatments were consuming the
same diet, there was no indication of a residual affect of the SBH diet on digestibility
(Table 14). Both control and SBH sows digested Similar amounts of dietary nutrients.
During parity 2 gestation, SBH sows digested less (P<.001) dietary GE and CP and less
(P<.08) EE (Table 14). However, the SBH sows digested more (P<.05) NDF and more
(P<.001) ADF than control sows. These results agree with others (Komegay, 1981; Bray
et al., 1986; 1987) who have substituted SBH into the diet of gestating sows. The SBH
sows had a lower (P<.02) DC for lignin than that for control sows. In parity 2 lactation
there appeared to be a residual influence of the dietary soybean hulls that were fed during
gestation. The SBH sows digested less (P<.01) CP from d 7 to 12 of lactation than the
control sows. The SBH sows also had a higher (P<.06) DC for the dietary ash than the
control sows.

When the apparant digestibility data from parity 1 and 2 lactations were pooled,
the differences between treatments for CP and ash DC disappeared (Table 15). The
control and SBH sows digested similar amounts of dietary nutrients during lactation.
Results for the gestation DC for the pooled data was similar to the previous analysis;
SBH sows digested less (P<.001) GE, less (P<.10) CP, and less (P<.05) EE and ash.
However, the SBH Sows did digest more (P<.05) NDF and ADF than the control sows.

The DC measured for BB of this experiment are within the range (36.7% to
57.95%) of those reported by Graham et a1. (1985) for growing pigs. Additionally, the

11.7 unit decrease in EE digestibility for gestating sows on the SBH diet was similar to

48

Table 14. Nutrient digestibility coefficients (DC) for Experiment 2“

 

 

Soybean P

Treatment Control hull value
E . l l .

Gross energy 80.6 : .7 81.3 : .7 .47
Crude protein 79.7 : .8 79.9 : .8 .82
Ether extract 30.1 : 5.1 35.6 : 5.1 .46
Ash 39.3 : 2.1 37.8 : 2.1 .63
NDF 46.0 : 2.3 50.0 : 2.3 .23
ADF 46.9 : 3.1 52.8 : 3.1 .20
Lignin 40.4 : 2.1 43.5 : 2.1 .30
E . 2 .

Gross energy 84.7 : .6 79.8 : .4 .001
Crude protein ' 79.9 : 1.0 68.7 : .7 .001
Ether extract 64.8 : 10.7 41.4 : 6.2 .08
Ash 32.9 : 2.7 28.7 : 1.5 .19
NDF 67.5 : 2.0 72.4 : 1.1 .05
ADF 62.6 : 1.8 78.5 : 1.3 .001
Lignin 26.8 : 5.3 8.8 : 3.7 .02
E . 2 l .

Gross energy 81.6 : .6 80.7 : .4 .23
Crude protein 81.4 : .6 79.1 : .4 .01
Ether extract 34.5 : 5.2 29.0 : 3.7 .41
Ash 23.8 : 4.2 34.6 : 3.0 .06
NDF 55.9: 2.5 55.1 : 1.7 .78
ADF 57.4 : 3.3 52.8 : 2.3 .27
Lignin 22.5 : 4.3 27.3 : 3.1 .39

 

“Least square means : SE

49

Table 15. Comparison of digestibility coefficients (DC) by stage for

 

 

Experiment 2
Gestation Lactation

Soybean Soybean
Treatment Control hull Control hull
Gross energy 85.7 : .8b 80.1 : .6c 80.9 : .5“ 80.9 : .4“
Crude protein 82.0 : 1.3d 69.5 : .9“ 80.3 : .8df 79.5 : .6f
Ether extract 55.8 : 8.4“ 44.1 : 5.98" 32.7 : 5.0" 32.9 : 4.3h
Ash 39.3 : 3.99 29.0 : 2.8h 33.7 : 2.3sh 36.4 : 2.0gi
NDF 64.1 : 2.88 72.4 : 2.0h 49.1 : 1.7i 52.4 : 1.4i
ADF 62.6 : 3.4g 78.5 : 2.4h 50.7 : 2.11 52.8 : 1.8i
Lignin 26.8 i 7.18 8.8 : 5.0h 32.7 i 4.38 34.0 -_+_~_ 3.78

 

. “Least square means : SE.
b“Means within a row lacking a common superscript are different (P<.001).
d“'Means within a row lacking a common superscript are different (P<.10).
ghiMeans within a row lacking a common superscript are different (P<.05).

the reduction in digestibility from adding fiber that was reported by Den Hartog et a1.
(1985), and lust et a1. (1985) (10.8 and 8.0% respectively) for growing pigs.

When comparing stages (gestation verses lactation), control sows digested more
(P<.001) GB in gestation than in lactation, while the DC of SBH sows for GE did not
differ between gestation and lactation (Table 15). The SBH sows digested more (P<. 10)
CP in lactation than in gestation, while control sows digested similar amounts of CP in
the two periods.

The DC for BB was depressed (P<.05) in lactation as compared to gestation for

both the control and the SBH sows. Ash digestibility was lower ((P<.05) in lactation for

50

control sows, but ash digestibility improved (P<.05) in lactation for SBH sows.

The digestibility of structural carbohydrates was reduced in lactation. The DC
for NDF and ADF were lower (P<.05) for both treatments during lactation. While the
lignin DC in lactation improved (P<.05) for the SBH sows, the extent of lignin
digestibility did not change between gestation and lactation for the control sows. Some
of the differences observed between gestation and lactation in the degree of digestibility
may be partially explained by the differences in the rate of passage between the two.
periods. As the sows were switched from the restricted amount of feed intake in gestation
to the ad-libitum rate in lactation, the rate of transit through the GI tract may have
increased. Digestive enzymes in the small intestine had less time to do their job. as did
the microbes of the colon.

C 10

In many respects data from this experiment mirror observations made in
Experiment 1. However, because it was only conducted over 2 parities (in contrast to the
3 in Experiment 1) some relationships may not have been as apparent. SBH sows did
consume more feed in parity 1, and had less constipation. The elevated ADFI of SBH of
SBH sows may in fact have been the result of the lower incidence of constipation which
may be attributed to the shorter transit time of the digesta of SBH sows. This may have
enabled SBH sows to consume more feed per unit of time.

During gestation, SBH sows digested less (P<.001) dietary gross energy and
crude protein and less (P<.10) ether extract. However, SBH sows digested more (P<.05)

neutral detergent fiber and more (P<.001) acid detergent fiber than control sows during

51

gestation. This observation may reflect the adaptability of the microflora within the GI
tract of the SBH sows. The SBH sows may have cultivated a greater number of
cellulolytic bacteria in their GI tract, similar to those sows of Varrel and Pond (1985). As
sows on each treatment were switched to a common lactation diet, differences in
digestibility disappeared. There was no residual effect of the treatment imposed in
gestation, on dietary DC measured in lactation. Even if the higher number of cellulolytic
bacteria could survive the new environment, the rate of feed passage of the ad-libitum fed
sows probably increased to such an extent that they were unable to exert any influence.

At this inclusion rate of SBH (19.1%), dietary digestibility of GE and CP willbe
reduced. Therefore as was done in the present experiment, actual feeding rates of the
gestation diet will need to be increased about 10% to supply similar daily nutrient intakes.

Although the differences in ADF I were significant in parity 1, the additional
amount of feed consumed by the SBH sows was not sufficient to significantly influence
changes in body weight or changes in body composition during that lactation.

Including SBH in sow gestation diets at the rate of 19. 1% will not adversely
affect, and may enhance sow performance during lactation. These observations may in
part be related to the reduction in the incidence rate of constipation at or near the time of

parturition.

CHAPTER 4

REVIEW OF THE LITERATURE PERTAININ G TO CHOLECYSTOKININ (CCK)

Introduction

Consumer demand for lean pork has forced the swine industry to select for
animals with reduced fat reserves. Inadvertently, there has been a concurrent reduction in
voluntary feed intake (Riley, 1989). Although daily voluntary feed intake increases as
lactation progresses, most lactating sows do not eat enough to maintain body weight
during-an average 3 to 4 week lactation period. Lactation weight loss is most rapid
during the later stages of lactation as milk output peaks. Some sows have been observed
to lose more than 1.5 kg body weight/day from days 21 to 28 of lactation (Chapter 2).
Excessive weight loss can lead to delayed return to estrus intervals (> 10 days) following
weaning, especially in the primiparous sow.

In lactating sows (Matzat, 1990) and in growing pigs (Pekas, 1985), voluntary
feed intake limits productivity. Lactating sows which were superalimentated (SA) via a
gastric fistula and received 20% more feed than those sows allowed ad libitum access to
feed were able to preserve a greater amount of body mass and were also able to

synthesize greater quantities of milk (Matzat, 1990).

52

53

Increasing voluntary feed intake of lactating sows could augment sow productivity and
increase profit potential for the producer.
Rogulationuflxoluntamfooiintoko

Voluntary feed intake is regulated by many unknown mechanisms collectively
referred to as appetite (Pekas and Trout, 1990). Appetite and hunger are not one and the
same. Whereas appetite is satisfied by palatability, hunger is satisfied by calories (ie;
animals stop eating long before digestion makes calories available to the animal) (Houpt
and Houpt, 1991). Although exact mechanisms of appetite regulation are poorly
understood, many endogenous compounds are known to inhibit feed intake.
Cholecystokinin (CCK) is one such compound that is known to suppress voluntary feed

intake. Other endogenous agents are known to stimulate voluntary feed intake.

Table 16. Endogenous inhibitory and stimulatory agents of appetite.

 

 

Inhibitory Stimulatory

Cholecystokinin Gamma amino butyric acid (GABA)
Calcitonin Growth hormone releasing factor (GRF)
Dopamine Norepinephrine

Epinephrine Opoid peptides (ie: B-endorphin)
Glucagon Pancreatic peptides (ie: Neuropeptide Y)
Insulin

Neurotensin

Serotonin (5-OHT)

Somatostatin

 

54

A partial list (Leibowitz, 1986) of inhibitory and stimulatory agents of appetite are listed
in Table 16.
k' ' ° c ' i

Cholecystokinin exists in at least 5 molecular forms containing from 4 to 39
amino acids. The sulfated form of the C-terminal octapeptide (CCK-8) is the most
abundant and potent in the circulation (Granner, 1988). CCK peptides stimulate
gallbladder contractions and pancreatic enzyme secretions, and inhibit gastric emptying
in some species. However, neither endogenously released nor exogenously infused CCK
inhibit gastric emptying in pigs (Rayner and Gregory, 1989). Cholecystokinin is,
synthesized in the I cells of the mucosa of the duodenum and proximal jejunum and is
released into the portal blood after a meal in response to peptides, amino acids, long chain
fatty acids. calcium and acid entering the small intestine (Granner, 1988). Fat
components entering the small intestine are the most potent stimulants of meal-induced
CCK release (Walsh, 1981). The release of peripheral CCK is under vagal control, and
abdominal vagotomy abolishes the effect of meal-induced release of CCK (Smith et al.,
1981; Verbalis et al., 1986; Linden et al., 1989). In addition to peripheral CCK-8, CCK-8
has also been found in central locations in brain and cerebral spinal fluid (Walsh, 1981).
Similar to the meal-induced peripheral release of CCK, hypothalamic concentrations of
CCK have been elevated post-prandialy in primates and cats (Schick et al., 1987; 1989,
respectively). Because CCK does not cross the blood-brain barrier (Walsh, 1981) it may
be assumed that there is production and release of CCK in the brain and cerebral spinal

fluid.

55

Mochanismuflfliusasatietxiactor

CCK participates in rapid, preabsorptive satiety in pigs (Anika et al., 1981) and
other species. Intraperitoneal injections of partially purified CCK produced a dose
related suppression of feed intake (Gibbs et al., 1973; Stallone et al., 1989), while lateral
cerebral ventricle injections of CCK also depressed feed intake (Tsai et al., 1984) of rats.
Systemic infusions of CCK-8 have reduced meal size of pigs (Houpt, 1983). Although
the mechanism of CCK action in appetite suppression is not fully understood, evidence
indicates that the satiety effects of exogenous (and perhaps endogenous) CCK depend on
the activation of central monoaminergic systems (Tsai et al., 1884). Linden (1989)
reported that intraperitoneal administration of CCK-8 restored levels of dopamine in the
cerebral spinal fluid of rats mildly deprived of food. Dopamine is a known anorexergic
(Leibowitz, 1986). Therefore, the satiety effect of CCK may in part result from central
release of dopamine in response to peripheral CCK release. Stallone et a1. (1989)
proposed that exogenous CCK-8 acts at peripheral sites and exerts a satiety effect by
increasing serotonin (5-HT) activity in the brain. Serotonin is also an anorexergic
(Leibowitz, 1986). Cooper and Dourish (1990) proposed that CCK and 5-HT are
mutually interdependent on each other in mediating satiety. Evidence from Kow and
Pfaff (1986) suggest that the role of CCK-8 in satiety induction is two-fold: CCK-8
serves as a satiety agent in the periphery, mediated through vagal afferent nerves to the
brain, and as a neurotransmitter in the brain to convey the information originating in the
periphery. The results of Linden (1989) concur with these findings, indicating that

peripheral CCK receptor mechanisms induce a release of CCK in the brain. In the brain

56

and in the periphery CCK may serve as a neurotransmitter (Kow and Pfaff, 1986; Linden,
1989). Ultimately, the satiety center of the hypothalamus is stimulated by these
endogenous compounds, and the animal terminates the meal.

To date, two types of CCK-receptors have been identified. An A-type found in
many peripheral locations, and a B-type found in the brain (Cooper and Dourish, 1990).
To understand the role of each receptor type in the induction of satiety, Cooper and
Dourish (1990) administered CCK receptor antagonists to rats. Devazepide, a selective
CCK-A receptor antagonist, and L-365,260, a selective CCK-B/gastrin receptor
antagonist, both increased feed intake in well-satiated rats. The CCK receptor antagonist
L-365,260 was more potent. Only DeVazepide was able to block the satiating effects of
exogenously administered CCK. These data provide further evidence that satiety results
from a combination of both peripheral and central CCK action.

In lactating rats and sows, daily ad libitum feed intake generally increases as
lactation length progresses. Fleming (1976) suggested that the energy expenditure
associated with increasing amounts of milk synthesis is responsible for a large portion of
this hyperphagia. However, other mechanisms, may also be involved in stimulating the
hyperphagia of lactating rats and sows.

Daily feed intake is the sum of the number of meals eaten within a day and the
size of each meal. In turn, meal size is a combination of the duration of time spent eating,
and the rate of feed ingestion. CCK mediates its anoretic effects by reducing meal size

(Leibowitz, 1986). Interestingly, the gradual increase in feed intake of lactating rats

57

occurs primarily because of an increase in meal size (Strubbe and Gorissen, 1980;
McLaughlin et al., 1983) rather than an increase in meal frequency. Meal size becomes
larger in lactating rats in spite of the fact that plasma CCK levels are elevated (Linden,
1989; McLaughlin et al., 1983). The elevated CCK concentrations could be partially
explained by the fact that peripheral CCK is released in lactating rats (and dogs) in
response to suckling stimulus (Linden et al. 1990). This release of CCK is immediate and
short lasting, and in rats it is of a smaller magnitude than that seen after feeding (Linden
et a1. 1990). Nevertheless, even with elevated CCK concentrations, feed intake gradually
increases as lactation progresses. Therefore, Linden (1989), McLaughlin et a1. (1983),
and Wager-Srdar et a1. (1986) speculate that the marked hyperphagia of lactating rats
reflects an insensitivity of the animals to the inhibitory effects of CCK on food intake as
lactation length progresses; CCK has greater anoretic effects during earlier phases of
lactation than during later stages of lactation. However, pancreatic hypertrophy, a known
biological response to elevated CCK concentrations still occurs in lactating rats
(McLaughlin et al., 1983). Therefore, CCK-insensitivity during lactation appears to be
restricted to satiety. In contrast to others, Helmreich et a1. (1991) did not observe a
decrease in sensitivity to the anorexigenic effects of CCK as lactation progressed in rats.
CCK treatment has been shown to stimulate pituitary oxytocin secretion in
lactating rats (Verbalis et al., 1986; Helmreich et al., 1991). The effect of CCK
administration on oxytocin release was blunted by gastric vagotomy (V erbalis et al.,
1986; Linden et al., 1990). Gastric vagotomy also blocks the inhibition of food intake

induced by CCK (Smith et al., 1981; Verbalis et al., 1986). Collectively these

58

observations suggest that peripheral CCK elicits its effects on satiety and oxytocin release
through gastric vagal fibers, and not directly on the brain in rats. Because the suckling-
induced release of CCK precedes milk-ejection and the associated release of oxytocin in
the rat, Linden (1989) speculated that the release of CCK in response to suckling may
facilitate the milk-ejection reflex. CCK has also been implicated to play a physiological
role in the maintenance of good maternal behavior as intracerebroventricular CCK
infusions have been observed to block the disruptiOn of maternal behavior in rats caused
by the simultaneous injection of Beta endorphin (Felicio et al., 1991).
WWW

To demonstrate that endogenous CCK participates in feeding behavior several
research groups have injected CCK antibodies (CCK-AB) into animals. Circulating
CCK-AB do not cross the blood-brain banier, but are believed to sequester the
peripheral, free circulating endogenous CCK making it unavailable to the CCK receptor.
This phenomenon is known as immunoneutralization.

Exogenous CCK-AB administered via continuous lateral cerebral ventricular
injection increased the feed intake by wethers (Della-F era et al., 1981). Feed intake of
Zucker rats has been increased by exogenous CCK-AB administration, and also by
endogenous CCK-AB following active immunization against CCK (McLaughlin et al.,
1985). Similarly, the feed intake and growth rate of growing pigs has been elevated (8.2
and 10.6% respectively) following immunization against desulfated CCK-8 conjugated to
human serum globulin (Pekas and Trout, 1990). In another experiment Pekas (1993)

reported elevated feed intakes of growing pigs following immunization against desulfated

59

CCK-8 conjugated to one of four different haptens; bovine serum albumin (BSA), human
serum globulin (HSG), Keyhole limpet hemocyanin (KLH), or purified protein derivative
(PPD).

Antibody titers to CCK-8 have also been successfully raised in sheep immunized
against CCK-8, however feed intake of the immunized animals was not. different than
controls (Trout et al., 1989; Spencer, 1992). Spencer (1992) gave several possible

explanations for this anomaly;

1) Although CCK-AB were raised, the amount may have been insufficient to
effectively irnmunoneutralize the endogenously produced CCK.

2) The CCK-AB raised may have had too low an affinity to effectively prevent the
hormone from binding to the receptor.

3) There may be an elevated production and secretion rate of endogenous CCK due to
decreased negative feedback as CCK-AB sequestered CCK.

4) If endogenous CCK action is primarily by autocrine or paracrine means, it will be
less affected by circulating AB.

In both experiments (Trout et al., 1989; Spencer, 1992), feed intake was actually
slightly depressed on the days following booster vaccinations. Pekas and Trout (1990)
reported similar observations in pigs. Spencer (1992) offered three explanations why the
immunization procedure may actually potentiate rather than neutralize C CK action;

1) The CCK-AB may act like a plasma binding protein and protect the hormone from
degradation thereby extending it's biological half-life.

2) The AB-bound hormone may be presented to the receptor in such a way as to enhance
it's orientation at the receptor binding sites.

60

3) The AB-bound hormone may extend the hormone's transmembrane effectiveness by
inhibiting internalization and clearance of the hormone.

Summer

CCK reduces feed intake both directly and indirectly by stimulating the satiety
center of the hypothalamus. Active immunization against CCK increased feed intake in
growing rats and pigs presumably through immunoneutralization of the endogenous
CCK. Active immunization of lactating animals against CCK has not been examined to
date.
Implications

If gestating sows were vaccinated against CCK, the resulting CCK-AB may
increase voluntary feed intake during lactation. If so, sows may respond similarly to
super-alimentated sows (Matzat, 1990) which synthesized greater quantities of milk while
simultaneously preserving a greater amount of body mass. Elevated milk production may
aid in piglet survival and enhance litter weaning weights. Preservation of body mass
during lactation may contribute to timely rebreeding following weaning. If active
immunization against CCK increases voluntary feed intake of lactating sows, it may be

adopted by producers and become a standard management procedure in the future.

CHAPTER 5

ACTIVE IMMUNIZATION OF GILTS AGAINST CHOLECYSTOKIN IN
(CCK) IMPROVES LACTATION PERFORMANCE

Abstract

On d 64 of gestation, 20 York x Landrace gilts were immunized with the
desulfated C-terminal octapeptide of CCK conjugated to Keyhole limpet hemocyanin
(KLH) and 4 gilts with KLH alone (control). Three booster doses of immunogen (B1,
B2, B3) were administered at 14 d intervals after the primary dose. Prior to each
vaccination and on d 7, 14 and 21 of lactation (LD7, LD14, LD21 respectively), blood
samples were taken from all sows via external jugular vein puncture. Serum was
harvested and frozen for later determination of CCK antibody (AB) titer. The CCK-AB
titer determination followed procedures described by Pekas (1996a). At parturition a
colostrum sample was collected from each sow and litter size was standardized to 10
piglets. Blood samples were collected from 2 piglets of each litter at LD7. Milk samples
were collected from all sows on LD7, LD14, and LD21. During lactation, sows were fed
twice daily and orts were discarded once daily. The average daily feed intake (ADFI)
was summarized weekly. Sows and piglets were weighed weekly. The deuterium
dilution technique was employed to ascertain if changes in body composition that occur

during a parity were influenced by CCK immunization. For a 12 day period in late

61

62

gestation and again in early lactation, chromium oxide (CrzO7) was added to the diet of
all gilts to provide a ration concentration of .05% chromium oxide. In both gestation and
lactation, a 5 day fecal collection period followed a 7 day adjustment period. Dietary
nutrient digestibility coefficients (DC) were estimated by the indicator method.

Four CCK immunized sows were removed due to poor health in lactation.
Antisera titers reached values greater than 1:10 in 15 of the remaining 16 treated animals.
In lactation, specific binding of radio-iodinated CCK was not demonstratable at 1:10
dilution of serum from control sows or their piglets. Mean log titers of sow serum at B2,
B3, LD7, LD14, and LD21 was .27, 1.10, 1.77, 1.67, and 1.58 respectively. Mean log
titer of colostrum was 2.42, and that of milk was .50, -.003, and -.20 at LD7, LD14 and
LD21 respectively. These log titers were highly correlated to each other (P<.005 in all
cases). Mean log titer of piglet serum (n=30) at LD7 was 1.22. Regression analysis
revealed that the ADFI of sows in week 3 (W3) of lactation was correlated with each sow
serum titer value and with the titer of colostrum (r2 = .43 to .67, P<.01 in all cases). The
ADF I in W3 was highly correlated (r2=.68, P<.001) with the average of the seven samples
taken from the sow during lactation (ALTSS). The ADFI for the entire 21 d lactation was
also correlated (r2: .37, P<.05) with the ALTSS. Total litter gain was related (r2=.3 l ,
P<.05) to and increased with the ALTSS and with the ADF I of sows in W3 (r2=.28,
P<.05) . Body compositional changes were not equivocally related to anti-CCK titer.
Nutrient digestibility was not impaired as a result of CCK immunization, and appeared to

be enhanced, and increase with titer in gestation.

63

It is concluded that the elevated feed intake expressed by sows with higher anti-
CCK titers was used by the sow to produce more milk which resulted in greater litter
weight gain.

Introduction

Studies conducted with swine have shown that performance is limited by
voluntary feed intake. Superalimentation (through a gastric fistula) at 20 % above ad
libitum intake increased rate of gain in growing pigs (Pekas, 1985), and increased milk
secretion and litter weight gain while simultaneously reducing sow weight loss during
lactation (Matzat, 1990). A series of studies with young rapidly growing pigs showed
that active immunization against CCK stimulated feed intake and/or growth (Pekas and
Trout, 1990; Pekas, 1993) in proportion to the titer elicited by the animal. Recently,
Pekas (1996) reported that some CCK-antigens increase CCK antiserum titers in swine
better than others.

Clinical studies with CCK have shown that exogenous CCK decreases the transit
time of digesta through the GI tract (Mutt, 1980). Cholecystokinin is also responsible for
pancreatic release of amylases, proteases and lipases. These enzymes function within the
small intestine to facilitate digestion of dietary proteins, lipids and starches. The premise
behind CCK immunization is that the anti-CCK antibodies generated by the animal
sequester endogenous CCK. Therefore, we hypothesized that the nutrient digestibility of
gilts with higher anti-CCK titers may be altered. There is no evidence in the literature
pertaining to this question. Therefore, the apparent digestibility of several nutrients was

estimated inlate gestation and again in early lactation in primiparous gilts that had been

immunized against CCK.

Immunization of gestating and lactating swine against CCK has not been
reported. Therefore, the present study was undertaken.
Cl . .

1) To determine the effect of active immunization against CCK on the voluntary
feed intake and productivity of lactating sows and their litters.

2) To determine if CCK antibodies are present in the colostrum and milk of
vaccinated sows, and if CCK-AB are passed on to their piglets.

3) To determine if active immunization against CCK affects the magnitude
of changes in sow body composition during gestation and lactation.

4) To determine if an active CCK-AB titer would modify nutrient digestion.
t ri l

Vaccino Ereoarotion

Although the sulfated form of CCK-8 is needed for biological activity, antibodies
have been raised to the unsulfated form (Pekas and Trout, 1990) at much lower expense.
Therefore, the purified C-terrninal octapeptide of desulfated cholecystokinin (CCK-8)
was used in this study. The conjugated CCK8:KLH antigen was custom prepared
(Cambridge Research Biochemicals Inc., Wilmington, DE) using the glutaraldehyde
condensation reaction (Harlow and Lane, 1988). Prior to each vaccination, the antigen
was allowed to soak overnight in phosphate buffer. The antigen/buffer was then
emulsified in Freund's adjuvant with the use of two, hypodermic syringes connected via a

double-hub needle.

65

Sow

1n lactating swine, depressed feed intake, extensive weight loss and delayed return
to estrus following weaning are more common in primiparous sows. Therefore, thirty-
three crossbred gilts were used and were housed in three groups (~10 gilts/group). To
synchronize estrus among gilts, eachgro'up was moved from inside a finishing barn to an
outdoor Cargill unit and were artificially inseminated (A1) with the semen of purebred
Hampshire boars on expression of their second estrus. Gilts were heat-checked with
boars twice daily and inseminated 12 hours after the onset of standing heat, and at 12
hour intervals thereafter for a total of three inseminations/ gilt. Gilts were housed indoors
in individual gestation crates over partially slotted floors throughout gestation.

At about (1 64 (: 3 d) of gestation gilts were given their primary Vaccination. The
primary dose contained CCK-8-KLH conjugate emulsified in a solution of 50% Freund's
complete adjuvant and 50% phosphate buffer (Pekas and Trout, 1990). Each 1 mL dose
contained about 1 mg of antigen. Each dose was administered by subdermal-
subcutaneous infusion at three loci in an infusion site on the neck posterior (5 to 10 cm)
to the base of the ear. On days 78, 92, and 106 of gestation gilts received booster
vaccinations (B1, B2, B3, respectively) containing the same amount of the conjugate,
albeit administered in Freund's incomplete adjuvant. The order of vaccine administration
among gilts within a group was reversed at each vaccination date. This was done in an
attempt to reduce the possibility of the antigen settling-out of the adjuvant within the
syringe, thereby resulting in a situation where some gilts would get less or more of the

antigen. The time line of this experiment is summarized in Figure 2. Booster doses were

66

alternately administered on the opposite side of the neck. These vaccination intervals are
similar to those used by Pekas and Trout (1990) in growing-finishing pigs. Our
hypothesis was that the immune response of the gilts would be similar to that of the
growing pigs used by Pekas (1993). Therefore, peak antisera titer should occur about 60
days after the primary vaccine or about 7 days post-parturition. As lactating laboratory
animals (rats) become desensitized to the effects of CCK as lactation progresses, our goal
was to minimize the inhibitory effect of CCK when it is greatest (early lactation). In
support of this strategy, research shows that total lactation feed intake is greater for sows
that eat more during early lactation (Moser, 1985). The control gilts were immunized
following the same protocol used for the CCK-8 immunized group and the control
vaccines were prepared in the same manner to provide equivalent amounts of KLH
without conjugation to CCK-8. Both the CCK immunized and the KLH immunized
control sows experienced the severe tissue reactions from the Freund's adjuvants.
Therefore, values obtained from the KLH immunized control sows would reflect
detrimental effects of the vaccination protocol on performance. Prior to each vaccination,
and also on d 7, 14 and 21 of lactation (LD7, LD14, LD21), blood samples were taken
from all sows via external jugular vein puncture. Serum was harvested and frozen for
later determination of CCK antibody titer.

Because active immunization against CCK had not been reported in sows, it was
not known if CCK-AB were passed in the colostrum and milk with other
immunoglobulins. Therefore, a colostrum sample was taken from all sows within 12

hours post parturition to determine CCK antibody titer. Milk samples were collected

67

 

 

 

 

 

 

 

Booster Bonnier Booster
Primry l 2 J
Vaccinations --.> = . = - = = ; .
(1L G GDZ G106 -"- LDO ln'I—Inu LD21:
Blood Collections-bl a 7 ~ _~ '
Milk Corrections —> 'aflg‘; , "if 1’ .
Piglet Blood Corrections-5' " , j' ' _ f I j , j "
Gestation Lactation} . .
”Wm 888888885. 1 .
Intrusion , Infusion'_-_*,Ij: Infusion

Figure 2. Experimental Time Line“I

“Time code: G0=breeding; G64=d 64 gestation; G78=d 78
gestation; G92=d 92 gestation; G106=d 106 gestation;
LDO=farrowing; LD7=d 7 lactation; LD14=d 14 lactation;

LD21=d 21 lactation.

from all sows on LD7, LD14, and LD21. Milk samples were collected via manual

expression approximately 5 minuets following a 3 mL dose of oxytocin administered

intrarnuscularly.
Throughout gestation, gilts were fed 2.27 kg per head once daily of a common
com-soybean meal diet (See Control diet, Chapter 2, Table 2). In lactation all sows had

ad libitum access to a com-soybean meal diet (Table 12) and water. During lactation,

sows were fed twice daily and feed disappearance was recorded at each feeding. Orts

68

were discarded once daily.

Sows were weighed on day 1 and 110 of gestation, within 12 hr post-parturition
and on LD7, LD14, and LD21.

Apparent digestibility of several nutrients was estimated in late gestation and
again in early lactation. Beginning at an average gestation length of 100 days and lasting
for 12 days, chromic oxide was added to the gestation diet of all gilts so as to provide a
ration concentration of .05% chromic oxide. Chromic oxide (.05%) was again added to
the lactation diet beginning on day one of lactation and lasting until day 12 of lactation.
In both gestation and lactation a 7 day adjustment period was followed by a 5 day
collection period. In the collection period a grab sample of fresh manure was collected
twice daily (AM and PM) from each sow. .All the. fecal grab samples from a sow within a
collection period were pooled and frozen during the collection process. Prior to analysis,
samples were dried for 72 hours at 55° C and ground in a blender. Digestibility
coefficients (DC) for gross energy (GE), starch, crude protein (CP), ether extract (EB),
ash, neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin were
determined using the indicator method equation described in Chapter 3, Figure 1.

Body composition of gilts was estimated at three different time points via the
deuterium oxide dilution technique. Body composition was estimated near breeding (d 7
to 14 of gestation), post-parturition (d 2 lactation) and post-weaning (7 d post-weaning).
The amount of empty body water, empty body fat, empty body protein, and empty body
ash that were estimated at each time point were then used to calculate the change in body

composition during gestation and lactation (This technique is described in Chapter 3).

69

Rights

Stillbom and live piglets were weighed individually within 12 hr of birth, and at
LD7, LD14, and LD21. Because suckling stimulus induces release of both CCK and
oxytocin, we attempted to equalize suckling intensity via standardization of litter size
among sows. Therefore, the number of pigs per litter was standardized by day 2 of
lactation to 10 piglets. Nonexperimental sows provided extra piglets when needed. A
blood sample was collected from two piglets (one male and one female) of each litter at
LD7. This was done to determine if CCK-AB that may have been present in colostrum
were absorbed by the pig. Serum was harvested and frozen for later determination of
CCK antibody titer. Creep feed was not offered to piglets during lactation. Litter weight
gains were used to assess sow milk production.

mm

Serum, colostrum and milk anti-CCK serum binding titers were estimated by
radioimmunoassay (Pekas, 1996). Bolton-Hunter 125I-labeled CCKSS (sulfated; 2,200
Ci/mmol; Dupont, Boston, MA) was the radiolabeled antigen. Each assay tube contained
4,885 dpm or 1.0 fmol of Bolton-Hunter 125I-labeled CCKSS. Antiserum titers were
computed from the specific binding of Bolton-Hunter 125I-labeled CCK8S at four dilutions
(1 :10, 1:100, 111,000, and l:10,000) in phosphate buffered saline (.01 M, pH 7.2)
containing .05% gelatin (Sigma; #G-2500). The antiserum titer is defined as that serum
dilution that gives 50% specific binding of Bolton-Hunter 125I-labeled CCKSS, and was
computed using a linear regression of the logit of specific binding percentage verses the

log of the serial dilution factor. For the specific protocols detailing laboratory procedures

70

used in the analysis of CCK-AB titer and feed and fecal nutrient analysis, see the
Appendices.

2mm

Linear regression (SAS for Windows, 1994) was used to estimate the association
of CCK-AB titer with sow and litter production data, with sow nutrient digestibility, and
with the change in body composition data. This was based on previous reports (Pekas
and Trout, 1990; Pekas, 1996) that showed that feed intake and growth were proportional
to the anti-CCK-AB elicited, and that comparison of immunized to control animals by
analysis of variance was not meaningful because a proportion of immunized pigs had
undetectable or very low antiserum titers. The number of KLH immunized (control)
sows were few and were included principally to verify that regression intercept
coefficients derived from CCK immunized sows were reasonable.
WW

Twenty four gilts conceived and farrowed (20 CCK-KLH immunized gilts and 4
KLH-KLH control gilts). Of the 20 CCKzKLH immunized gilts, four became ill and one
did not elicit a detectable antiserum titer. These five sows were removed from the data
base. Therefore, unless stated otherwise, n=15 in all analyses.

5 352K . E l l .n_

Basal nonspecific binding of the radiolabelled CCK8 antigen, by serum collected
before the primary injection of immunogen, was scarcely detectable and only at the 1 :10
dilution. This was true for both the KLHzKLH control gilts and the CCKzKLH

immunized gilts. Specific binding of [EH-'25I]CCK8S in serum collected from

71

CCKzKLH sows immediately before the primary (P) dose injection was less than 2% at

1 :10 dilution and was not detectable at 1:100, 1:1000, or 1:10,000 dilution; consequently
antiserum titers could not be computed. Similarly, antiserum titers could not be
computed for serum collected from most CCK8:KLH immunized sows at the first booster
(Bl). The progression of serum, colostrum and milk titers of the 15 CCK8:KLH
immunized sows and the average piglet titer is illustrated in Figure 3. Mean (:SEM) log
titers of serum at BZ, B3, LD7, LD14, and LD21 was .27 (;|-_ .07), 1.10 (i .28), 1.77 (j;
.46), 1.67 (i .43), 1.58 (i .41) respectively. Variation of serum log titer values was high,
with the coefficient of variation ranging from between 67% and 73% during lactation.
This wide variation of titer was typical of the variation observed in young pigs
immunized against CCK (Pekas and Trout, 1990, 1993; Pekas 1996). The peak average
log titer of sow serum, 1.77 (equivalent to linear titer 1:59) occurred at LD7; thereafter
the log titer decreased to 1.58 (equivalent to linear titer 1:38) at LD21. The pattern of
immune response elicited by these primiparous sows was similar to that of growing pigs
where peak titers were observed about 60 days after the primary dose of immunogen
(Pekas and Trout, 1990, 1993; Pekas, 1996). Similarly, the peak log titer obtained with
the CCK8:KLH immunogen in young pigs was 1.9, (equivalent to linear titer 1:80, Pekas,

1996).

72

 

 

 

 

 

 

 

 

 

2.5 1
2
1.5 A
I-
,3: 1 _ _ I Saw serum
:0 ‘ I Piglet serum
'3 0.5 EJColostrum and milk
0 _—

 

 

-0.5 . . . . . v .
B2 B3 LDO LD7 LD14 LD21

Figure 3. CCKAB titers for sow and piglet serum, colostrum and milk

The mean log titer of defatted colostrum was 2.42 (3; .624, CV = 47.9%) which
was 2.3-fold higher than the log titer of sow serum at B3, and about 1.3-fold higher than
the log titer of sow serum at LD7. On a linear titer basis, where the titer of colostrum is
1:263 and of serum at LD7 is 1:59, colostrum titer is 4.46-fold higher than the peak sow

serum titer.

73

    

 

g 1'
'5

so

.9.

a“

5

a

in

-§' r2= .69, P<.001

0.5
0‘ A Y=.1.68+.167X
-1 -0.5 k 1 2 3

Sow serum at BB, log titer

Figure 4. Relationship between log titer of
sow serum and colostrum.

The regression equations show that colostrum titer increased .65 log unit for each log unit
increase in sow serum titer measured at B2 (R2: .53, P<.Ol), and .67 log unit for each log
unit increase in sow serum titer measured at B3 (R2: .69, P<.001) (Figure 4).

Titers of the defatted" milk samples were relatively low compared to titers of
colostrum or to those of sow serum (Figure 3). Mean log titers of defatted milk at LD7,
LD14, and LD21 were .50 (_+_ .130), -.003 (j; .009), and -.20 (j; .05) respectively. Milk
titer at LD7 increased .77 log unit for each log unit of increase of sow serum titer
measured at LD7.

Table 17 summarizes the correlation coefficients, r, of the log titers of all sow
samples. The correlation coefficients between log titers of serum, colostrum, and milk
samples were high, ranging from .67 to .99 (P <.005 in all cases), and demonstrate that
the CCK-AB titer of all samples taken from sows are highly interdependent.

These data imply that the concentration of CCK-AB in colostrum and milk are

74

dependent on the concentration of CCK-AB in sow serum. The CCK immunoglobulins
are sequestered by the mammary gland and concentrated in colostrum at considerably
greater concentrations than that observed in the circulating serum. The role that anti-
CCK immunoglobulins may play in CCK immunoneutralization before being transferred
and accumulated in colostrum and milk is not known. For this reason the
immunoglobulins in colostrum and milk, accounted for by titer, are included in these
analyses. For comparative purposes, the log titer (mean=] .22, CV = 125.3%) of piglet

serum (n=30) at LD7 is also presented in Figure 3.

75

Table 17. Matrix table of correlation coefficients, r between all pairs of log titer values
for sow serum (SS), colostrum (C) and milk (M)'I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample SS M

LD LD LD LD
Time” 32 B3 LD7 14 21 14 21
SS-BZ 1.00 .88 .69 .74 .70 ; .76 .67
SS-B3 1.00 .84 .89 .84 l .78 .74
SS-LD7 1.00 .98 .99 ’ .81 .78
SS-LD14 1.00 .97 f .83 .80
SS-LD21 1.00 3 .84 .81
C I .76 .74
M-LD7 g _ .93 .90
M-LD14 I 1.00 .98
M-LD21 I 1.00

 

 

 

 

 

 

 

 

aProbability of greater r value is P <.005 in all cases listed.
t’I‘ime code: B2=Booster 2; B3=Booster 3; LDO=Farrowing; LD7, LD14,
and LD21: d 7, 14 and 21 of lactation respectively.

53CCK°rS [.1
Anti-CCK-AB titers in the serum of piglets (two piglets per litter), were observed
(mean log titer =1 .22) and believed to have been derived passively through absorption
following transfer of anti-CCK immunoglobulins from the sow to colostrum. Although
piglet blood samples were not taken prior to nursing, it is assumed that the CCK-AB were
passively absorbed in the first 12 to 24 hours after birth. Specific binding of radio-

iodinated CCK was not demonstratable at 1:10 dilution in serum from piglets nursing

76

control sows or from non-experimental piglets that had been transferred on to immunized
sows at 1 or 2 d of age.

Piglet titers were highly correlated (r2: .79, P<.001) with the colostrum titers from
the sow being suckled (Figure 5), and increased 1.16 log units for each log unit of

increase in colostrum .

   
 

r2 = .79, P<.001
Y = -l.59 +1.16X

Piglet serum, log titer
3

-2 .45-41

 

Colostrum, log titer

Figure 5. Relationship between log titer of
colostrum and piglet serum.

As stated earlier, the titer of colostrum was closely associated with titer of sow
serum (Table 17). Therefore, titer of piglet sermn was expected to, and found to .be
associated with sow serum titer measured at each time point (r2 = .62 to .89, P<.001 in all
cases). Because piglets derived their titer from colostrum, which was supplied
immunoglobulins from sow serum, the B3 serum titer of the sow should best reveal
relationships of sow titer to that of piglet titer. Regression analysis disclosed that this
was true. Piglet titer increased 1.0 log unit for every one unit increase in B3 sow serum

titer (Figure 6).

\l
\l

  
  

r2 = .89, P<.001
Y = .12 + 1.0x

glet serum, log titer
N

o _'. :FPi
.0. .
H :

i

1 2 3

 

e 11
Sow serum at B3, log titer

Figure 6. Relationship between log titer of
sow serum and piglet serum

E l . l l C :1; . . Z3119!)

The ADFI (: SE) during the first, second, and third week of lactation, and for the
entire 21 (1 period was 4.19 (:21), 4.98 (1.36), 5.62 (:34) and 4.93 (35.24) kg/day for the
four control sows, and 4.24 (35.10), 4.93 (i .18), 5.07 (i .19), and 4.75 (35.13) kg/day for
the CCK immunized sows.

Correlation coefficients, r, were computed to relate ADFI during the first, second,
and third week, and for the total 21-day lactation period with the log titers of serum,
defatted colostrum, and with the average log titer of the seven samples (ALTSS) taken
from each sow during lactation (Table 18). The seven samples included the three
samples of sow serum, the one sample of defatted colostrum, and the three samples of
defatted milk. The ALTSS is an indicator of the overall immune response executed by
sows during lactation.

The ADFI during week 1 and week 2 of lactation were not significantly correlated

78

with log titer of any of the serum samples, but was correlated with log titer of colostrum
(P <.05 for week 1; P<.10 for week 2). However, the ADFI during week 3 was highly
correlated with log titer of each sample of serum (P<.01), and especially with colostrum
and the ALTSS (P<.001). This evidence indicates that the feed intake response to CCK
immunization is either dependent on the peak titer, which was not expressed until the
second week of lactation, or is subject to a time delay after a minimal threshold titer was
expressed. The ADF I over the entire 21-day lactation was correlated with log titers of
samples of serum (P<.10), and especially with colostrum (P=.002), and with the ALTSS
(P=.02). Although the strongest relationships between ADFI and CCK-AB titer were
observed during week 3, the ADFI over the 21-day lactation did increase with CCK-AB

titer.

79

Table 18. Correlations, r of ADF I with the log titer of sow
serum and defatted colostrum.

 

 

ADELJse
Weeks

Period Week 1 Week 2 Week 3 1,2,3
Sow serum

Booster 2 .157 .177 .655“ .432

Booster 3 .172 .167 .718“ .462c

Lactation, (1 7 .056 .132 .716“ .415

Lactation, d 14 .128 .123 .734“ .440b

Lactation, (1 21 .065 .143 .705“ .417
Colostrum .543c .433b .819“ .725“
ALTSS“ .294 .312 .822“ .608c

 

“Probability of greater r value, P<.01.

bProbability of greater r value, P<.10.

“Probability of greater r value, P<.05.

“Probability of greater r value, P<.001.

°ALTSS=Average log titer of seven samples taken
during lactation; sow serum at d 7, d 14, and d 21 of
lactation, colostrum, and milk at d 7, d 14 and d 210f
lactation.

 

 

 

80

 

 

7 ... o
6 -- . .
'2 24”.: °
3 3 ~-
II-
3 2 - r2 = .68, P<.001
1 -- Y = 4.40 + .61X
-1 0 1 2 3

ALTSS, log titer

Figure 7. Relationship between average log
titer of seven sow samples (ALTSS)
and ADFI in week 3.

The relationship between feed intake during week 3 of lactation and the ALTSS taken
from sows during the 21-day lactation was examined by linear regression. The quantity
of ADF I during week 3 of lactation (Y; kg/day) as a function of ALTSS (X) is described
by the equation: Y = a + bX, where a = 4.40 kg/day (intercept) and b = .615 (slope
coefficient), illustrated in Figure 7. Using the ALTSS mean (1 .1) and standard deviation
(SD=1.0), this equation estimates that ADFI during week 3 would be about 4.4, 5 .1, 5.7,
or 6.3 kg/hd when the ALTSS was 0 (no titer), 1.1 (the mean), 2.1 (mean + 1 SD), or 3.1
(mean + 2 SD) respectively. These estimates represent a 0, 16, 30, or 43% increase of

feed intake compared to sows with an anti-CCK titer of zero.

81
w ' n ' i
Means (3: SEM) of body weight change of sows during week 1, 2, 3, and for the
entire 21-d lactation are presented in Table 19. The CCK immunized sows gained an
average 1.67 kg/hd body weight during the 21-day lactation period. This net weight gain
was the result of 4.75 kg/sow gain in Week 1 “of lactation, followed by weight loss in

week 2 and week 3 of lactation.

Table 19. Summary of sow body weight changes during lactation by weekly periods and
correlations, r with the log titer of serum, colostrum and milk.

 

Body weight change (kg/period)

 

Period Week 1 Week 2 Week 3 Weeks 1,2,3
Means 4.75 -2.89 -0.18 1.67
SEM . 1.41 1.03 .88 1.76
mm

SS-B2b .276 .099 .374 .464“
SS-B3 .101 .114 .242 .268
SS-LD7 -.173 .002 .222 -.027
SS-LD14 -.084 -.106 .288 -.014
SS-LD21 -.174 -.019 .221 -.040
Colostrum -.064 .156 .242 .159
ALTSSc -.086 .057 .302 .1 15

 

“Probability of greater r value, P<.10

lyTime code: B2=Booster 2; B3=Booster 3; LDO=Farrowing; LD7, LD14,
and LD21= d 7, 14 and 21 of lactation respectively.

“ALTSS=Average log titer of seven samples taken during lactation; SS-LD7,
SS-LD14, SS-LD21, Colostrum, M-LD7, M-LD14, MLD21.

82

Body weight loss of sows during lactation is normal under conventional husbandry
practices. The possibility that CCK immunization, and the associated increase in feed
intake would suppress, or even reverse body weight loss during lactation was examined
by correlation analysis. As for the analysis of feed intake response, correlation
coefficients between body weight change variables and log titer variables were computed
(Table 19). Although trends of total body weight change during lactation indicated that
the weight losses during week 2 and especially those during week 3 were suppressed, the
effect was not clearly associated with anti-CCK titers of serum, colostrum or milk. Only
total sow body weight change over the 21-d lactation was associated (P<.10) with the log
titer of sow serum, and then only that titer measured at the second booster (82).

i‘ 00160 I“. Cllll900n1J10-C ,-. ° .i' o __iu,1.-1l 101

Body composition of sows was determined both during gestation and lactation.
Net change of the various body compartments (including: body weight, empty body
weight, empty body water, empty body protein, empty body fat, and empty body ash) is
summarized in Table 20. It is imperative to note that the published procedures and
associated equations for the deuterium dilution technique of estimating body composition
were established for and mandate specific infusion times relative to the life-cycle of the
sow. Therefore, results of sow weight changes for a particular period, derived from the
D20 procedure may not mirror previously described sow body weight changes within this
text. Nevertheless, as all sows were treated similarly, the collected data are considered

informational. All body compartments were enlarged during gestation and all were

reduced during lactation.

83

Table 20. Body weight change and empty body (EB) composition changes of CCK-
immunized sows during gestation and lactation, kg“

 

 

 

Time Weight weight water protein fat ash

Gestation 39.4 i 6.4 33.0 i 5.9 11.7 i 4.0 2.7 j; 1.1 12.4 i 4.8 .9 i .2

Lactation -20.1 i 7.3 -18.5 i 6.8 -6.2 i 3.9 -1.3 i .5 -9.7 i 5.0 -.2 i .1
“Mean : SD.

This trend was expected. An important objective was to establish if CCK immunization
might influence the magnitude of these changes. Therefore, correlation coefficients
between the body compartment mass changes vs. the log titers of a variety of samples
were computed. With rare exception (sows with higher LD7 and LD14 serum titers lost
more [r=.44, P<.10] EB ash in lactation), there was no association between compartment
mass change and log titers, either during gestation nor lactation. This evidence,
combined with evidence that body weight change was not associated with log titer,
indicates that immunized sows with higher titers utilized the nutrients from their
additional feed intake for the synthesis and secretion of milk to feed their piglets.

The possibility that CCK immunization, and the associated increase in feed intake
would change the rate of backfat depth dynamics (depletion or deposition) was examined
by correlation analysis. Correlation coefficients were computed for backfat depth
(measured via ultrasound), and change verses the log titers of serum, colostrum, and

ALTSS. The results are summarized in Table 21, and reveal that backfat depth at LD21

84

Table 21. Sow backfat depth and change, mm and correlations, r
with the log titers of serum, colostrum and milk.

W

Lactation Parity

 

 

 

Item Breeding (1 21 change
Means 22.6 20.4 -2.1
SD 2.4 3.5 2.6
_E_
Sow serum
Booster 2 .12 -. 1 7 -.34
Booster 3 .05 -.32 -.49“
Lactation, (1 7 -.03 -.55b -.73c
Lactation, (1 14 -.06 -.52b -.65c
Lactation, (1 21 -.08 -.60b -.74°
Colostrum .03 -.30b -.44
ALTSS“ .01 -.51b -.71c

 

“Probability of greater r value, P<.10.

bProbability of greater r value, P<.05.

“Probability of greater r value, P<.01.

“ALTSS=Average log titer of seven samples taken
during lactation; sow serum at d 7, d 14, and d 21 of
lactation, colostrum, and milk at d 7, d 14 and d 210f
lactation.

is negatively associated with the log titer of samples collected during lactation. The
association of backfat depth change and CCK-AB titer was explored by linear regression
analysis to obtain an estimate of quantitative response expressed in terms of the log titer.

Using sow serum at LD21, the regression equation and coefficients arezY = a + bX,

where Y = backfat loss (mm), a = .49, b = -1.67, and X = log titer of serum at LD21, (r2

85

= .55, P < .01). Thus sows lost an average 1.67 mm of backfat for each one unit increase
in log titer of sow serum at LD21. Substituting the ALTSS in the equation; a = -.09, b =
-1.85 (r2 = .51, P<.01), predicts that sows lost an average 1.85 mm for each 1 unit

increase in log titer (Figure 8).

A”?

r1 = .51, P<.01
' Y = -.09 + 1.85x

'
d "

 

Parity backfat change, mm
4a b (a L b t'o .'. 1

ALTSS, log titer

Figure 8. Relationship between average log titer
of seven sow samples (ALTSS) and
backfat depth change.

86

LAW

Means of litter weight gains and correlation coefficients between litter gain
variables and log titer variables of the sow and of the piglets are summarized in Table 22.
The litter weight gain that occurred in week 1 and in week 2 of lactation was not
associated with any single titer measurement derived from the sow. However, week 3
litter gains were correlated with every sow serum titer, colostrum, and with the ALTSS.
Week 3 litter gains were highly correlated (r=.84, P<.001) with sow serum titers
measured at LD7, LD14, and with that measured at LD21. Total litter weight gain was
related to every lactation sow serum titer and to the ALTSS, but not to colostrum. Linear
regression analysis showed that total litter weight gain increased 4.0 kg for each one unit

increase in ALTSS (13:.41, P<.01, Figure 9).

87

Table 22. Summary of litter weight gains during lactation by weekly periods and
correlations, r with the log titer of serum, colostrum and milk.

 

Litter weight gains (kg/period)

 

 

Period Week 1 Week 2 Week 3 Total
Means 10.1 12.8 13.1 35.9
SD 2.9 1.8 3.3 6.2

 

Correlation coefficients

 

Sow serum
Booster 2 .04 .10 .59“ .27
Booster 3 .15 .07 .70b .47c
Lactation, d 7 .39 .36 .84“ .73b
Lactation, d 14 .36 .24 .84“ .69b
Lactation, (1 21 .40 .32 .85d .73b
Colostrum .29 .06 .51“ .39
ALTSS“ .35 .23 .77d .64“
Piglet titer, d 7 .26 .16 .73b .56“

 

“Probability of greater r value, P<.05

bProbability of greater r value, P<.01

“Probability of greater r value, P<.10

“Probability of greater r value, P<.001

“ALTSS=Average log titer of seven samples taken during lactation; sow serum at
d 7, d 14, and d 21 of lactation, colostrum, and milk at d 7, d 14 and d 210f
lactation.

 

,0 r2= .41, P<.01
5 Y = 31.5 + 4.03x

 

Total litter weight gain, kg

-1 0 1 2 3
ALTSS, log titer

Figure 9. Relationship between average log
titer of seven sow samples (ALTSS)
and litter weight gain

Although confounded with other variables, litter gain in week 3 of lactation was
correlated (r=.73, P<.01) with the log titer of serum from the piglets themselves, as was
the total gain of the litter (r= .56, P<.05) (Table 22). Regression analysis showed that
litters gained an additional 2.2 total kg in lactation for every 1 log unit increase in their
serum titer (Figure 10).

Litter weight gain during week 3 of lactation was also correlated (r2 =.44, P<.01)
with sow feed intake during week 3 of lactation. The linear regression equation shows
that litter weight gain increased 3.0 kg per week (.43 kg/d) for each 1 kg increase in ADFI
during week 3 (Slope = 3.0). Total litter weight gain over the 21 d lactation period was
also correlated (r2 = .28, P<.05) with sow feed intake during week 3 of lactation (Figure
11) and similarly increased 4.4 kg for every 1 kg increase in ADFI of the sow during

week 3 of lactation.

89

 

10 r2 = .31, P<.05
5 Y = 33.20 + 2.24X

j I r I

-1 0 1 2 '3

Total litter weight gain, kg

Piglet serum, log titer

Figure 10. Relationship between log titer of
piglet serum and litter gain

so 4
45 «1 °
40 ~~
35 ~-
30 4
25 4
20 «-
15 4
1° ~~ r2= .28, P<.05

g ' Y 1= 13-43.+4~43?‘

 

 

 

1
fl

Total litter weight gain, kg

0 2 4 6 8
Sow ADFI week 3 of lactation, kg

Figure 11. Relationship between sow ADF]
and litter weight gain

90

As stated previously, the feed intake of sows was elevated in proportion to the
CCK-AB titer. Because of the co-variation between feed intake of the sow and the CCK-
AB titer of the sow, the regression analysis of litter weight gain with either sow feed
intake or sow CCK-AB titer is confounded. There is also co-variation between piglet
titer and sow titer. Therefore, it can not be said if the elevated rate of litter weight gain
can be attributed to the higher rate of ADF I by the sow, to the CCK-AB titer level of the
sow, or to the CCK-AB titer of the pig.

However, when considering the strength of the different calculated relationships,
several observations can be made: The best association of week 3 ADF I of the sow and
sow serum titer is that measured at LD14 (r2=.54, P<.01). Similarly, the sow serum titers
measured at LD14 and LD21 showed the strongest relationships (r2=.71, P<.001) with
week 3 litter gains. The sow's ability to synthesize milk will ultimately limit the growth
rate of the pig, no matter what the piglet’s appetite may be. Nevertheless, the associations
between week 3 litter gain and colostrum (from which the pigs derived their titers)
(r2=.26, P<.05), and week 3 litter gain and piglet titer (r2=.54, P<.01) are not as robust as
the relationship between sow serum titer and litter weight gain. Because the ADFI of the
sow was dependent on, and increased as log titer of the sow increased, it seems logical
that the increase in litter weight gain is a direct result of the elevated feed intake of the
sow. Since piglets in this study did not have access to creep feed, it is assumed that the
additional litter gain was derived from an elevated secretion rate of milk by the
immunized sows.

Interestingly, litter weight gain was positively associated with feed intake and sow

91

titer, while sow weight during the same period changed little and was not associated with
titer. This implies that the additional nutrients derived from the elevated ADFI were
prioritized for milk synthesis over maintenance. Litter weight gains and sow weight
change and composition changes are associated negatively. Sows that produced heavier
litters at LD21 accumulated less weight themselves in lactation (r2 = -.35, P<.05).
Additionally, sows producing heavier litters lost more (r2 = -.33, P<.05) EB protein, more
(r2 = -.42, P<.01) EB ash, more (r2 = -.26, P<.10) EB fat, and more (r2 = -.81, P<.001)
subcutaneous backfat. It appears that the elevated ADFI and associated higher levels of
some dietary nutrient(s) may have stimulated milk synthesis. However, other dietary

nutrients were insufficient to prevent body catabolism.

 

The gestation fecal samples for one sow were inadvertently not collected.
Therefore, gestation DC data include only 14 observations. The relationship between
CCK-AB titer and the digestibility coefficients obtained during gestation were best
described using those titers obtained in gestation; B2 and B3. For a time reference, the
BZ and B3 serum titer values were measured approximately 16 and 1 (1 respectively, prior
to the beginning of the gestation digestibility collection period. The DC measured during
gestation for GE, starch, CP, EE, and ash were correlated with CCK-AB titer level and
increased proportionably with the titer of the gilt (Table 23). As CCK-AB titer at B3
increased, sows were able to extract more (r2=.26, P<.10) GE and CP, more (12:21,
P<.10) EE, and more (r2=.25, P<.10) ash from the gestation diet. Similarly, sows with

higher anti-CCK-AB titers at B2 and B3 were able to utilize more dietary starch (r2 =.30,

92

P<.05; r2=.36, P<.05 respectively). The ability of sows to utilize fibrous components of
the diet was not influenced by CCK-AB titer level. The DC calculated during gestation
for NDF, ADF, and lignin were not correlated with any CCK-AB titer of the gilts. This
observation seems logical as the enzymes responsible for fiber digestion are not
synthesized by the pancreas but are rather microbial in nature. During lactation (LD8 to
LD12) dietary nutrient digestion was not influenced by CCK-AB titer. However, there
was a trend (r2 =.19, P=.103) for the ash DC to increase as CCK-AB titer measured at d

14 of lactation increased.

Table 23. Regression coefficients, r2 for CCK-antibody titer and dietary DC
in gestation.

 

Digestibility coefficients GE Starch CP EB“ Ash
Booster 2 NSb .30“ NS NS NS

Booster 3 .26“ .36“ .26“ .21“ .25“

“Ether extract

bNot statistically significant
cP<.05.

dP<.10.

 

 

The mean calculated DC measured during gestation and lactation for CCK
immunized sows are presented in Table 24. In both gestation and lactation, the digestion
of dietary starch was nearly complete (DC = 98.9 and 98.3 respectively). These values
are close to those observed by Graham et al. (1985) who reported fecal starch DC ranging

from between 98.9 to 99.4 in growing pigs. Others (Jorgensen et al., 1985; Just et al.,

93

1985) have reported whole tract starch DC of 100. During lactation, sows tended (P=.15)
to digest less of the dietary GE than they did in gestation, while digestibility of starch was
significantly lower (P<.02). These data may reflect increased rate of digesta passage
through the GI tract as sows are switched from restricted to ad-libitum feed intake in
lactation. Pekas and Trout (1990) did not observe a depression in digestibility (measured
by G/F) in pigs which expressed a greater ADF I as a result of immunization against
CCK. Whereas Pekas and Trout (1990) looked at the efficiency of utilization of the
entire diet, the apparant digestibility method used in this study measured the efficiency of
utilization of individual nutrients, and may have been more sensitive. Additionally,
whereas Pekas and Trout (1990) reported G/F for the entire trial, thereby allowing time
for adaptability, the measurements in this trial were taken in early lactation (LD8 to
LD12), probably before a higher rate of digestive enzyme synthesis could manifest itself.
Digestibility of ash was greater (P<.02) in lactation than in gestation and may reflect the
large mineral demands of milk synthesis. Digestibility of lignin was also greater (P<.02)

in lactation.

Table 24. Digestibility coefficients measured in gestation and lactation for CCK
immunized sows“

 

Stage GE Starch CP EE" Ash NDF ADF Lignin

 

Gestation 8291.9 98.91 .2c 77.71l.0 24216.0 31.7113c 53912.0 53913.0 48.1112c

Lactation 81.019 98.312“ 79911.0 32315.8 44611.9“ 51512.0 53212.9 54211.2“

“1 SEM.
bEther extract.
c“Means within the same column with different superscripts are different, P<.02.

 

94

Nutrient digestibility of gilts vaccinated against CCK does not appear to be
impeded either in gestation nor in lactation as serum CCK-AB titers increase. These data
would suggest that the CCK-AB of the vaccinated gilts are not impairing pancreatic
release of digestive enzymes. In gestation, the digestibility of GE, starch, CP, EE, and
ash was actually enhanced as CCK-AB increased. Spencer (1992) offered three
explanations as to why the immunization procedure may actually potentiate rather than
neutralize CCK action;

1) The CCK-AB raised may have had too low an affinity to effectively prevent the
hormone from binding to the receptor.

2) The AB-bound hormone may be presented to the receptor in such a way as to
enhance it's orientation at the receptor binding sites.

3) The AB-bound hormone may extend the hormone's transmembrane
effectiveness by inhibiting internalization and clearance of the
hormone.

Within this discussion these mechanisms would be limited to the receptors of the
pancreas, because as previously described, the lactation ADFI and litter weight gain of
these CCK immunized sows paralleled increases in CCK-AB titer.

It is also possible that production of CCK may have been up-regulated as a result
of immunoneutralization. As the pancreas is in close proximity to the site of CCK

release, it may have had greater exposure to CCK before it was diluted and neutralized in

the periphery.

95

We

As done in Experiment 1 and 2, an assessment was made of the incidence of
constipation among sows. Due to missing observations, the number of sows in this data
set totaled nine (n=9). Nevertheless, some interesting observations were made: Using
regression analysis it was observed that sows with softer feces at LD3 had a greater
(r2=.39, P<.10) week 2 ADFI. Similarly, sows with sofier feces at LDlO had a greater
(r2=.46, P<.05) week 2 ADFI and a greater (11:.40, P<.10) overall ADFI. Interestingly,
sows with firmer feces at LD3 were able to digest more (r2=.45, P<.05) starch, and more
(r2=.47, P<.05) CP, and tended (r2=29, P=.13) to digest more dietary GB in lactation.
These observations may be related to the differences in the rate of digesta transit time
through the GI tract. It is presumed that sows with firmer feces (lower water content) had
a longer transit time, allowing greater time for digestive enzymes to work.

The relationship between the actual vaccination order of the gilts and their
resulting titer was determined via regression analysis. This analysis included complete
records on 10 gilts (n=10). The results demonstrated that gilts vaccinated closer to the
end of the vaccination order at time B2 had higher (r2=.43, P<.05) B3 titers, higher
(r2=.31, P<.10) colostrum titers, and higher (r2=.30, P<.10) M-LD21 titers. Gilts
vaccinated closer to the end of the vaccination order at time B2 also tended (r2=29,
P=.11) to have higher M-LDl4 titers, and their pigs tended (r2=.24, P=.15) to exhibit
greater titer values. These limited observations would at least suggest that vaccine
preparation and/or vaccination order should be carefully considered prior to execution of

the next experiment.

96

W

This experiment was designed to be analyzed via linear regression using CCK-AB
titer as the dependent variable. Many strong relationships between CCK-AB titer and
sow performance and CCK-AB titer and pig performance during lactation were
discovered. However, some relationships appeared to lack continuity; consisting of low
titer (mean lactation sow serum titer < 1.0), and high titer (mean lactation sow serum titer
> 1.0) groups of sows. Therefore, the data was reanalyzed via the GLM procedure of
SAS, using only treatment in the model statement. Using this analysis, high titer (HT)
sows consumed more (P<.01) feed than low titer (LT) sows during week 3 of lactation
(ADFI = 5.41 and 4.40 kg respectively). The mean ADF I for the HT sows was also
greater (P<.06) than LT sows throughout the 21 d lactation period (4.93 vs 4.39 kg
respectively). Similarly, litter weight gains during week 3 of lactation were greater
(P<.03) for HT sows than LT sows (14.38 vs. 10.56 kg respectively), and total litter
weight gains were greater (P<.10) for HT sows than LT sows (32.18 vs. 37.83 kg
respectively).
I l' . [E . 3

These results show that CCK immunization has the potential to improve
performance of primiparous gilts. CCK immunization increased ADF I during the third
week of lactation by about .62 kg for each unit of log titer increase, or about 1.8 kg/hd/d
for high titer sows. Effects on sow body weight change and body compositional changes
were equivocal. Although changes in body composition of the sows was not clearly

influenced by CCK immunization, backfat depth was reduced as anti-CCK titer

97

increased.

Immunization of first parity gilts against CCK has potential in the swine industry
to increase feed intake resulting in increased milk secretion and litter weight gain. This
new technology has potential to (1) produce heavier piglets at weaning which would be
expected to maintain this weight advantage and attain market weight in less time, or (2)
produce heavier piglets which could be weaned earlier (segregated early weaning
[SEW]), with less incidence of mortality and morbidity. At this time it is not known what
direct (if any) effect the passively derived CCK immunoglobulins may exert on the
piglet. Although the half-life of passively derived immunoglobulins is short (3 to 4
weeks), they may have tremendous potential in SEW situations.

The potentially negative affect of impaired or reduced nutrient digestibility as a
result of CCK vaccination was not observed in this trial. In gestation, it appeared that
nutrient digestibility was actually improved in gilts with higher CCK-AB titers.
Additional studies which follow similar protocols should certainly be conducted to

confirm these observations.

APPENDIX

APPENDIX

W
D .. [5. T' 9' :11 1..
393291115
° i vi

2.68 g NazHPO4 H20 (.01 M) (Sigma cat.# S-9390)

0.37 g EDTA Naz (.001 M) (Sigma cat.# ED2SS)

1.00 g Na azide (.1% w/v) (Sigma cat. #S-2002)

8.10 g NaCl (.14 M) (Sigma cat. # S-9888)

1.00 g Gelatin (.1% w/v) (Sigma G-2500)
Place in 1000 mL volumetric flask and partially fill to volume with deionized distilled
water, Stir on low heat on magnetic stirrer in order to dissolve gelatin. After gelatin is
dissolved, solution is cooled to room temperature. Solution is then adjusted to pH 7.5
with 1 N HCL. Finish filling the flask to volume with deionized distilled water. Check
to be sure pH is still 7.5.

2 “V a t r

2.68 g NazHPO4 H20 (.01 M) (Sigma cat.# S-9390)

0.37 g EDTA Na2 (.001 M) (Sigma cat.# ED2SS)

1.00 g Na azide (.1% w/v) (Sigma cat. #S-2002)

8.10 g NaCl (.14 M) (Sigma cat. # S-9888)

0.50 g Gelatin (.1% w/v) (Sigma G-2500)
Place in 1000 mL volumetric flask and partially fill to volume with deionized distilled

98

99

water, Stir on low heat on magnetic stirrer in order to dissolve gelatin. After gelatin is
dissolved, solution is cooled to room temperature. Solution is then adjusted to pH 7.5
with 1 N HCL. Finish filling the flask to volume with deionized distilled water. Check
to be sure pH is still 7.5.
% - ' ° fe

1.25 g Norit A charcoal (Fisher cat. # C176-500)

0.125 g Dextran, average molecular weight = 79100 (Sigma cat. # D-4751)

100 mL of 0.05% Gel Buffer, pH 7.5
Place in 150 mL beaker and stir at room temperature on magnetic stirrer for 30 minuets.
Store in refrigerator. Stir continuously on magnetic stirrer over crushed ice while using
during the assay procedure.

41.11329:

['251]-Bolton Hunter-CCK-8s (DuPont NEN cat. # NEX-203)
The ul of concentrated tracer used will depend on the size of the assay, but will be diluted
with 0.1% Gel Buffer, pH 7.5 to give a final radio concentration of 4884 dpm/ 100 111
assay (1 frnol CCK-8s/ 100 111 assay). Mix on rocker table for 30 minuets at room
temperature. Tracer is then quantitatively transferred to another tube to prevent any
bound tracer on walls of tube from leaching into the solution. Fifty ul aliquots are then
pipetted into 12 X 75 mm tubes and counted to determine actual dpm being put into assay

tubes.

100
mm
For assays in Dr. J.C. Pekas' lab (USDA, ARS, MARC), 25 ul of pig 42307 (Expt.
#JP8704, CCK immunized, blood collected on 1/28/88) is diluted in 25 mL of 0.05% gel
buffer, pH 7.5 (1 :1000 dilution) )which gives specific binding of approximately 50%.
Diluted antisera is then aliquoted into 300 ul amounts into 12 X 75 mm assay tubes and
stored at -20°C. These tubes are used directly in each assay run.
' er ar ' il '
Defatted colostrum and milk samples were prepared by initially aliquoting 1 mL of each
sample into a 12 X 75 glass test tube. Samples were centrifuged at 2,000 X G for 30
minuets at 4°C. The resulting fat "layer" on the top of each sample was gently pierced,
and the supemate was poured into another 12 X 75 glass test tube. Raw antisera and
defatted colostrum and milk were diluted as follows:
A) 1:10 111 ul of sample + 1,000 111 of 0.05% gel buffer
B) 1:100 111 111 of "A" + 1,000 ul of 0.05% gel buffer
C) 1:1,000 111 ul of "B" + 1,000 111 of 0.05% gel buffer

A) 1:10,000 111 ul of "C" + 1,000 ul of 0.05% gel buffer

AW

Total Counts (TC) ' 100 111 of tracer

Non Specific Binding (N SB) 300 ul of 0.05% gel buffer + 100 ul of tracer
Reference Antisera (RA) 300 ul of reference antisera + 100 111 of tracer

Unknowns 300 ul of unknowns + 100 111 of tracer

8.

101

. Pipette 300 ul of each antisera dilution in triplicate into 12 X 75 mm glass test tubes.

For "NSB" tubes use 300 ul of 0.05% gel buffer.

. Add 100 u! of tracer to each tube and gently vortex.
. Cover racks of assay tubes with alurrrinurn foil and incubate for 48 hours at 4°C.

. After 48 hours add 250 ul of ice-cold dextran-coated charcoal to tubes. Shake rack of

tubes vigorously to mix. Incubate for 10 minuets at 4°C. Do not add charcoal to TC
tubes.

Afier the 10 minuet incubation, add 300 ul of ice-cold 0.05% gel buffer to tubes and
shake the rack of tubes vigorously to mix. Incubate an additional 10 minuets at 4°C.
After the second 10 minuet incubation, centrifuge tubes for 5 minuets in 4°C
centrifuge at 2,000 X G.

Remove tubes from centrifuge and immediately remove supemate from charcoal
pellet using disposable 9" Pasteur pipettes. Place supematants in corresponding 12 X
75 mm glass test tubes. Keep other tubes on ice until they are ready to be separated.

Count both the supernates and charcoals in a gamma counter for 2 minuets.

Calculations

Nonspecific binding = cpm of NSB/cpm of TC

Specific binding = (cpm of unknown supemate - cpm of NSB) / (cpm of TC - cpm of

NSB)

102
W

1) Weigh 1.0g (1.05g) air-dry sample into a Berzelius beaker.

2) Add 100 mL of room temperature neutral detergent (ND), 2 ml of a 20% solution of
amylase (N ovo Termamyl), and .Sg sodium sulfite. Sodium sulfite was used to
break disulfide bonds present in the feces.

3) Boil beaker contents under reflux for 60 minuets. As beakers are removed from the

heat source, add an additional 2 mL of a 20% solution of amylase (Novo Termamyl).

4) Filter beaker contents under suction through fritted glass crucibles.

A) While on the suction manifold, rinse the filtrate 3 times with hot water
(90-100° C). To ensure that all detergent is completely removed, rinse the outside
and bottom of each crucible with hot water as it is removed from the filter
manifold.

5) While under an exhaust hood, apply suction to filtrates and rinse with acetone to

facilitate drying. Allow acetone to completely evaporated.

6) Dry crucibles at 106° C for a minimum of 8 hours and hot-weigh.

The NDF can now be subjected to acid-detergent fiber analysis procedures.

l 112 El :lDE:

l) Loosen the fiber mat remaining in the bottom of each crucible by placing crucibles in

a saucepan containing an amount of acid detergent (AD) sufficient enough to cover

the NDF mat. Boil for 5 minutes.

103

2) Rinse the crucibles two times each with 50 mL room temperature AD into a Berzelius
beaker. Particles adhering to the crucible can be loosened with the help of a rubber
policeman.

3) Place beakers on a heat source and allow to boil under reflux for 55 minutes.

4) As beakers are removed from the heat source, empty beaker contents back into their
respective crucibles and filter under suction.

A) While on the suction manifold, rinse the filtrate 3 times with hot water
(90-100° C). To ensure that all detergent is completely removed, rinse the outside
and bottom of each crucible with hot water as it is removed from the filter
manifold.

5) Under an exhaust hood apply suction to filtrates and rinse with acetone to facilitate
drying. Allow acetone to completely evaporate.

6) Dry crucibles at 106° C for a minimum of 8 hours and hot-weighed.

The ADF can now be subjected to lignin analysis procedures.

WWW

Prior to starting this procedure, place 72% H2804 on ice and allow it to cool to about

15° C.

1) Place crucibles containing the ADF in shallow Pyrex pans and add an amount of
cooled 72% H2804 sufficient enough to cover the ADF fiber mat.

2) Place a Teflon rod into each crucible and stir the contents into a paste.

3) Add fresh 72% H2SO4 to the samples as it drains through the crucibles.

4) Allow samples to soak in the 72% H2804 for 3 hours at room temperature.

104
5) After 3 hours, fill crucibles 2/3 full with hot water (90-100° C).

6) Rinse material adhering to the Teflon rods into the respective crucible, and remove the

rods.
7) Filter crucible contents under suction and rinse 3 times with hot water (90-100° C).

A) To ensure that all acid is completely removed, rinse the outside

and bottom of each crucible with hot water as it is removed from the filter
manifold.

8) Dry crucibles at 106° C for a minimum of 8 hours and hot-weighed.
9) Place the lignin-containing crucibles in a muffle furnace at 500° C for a minimum of 6

hours to ash. Allow crucibles to cool to 200° C, place in drying oven and hot-weighed

at 106° C.

c l ' f ' i
Fiber fractions were calculated as a percent of the dry matter of the sample in the
following manner where;
N = crucible and NDF residue
A = crucible and ADF residue
L = crucible and LDF residue
X = crucible and ash residue
DM = g of sample dry matter
NDF = (N-X)/DM
ADF = (A-X)/DM

ADL = (L-X)/DM

105

I} . . E 1 EB:

1) Using a pencil, sequentially number Whatrnan filter paper (#4, 11 cm) and place it in a
drying oven (106° C) overnight, and hot-weigh.

2) Allow the filter paper to cool to room temperature and weigh approximately 2 g of
sample onto the filter paper.

3) Fold the paper carefully so as to ensure that all of the sample remains contained.
Secure the ends with paper clips.

4) Place the filter papers containing the samples "Lincoln-log" style into the modified
Soxhlet ether extraction apparatus under a hood.

In this system, ether is heated to boiling, condenses and fills a container holding
the samples. Samples soak in the condensed ether and ether-soluble compounds are
released into the solvent. Polluted ether is frequently removed by siphon and replaced by
freshly condensed ether. Samples remained in this system overnight (approximately 16
hours).

5) Remove samples from the ether extraction devise, gently remove paper clips and
allow the ether to evaporate from the samples under a hood.
6) Place samples in a drying oven (106° C) overnight and hot-weighed.
The % EE (fat) of the samples is calculated as follows where;
DM = g of sample dry matter
EX = ether-extracted sample weight
PW = paper weight

EE = ((DM - (EX - PW))/DM) * 100

106
Sample CP concentration was determined using a modification of the procedure

described by Hach et. al (1987).

1) Weigh .25 g (1.005g) feed or .20 g (1.005g) feces onto a precut tissue paper
measuring approximately 6 X 6 cm.

2) Fold the tissue containing the sample and slip it gently into a 100 mL Digestahl flask.

3) Add 4 mL concentrated H2804 to each flask and swirl gently to allow the paper and
sample to become saturated with acid.

4) Attach aspirators with condenser columns and capillary funnels to each flask and
place on burners at 440° C.

5) After 6 minutes place 20 mL of 50% H202 into the capillary funnel of each
apparatus. Heat flasks continually and allow them to remain on the burners for an
additional 1-2 minutes after the H202 is completely emptied from the capillary funnel.

6) Remove the flasks from the burners and remove condensers from the flasks once

fumes are no longer visible in the flasks.

7) When the flasks have cooled to room temperature, diluted flask contents to 100 mL
with distilled H20. Stopper and mixed thoroughly.

8) Prepare a set of standards using NH3C1 and distilled H20 as follows:

 

' SLQ 11.9.1 11.112 11.95 9.96 11.9.8
mL stock solution 0.0 0.1 0.2 0.4 0.6 0.8

(0.3056 g NH3Cl/L distilled H20)
mL distilled H20 0.8 0.7 0.6 0.4 0.2 0.0

 

107

9) Add 25 mL of a .lg/L solution of polyvinyl alcohol and 1 mL of Nessler's reagent
to each standard and to each .8 mL aliquot of sample. Mixed thoroughly.
10) Read absorbance values via spectrophotometer at 460 nm visible light within 1 hour
of adding the Nessler's reagent.
1]) Estimate nitrogen concentration of the samples.

A) Use the linear equation derived from the regression of the standards where:

mg N/mL = absorbance-4mm
slope

B) Convert mg N/mL to a sample basis:
mg N/sample = mg N/mL * 100
C) Calculate percent N:
%N = ((mg N/sample) / (mg sample DM used)) * 100
12) Calculate crude protein of the samples:

%CP = %N * 6.25.

108

I; . . E l

l) Weigh a .2 g (1.01g) feed sample or a .6 g (1.01 g) fecal sample and place it into a
125 ml Erlenmeyer flask containing 20 mL distilled H20. Allow the samples to
hydrate for at least 4 hours.

2) Add .5 mL 50% NaOH to each flask while gently swirling. Allowed samples to
gelatinize for 15 minutes.

3) Add 15 mL distilled H20 and 10 mL acetate buffer and gently swirl.

4) Add .8 mL concentrated HCL and 250 ul Amylase (Diazyme L-200, Solvay Enzymes)
to each flask and gently swirled.

5) Seal with a cork stopper and set in a 55° C water bath for 14 hours.

6) After the incubation period, transfer the flask contents to a 200 mL volumetric flask
and diluted with distilled H20 to 200 mL. Mixing thoroughly.

7) Place a 5 mL aliquot into a centrifuge tube and spin at 26,000 X G for 20 minutes.

8) Transfer a 900 111 aliquot of the resulting supemate to a vial and analyzed for glucose
concentration by HPLC.

9) Convert sample glucose concentration to starch concentration.

109

f 'umtu o it. o 1.". .1 o_ ° 0 n c; talk hr i 100-

By adding distilled water to a known amount of ground corn stalk material it was
estimated that 1 g of air dry ground corn stalk material could absorb approximately 8.0 g
of water. Based on this information, a 3.4 % solution of NazCr207 (.034 g
NazCrzO7/mL distilled water) was prepared. When applied to the ground corn stalk
material as described below, this concentration of NaZCrzO7 provided a chromium
concentration equal to approximately 11% of the weight of the fiber (Uden et al 1980).

1129111111.:

1) Weigh the fiber material into a shallow aluminum pan.

2) Slowly pour the solution of NazCr207 over the fiber until the solution covers
the fiber completely. Allow ample time for the fiber to become completely saturated.

3) Cover the aluminum pan with aluminum foil, being careful to stretch the foil tight so
as not to have the foil touching the NazCrZO7 solution (otherwise the solution will run
out of the pan via capillary action).

4) Bake in an oven at 100°C for 24 hours. Make sure the foil is sealed tightly around all
edges so that the fiber is baked in a moist environment (otherwise the fiber may dry
out and burn).

5) Place the fiber in a pillowcase and tie it securely to a water faucet. Manually agitate

the fiber within the pillow case while letting the water run vigorously for at least an
houn

6) Remove the fiber from the pillowcase and suspend the fiber in tap water. Add an

amount of ascorbic acid equal to one half the weight of the dry fiber, and let stand for

110

one hour.

7) Again place the fiber in a pillowcase and tie it securely to a water faucet. Manually

agitate the fiber within the pillow case while letting the water run vigorously for at
least an hour , and until it is free of soluble green matter.

8) Dry the material in an oven at 65°C.

111

l) Weigh approximately 1 g feed or .5 g feces into a 250 mL Phillip beaker.

2) Add 20 mL of nitric acid (14M) and 4 mL of perchloric acid (10M) to each sample.

3) Digest samples on hot plates under a perchloric hood until approximately 1 mL of

volume remain in the flask.

4) Remove flasks from the hot plate and wash the interior sides with deionized water.

5) Once flasks have completely cooled, add a sufficient amount of deionized water to
attain a net weight of about 50 g. Samples which contained high Cr concentrations
were further diluted with distilled water.

6) The concentrations of Cr in the samples were determined by flame atomic absorption
spectrophotometry (model I1 951, Instrumentation Laboratory, Wilmington, MA). The
final concentration of Cr was calculated using the following formula where:

PPM = concentration of mineral in part per million
D = final dilution

F WF = final weight of the flask

F W = initial weight of the flask

C = concentration of Cr (ppm)

SW = sample weight

PPM = [(D)(FWF-FW)(C)] / sw

112
WW
1) Press approximately 0.8 g of sample into a pellet with the aid of a hydraulic jack, and
record weight.
2) Completely combust the sample in an Parr adiabatic bomb calorimeter (Parr
Instrument Company, Moline, IL)
3) Titrate the resulting residue with 0.725 N sodium carbonate to correct for heat
generated by nitric acid formation.
4) The final GE content was calculated with the following formula where:
A = temperature change recorded from bomb calorimeter
B = bomb adjustment factor
C = kcal energy contained in nitric acid produced during the combustion
D = kcal energy contributed by ignition wire

GE, kcal/g = ((A * B) - ( C + D))

LITERATURE CITED

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Allen, MS. and DR. Mertens. 1988. Evaluating constraints on fiber digestion by rumen
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